U.S. patent application number 12/184956 was filed with the patent office on 2009-03-26 for glycopegylated factor ix.
This patent application is currently assigned to Neose Technologies, Inc.. Invention is credited to Robert J. Bayer, Caryn Bowe, Shawn DeFrees, Krishnasamy Panneerselvam.
Application Number | 20090081188 12/184956 |
Document ID | / |
Family ID | 35910388 |
Filed Date | 2009-03-26 |
United States Patent
Application |
20090081188 |
Kind Code |
A1 |
DeFrees; Shawn ; et
al. |
March 26, 2009 |
GLYCOPEGYLATED FACTOR IX
Abstract
The present invention provides conjugates between Factor IX and
PEG moieties. The conjugates are linked via an intact glycosyl
linking group interposed between and covalently attached to the
peptide and the modifying group. The conjugates are formed from
glycosylated peptides by the action of a glycosyltransferase. The
glycosyltransferase ligates a modified sugar moiety onto a glycosyl
residue on the peptide. Also provided are methods for preparing the
conjugates, methods for treating various disease conditions with
the conjugates, and pharmaceutical formulations including the
conjugates.
Inventors: |
DeFrees; Shawn; (North
Wales, PA) ; Bayer; Robert J.; (San Diego, CA)
; Bowe; Caryn; (Doylestown, PA) ; Panneerselvam;
Krishnasamy; (Poway, CA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP (SF)
One Market, Spear Street Tower, Suite 2800
San Francisco
CA
94105
US
|
Assignee: |
Neose Technologies, Inc.
Horsham
PA
|
Family ID: |
35910388 |
Appl. No.: |
12/184956 |
Filed: |
August 1, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11166028 |
Jun 23, 2005 |
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12184956 |
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PCT/US2004/041070 |
Dec 3, 2004 |
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11166028 |
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60684729 |
May 25, 2005 |
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60527089 |
Dec 3, 2003 |
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60539387 |
Jan 26, 2004 |
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60592744 |
Jul 29, 2004 |
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60614518 |
Sep 29, 2004 |
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60623387 |
Oct 29, 2004 |
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Current U.S.
Class: |
424/94.64 ;
435/188; 435/68.1 |
Current CPC
Class: |
A61P 7/04 20180101; A61K
47/60 20170801; Y02A 50/471 20180101; A61K 38/4846 20130101; A61K
47/549 20170801; A61P 7/00 20180101 |
Class at
Publication: |
424/94.64 ;
435/188; 435/68.1 |
International
Class: |
A61K 38/48 20060101
A61K038/48; C12N 9/96 20060101 C12N009/96; C12P 21/04 20060101
C12P021/04; A61P 7/00 20060101 A61P007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 3, 2004 |
US |
PCT/US2004/041070 |
Claims
1. A Factor IX peptide conjugate comprising a glycosyl linking
group attached to an amino acid residue of said peptide, said
glycosyl linking group comprising a modified sialyl residue having
the formula: ##STR00064## wherein R.sup.2 is H, CH.sub.2OR.sup.7,
COOR.sup.7 or OR.sup.7 wherein R.sup.7 represents H, substituted or
unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
R.sup.3 and R.sup.4 are members independently selected from H,
substituted or unsubstituted alkyl, OR.sup.8, NHC(O)R.sup.9 wherein
R.sup.8 and R.sup.9 are independently selected from H, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl or
sialic acid; L.sup.a is a linker selected from a bond, substituted
or unsubstituted alkyl and substituted or unsubstituted heteroalkyl
R.sup.16 and R.sup.17 are independently selected polymeric arms;
X.sup.2 and X.sup.4 are independently selected linkage fragments
joining polymeric moieties R.sup.16 and R.sup.17 to C; and X.sup.5
is a non-reactive group.
2. The peptide conjugate according to claim 1, wherein the moiety:
##STR00065## has a formula that is a member selected from:
##STR00066## wherein Q is selected from H and substituted or
unsubstituted C.sub.1-C.sub.6 alkyl; e and f are integers
independently selected from 1 to 2500; and q is an integer from 0
to 20.
3. The peptide conjugate according to claim 2, wherein said moiety
has a formula that is a member selected from: ##STR00067## wherein
Q is selected from H and substituted or unsubstituted
C.sub.1-C.sub.6 alkyl; e, f and f' are integers independently
selected from 1 to 2500; and q and q' are integers independently
selected from 1 to 20.
4. The peptide conjugate according to claim 1, wherein said
glycosyl linking group has a formula selected from:
##STR00068##
5. The peptide conjugate according to claim 4, wherein said
glycosyl linking group has the formula: ##STR00069##
6. The peptide conjugate according to claim 5, wherein said
glycosyl linking group attached to said amino acid residue has the
formula: ##STR00070## wherein AA is said amino acid residue of said
peptide.
7. The peptide conjugate according to claim 6, wherein said amino
acid residue is a member selected from serine or threonine.
8. The peptide conjugate according to claim 1, wherein said peptide
has the amino acid sequence of SEQ. ID. NO: 1.
9. The peptide according to claim 8, wherein said amino acid
residue is a serine at position 61 of SEQ. ID. NO:1.
10. The peptide conjugate according to claim 1, wherein said
peptide comprises at least one glycosyl linking group comprising a
substructure having the formula: ##STR00071## wherein R.sup.15 is
said modified sialyl residue; and p is an integer from 1 to 10.
11. The peptide conjugate according to claim 10, wherein said at
least one glycosyl linking group attached to an amino acid of said
peptide has a formula selected from: ##STR00072## and combinations
thereof wherein AA is said amino acid residue of said peptide; t is
an integer equal to 0 or 1; p is an integer from 1 to 10; and
R.sup.15' is a member selected from H, OH, sialic acid, said
modified sialyl residue and Sia-Sia.sup.p wherein Sia.sup.p is said
modified sialyl residue, wherein at least one R.sup.15' is selected
from said modified sialyl residue and Sia-Sia.sup.p.
12. The peptide conjugate according to claim 11, wherein said amino
acid residue is an asparagine residue.
13. The peptide conjugate according to claim 12, wherein said
peptide has the amino acid sequence of SEQ ID NO: 1.
14. The peptide conjugate according to claim 13, wherein said
glycosyl residue is attached to a member selected from Asn 157, Asn
167 and combinations thereof.
15. A method of preparing a peptide conjugate according to claim 1,
said method comprising: (a) contacting a substrate Factor IX
peptide comprising a glycosyl moiety selected from: ##STR00073##
with a PEG-sialic acid donor having the formula: ##STR00074##
wherein c is 0 or 1; and (b) contacting said Factor IX and said
PEG-sialic acid donor with an enzyme that transfers PEG-sialic acid
from said donor onto said glycosyl moiety, under conditions
appropriate for said transfer.
16. The method of claim 15, further comprising, prior to step (a):
(b) expressing said substrate Factor IX peptide in a suitable
host.
17. The method of claim 16, wherein said host is selected from an
insect cell and a mammalian cell.
18. The method of claim 17, wherein said insect cell is a
Spodoptera frugiperda cell line.
19. A method of stimulating blood coagulation in a mammal, said
method comprising administering to said mammal a peptide conjugate
according to claim 1 in an amount sufficient to stimulate said
blood coagulation.
20. A method of treating hemophilia in a subject, said method
comprising administering to said subject a peptide conjugate
according to claim 1 in an amount effective to treat said
hemophilia.
21. A pharmaceutical formulation comprising the Factor IX peptide
conjugate according to claim 1, and a pharmaceutically acceptable
carrier.
22. A Factor IX peptide conjugate comprising a glycosyl linking
group attached to an amino acid residue of said peptide, said
glycosyl linking group comprising a modified sialyl residue having
the formula: ##STR00075## wherein R.sup.2 is H, CH.sub.2OR.sup.7,
COOR.sup.7, COO.sup.- or OR.sup.7 wherein R.sup.7 represents H,
substituted or unsubstituted alkyl or substituted or unsubstituted
heteroalkyl; R.sup.3 and R.sup.4 are members independently selected
from H, substituted or unsubstituted alkyl, OR.sup.8, NHC(O)R.sup.9
wherein R.sup.8 and R.sup.9 are independently selected from H,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl or sialic acid; s is an integer from 1 to 20; f is an
integer from 1 to 2500; and Q is a member selected from H and
substituted or unsubstituted C.sub.1-C.sub.6 alkyl.
23. The peptide conjugate according to claim 22, wherein said
modified sialyl residue has the formula: ##STR00076##
24. The peptide conjugate according to claim 23, wherein Q is
selected from H and CH.sub.3.
25. The peptide conjugate according to claim 22, wherein said
peptide comprises at least one glycosyl linking group comprising a
substructure having the formula: ##STR00077## wherein R.sup.15 is
said modified sialyl residue; and p is an integer from 1 to 10.
26. The peptide conjugate according to claim 25, wherein said at
least one glycosyl linking group attached to an amino acid of said
peptide has a formula selected from: ##STR00078## and combinations
thereof wherein AA is said amino acid residue of said peptide; t is
an integer equal to 0 or 1; p is an integer from 1 to 10; and
R.sup.15' is a member selected from H, OH, sialic acid, said
modified sialyl residue and Sia-Sia.sup.p wherein Sia.sup.p is said
modified sialyl residue, wherein at least one R.sup.15' is selected
from said modified sialyl residue and Sia-Sia.sup.p.
27. The peptide conjugate according to claim 22, wherein said amino
acid residue is asparagine.
28. The peptide conjugate according to claim 27, wherein said
peptide has the amino acid sequence of SEQ ID NO: 1.
29. The peptide conjugate according to claim 28, wherein said
glycosyl residue is attached to a member selected from Asn 157, Asn
167 and combinations thereof.
30. The peptide conjugate according to claim 22, wherein said
glycosyl linking group comprises the formula: ##STR00079## in which
b is 0 or 1.
31. The peptide conjugate according to claim 30, wherein s is 1;
and f is an integer from about 200 to about 300.
32. The peptide conjugate according to claim 22, wherein said amino
acid residue is a member selected from serine or threonine.
33. The peptide conjugate according to claim 32, wherein said
peptide has the amino acid sequence of SEQ. ID. NO:1.
34. The peptide conjugate according to claim 33, wherein said amino
acid residue is a serine at position 61 of SEQ. ID. NO:1.
35. A method of preparing the peptide conjugate according to claim
22, said method comprising: (a) contacting a substrate Factor IX
peptide comprising a glycosyl moiety selected from: ##STR00080##
with a PEG-sialic acid donor having the formula: ##STR00081##
wherein c is 0 or 1; r is 0 or 1; and (b) contacting Factor IX and
said PEG-sialic acid donor with an enzyme that transfers PEG-sialic
acid from said donor onto said glycosyl moiety, under conditions
appropriate for said transfer.
36. The method of claim 35, further comprising, prior to step (a):
(b) expressing said substrate Factor IX peptide in a suitable
host.
37. The method of claim 36, wherein said host an insect cell.
38. The method of claim 37, wherein said insect cell is a
Spodoptera frugiperda cell line.
39. A method of stimulating blood coagulation in a mammal, said
method comprising administering to said mammal a peptide conjugate
according to claim 22 in an amount sufficient to stimulate said
blood coagulation.
40. A method of treating hemophilia in a subject, said method
comprising administering to said subject a peptide conjugate
according to claim 22 in an amount effective to treat said
hemophilia.
41. A pharmaceutical formulation comprising the Factor IX peptide
conjugate according to claim 22, and a pharmaceutically acceptable
carrier.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 11/166,028, filed Jun. 23, 2005, which claims
priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Patent
Application No. 60/684,729, filed May 25, 2005; U.S. patent
application Ser. No. 11/166,028, filed Jun. 23, 2005, which is a
continuation-in-part of PCT Application No. PCT/US2004/041070,
filed Dec. 3, 2004, which claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Patent Application No. 60/527,089, filed
Dec. 3, 2003, U.S. Provisional Patent Application No. 60/539,387,
filed Jan. 26, 2004, U.S. Provisional Patent Application No.
60/592,744, filed Jul. 29, 2004, U.S. Provisional Patent
Application No. 60/614,518, filed Sep. 29, 2004, and U.S.
Provisional Patent Application No. 60/623,387, filed Oct. 29, 2004,
each of which are 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 best 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. Gas6 is a growth arrest hormone encoded by growth
arrest-specific gene 6 (gas6) 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 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 IX.sup.a. 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 X.sup.a 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. Chem. 272: 11863-11868 (1997). Active site modified APC binds
to factor Va and prevents factor X.sup.a 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 .mu.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 protein glycosylation is the
relatively short in vivo half life exhibited by these peptides.
Overall, the problem of shot in vivo half life means that
therapeutic glycopeptides must be administered frequently in high
dosages, which ultimately translate to higher health 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 two hours and at high dosages (45
to 90 .mu.g/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 pharmacokinetic properties have been produced by attaching
synthetic polymers to the peptide backbone. An exemplary polymer
that has been conjugated to peptides is poly(ethylene 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 (Davis 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. Pat. No. 4,088,538 U.S.
Pat. No. 4,496,689, U.S. Pat. No. 4,414,147, U.S. Pat. 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, random 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
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 in 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. Pat. No. 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 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 poly(ethylene glycol) moieties
affords a novel Factor IX derivative with pharmacokinetic
properties that are improved relative to the corresponding native
(un-pegylated) Factor IX (FIG. 3). Moreover, the glycoPEGylated
Factor IX retains its pharmacological activity (FIG. 4).
[0016] In an exemplary embodiment, "glycopeglyated" Factor IX
molecules of the invention are produced by the enzyme mediated
formation of a conjugate between a glycosylated or non-glycosylated
Factor IX peptide and an enzymatically transferable saccharyl
moiety that includes a poly(ethylene glycol) moiety within its
structure The PEG moiety is attached to the saccharyl moiety
directly (i.e., through a single group formed by the reaction of
two reactive groups) or through a linker moiety, e.g., substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
etc. An exemplary transferable PEG-saccharyl structure is set forth
in FIG. 7.
[0017] The polymeric modifying moiety can be attached at any
position of a glycosyl moiety of Factor IX. Moreover, the polymeric
modifying moiety can be bound to a glycosyl residue at any position
in the amino acid sequence of a wild type or mutant Factor IX
peptide.
[0018] In an exemplary embodiment, the invention provides an Factor
IX peptide that is conjugated through a glycosyl linking group to a
polymeric modifying moiety. Exemplary Factor IX peptide conjugates
include a glycosyl linking group having a formula selected
from:
##STR00001##
[0019] In Formulae I and II, R.sup.2 is H, CH.sub.2OR.sup.7,
COOR.sup.7 or OR.sup.7, in which R.sup.7 represents H, substituted
or unsubstituted alkyl or substituted or unsubstituted heteroalkyl.
The symbols R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.6'
independently represent H, substituted or unsubstituted alkyl,
OR.sup.8, NHC(O)R.sup.9. The index d is 0 or 1. R.sup.8 and R.sup.9
are independently selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl or sialic acid. At
least one of R.sup.3, R.sup.4, R.sup.5, R.sup.6 or R.sup.6'
includes the polymeric modifying moiety e.g., PEG. In an exemplary
embodiment, R.sup.6 and R.sup.6', together with the carbon to which
they are attached are components of the side chain of sialic acid.
In a further exemplary embodiment, this side chain is
functionalized with the polymeric modifying moiety.
[0020] In an exemplary embodiment, the polymeric moiety is bound to
the glycosyl linking group, generally through a heteroatom on the
glycosyl core (e.g., N, O), through a linker, L, as shown
below:
##STR00002##
R.sup.1 is the polymeric modifying moiety and L is selected from a
bond and a linking group. The index w represents an integer
selected from 1-6, preferably 1-3 and more preferably 1-2.
Exemplary linking groups include substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl moieties and sialic
acid. An exemplary component of the linker is an acyl moiety.
Another exemplary linking group is an amino acid residue (e.g.,
cysteine, serine, lysine, and short oligopeptides, e.g., Lys-Lys,
Lys-Lys-Lys, Cys-Lys, Ser-Lys, etc.)
[0021] When L is a bond, it is formed by reaction of a reactive
functional group on a precursor of R.sup.1 and a reactive
functional group of complementary reactivity on a precursor of the
glycosyl linking group. When L is a non-zero order linking group, L
can be in place on the glycosyl moiety prior to reaction with the
R.sup.1 precursor. Alternatively, the precursors of R.sup.1 and L
can be incorporated into a preformed cassette that is subsequently
attached to the glycosyl moiety. As set forth herein, the selection
and preparation of precursors with appropriate reactive functional
groups is within the ability of those skilled in the art. Moreover,
coupling of the precursors proceeds by chemistry that is well
understood in the art.
[0022] In an exemplary embodiment L is a linking group that is
formed from an amino acid, or small peptide (e.g., 1-4 amino acid
residues) providing a modified sugar in which the polymeric
modifying moiety is attached through a substituted alkyl linker.
Exemplary linkers include glycine, lysine, serine and cysteine.
Amino acid analogs, as defined herein, are also of use as linker
components. The amino acid may be modified with an additional
component of a linker, e.g., alkyl, heteroalkyl, covalently
attached through an acyl linkage, for example, an amide or urethane
formed through an amine moiety of the amino acid residue.
[0023] In an exemplary embodiment, the glycosyl linker has a
structure according to Formula I and R.sup.5 includes the polymeric
modifying moiety. In another exemplary embodiment, R.sup.5 includes
both the polymeric modifying moiety and a linker, L, joining the
modifying moiety to the glycosyl core. L can be a linear or
branched structure. Similarly, the polymeric modifying can be
branched or linear.
[0024] The polymeric modifying moiety comprises two or more
repeating units that can be water-soluble or essentially insoluble
in water. Exemplary water-soluble polymers of use in the compounds
of the invention include PEG, e.g., m-PEG, PPG, e.g., m-PPG,
polysialic acid, polyglutamate, polyaspartate, polylysine,
polyethyeleneimine, biodegradable polymers (e.g., polylactide,
polyglyceride), and functionalized PEG, e.g.,
terminal-functionalized PEG.
[0025] The glycosyl core of the glycosyl linking groups of use in
the Factor IX conjugates of the invention is selected from both
natural and unnatural furanoses and pyranoses. The unnatural
saccharides optionally include an alkylated or acylated hydroxyl
and/or amine moiety, e.g., ethers, esters and amide substituents on
the ring. Other unnatural saccharides include an H, hydroxyl,
ether, ester or amide substituent at a position on the ring at
which such a substituent is not present in the natural saccharide.
Alternatively, the carbohydrate is missing a substituent that would
be found in the carbohydrate from which its name is derived, e.g.,
deoxy sugars. Still further exemplary unnatural sugars include both
oxidized (e.g., -onic and -uronic acids) and reduced (sugar
alcohols) carbohydrates. The sugar moiety can be a mono-, oligo- or
poly-saccharide.
[0026] Exemplary natural sugars of use as components of glycosyl
linking groups in the present invention include glucose,
glucosamine, galactose, galactosamine, fucose, mannose,
mannosamine, xylanose, ribose, N-acetyl glucose, N-acetyl
glucosamine, N-acetyl galactose, N-acetyl galactosamine, and sialic
acid.
[0027] In one embodiment, the present invention provides an Factor
IX peptide comprising the moiety:
##STR00003##
wherein D is a member selected from --OH and R.sup.1-L-HN--; G is a
member selected from H and R.sup.1-L- and
--C(O)(C.sub.1-C.sub.6)alkyl; R.sup.1 is a moiety comprising a
straight-chain or branched poly(ethylene glycol) residue; and L is
a linker, e.g., a bond ("zero order"), substituted or unsubstituted
alkyl and substituted or unsubstituted heteroalkyl. In exemplary
embodiments, when D is OH, G is R.sup.1-L-, and when G is
--C(O)(C.sub.1-C.sub.6)alkyl, D is R.sup.1-L-NH--.
[0028] In another aspect, the invention provides a peptide
comprising a glycosyl linking group having the formula:
##STR00004##
[0029] In other embodiments, the group has the formula:
##STR00005##
in which t is 0 or 1.
[0030] In yet another embodiment, the group has the formula:
##STR00006##
in which the index p represents and integer from 1 to 10, and c
represents 0 or 1.
[0031] In another aspect, the invention provides a method of making
a PEGylated Factor IX peptide of the invention. The method
includes: (a) contacting a substrate Factor IX peptide comprising a
glycosyl group selected from:
##STR00007##
with a PEG-sialic acid donor having the formula:
##STR00008##
and an enzyme that transfers PEG-sialic acid from said donor onto a
member selected from the GalNAc, Gal and the Sia of said glycosyl
group, under conditions appropriate for said transfer. An exemplary
modified sialic acid donor is CMP-sialic acid modified, through a
linker moiety, with a polymer, e.g., a straight chain or branched
poly(ethylene glycol) moiety. The indices c and r independently
represent 0 or 1.
[0032] The peptide can be acquired from essentially any source,
however, in one embodiment, prior to being modified as discussed
above, the Factor IX peptide is expressed in a suitable host.
Mammalian (e.g., CHO), bacteria (e.g., E. coli) and insect cells
(e.g., Sf-9) are exemplary expression systems providing Factor IX
of use in the compositions and methods set forth herein. An
exemplary O-linked glycan that is glycopegylated is shown in FIG.
9. Exemplary glycans produced in an insect system and a mammalian
system, and subsequently glycoconjugated and or remodeled and
glycoconjugated to PEG are set forth in FIG. 10 and FIG. 11.
[0033] In another aspect, the invention provides a method of
treating a condition in a subject in need thereof. Exemplary
conditions include those characterized by compromised blood
clotting in the subject. The method includes the step of
administering to the subject an amount of the polymer-modified
Factor IX peptide of the invention effective to ameliorate the
condition in the subject.
[0034] In another aspect, the invention provides a method of
enhancing blood clotting in a mammal. The method includes
administering to the mammal an amount of the polymer-modified
Factor IX peptide of the invention effective to enhance clotting in
the mammal.
[0035] In another aspect, the invention provides a method of
treating a condition in a subject in need of treatment with Factor
IX. The method includes the step of administering to the subject an
amount of a polymer-modified Factor IX peptide of the invention
effective to ameliorate the condition of the subject.
[0036] In another aspect, the invention provides a pharmaceutical
formulation comprising a polymer-modified Factor IX peptide of the
invention and a pharmaceutically acceptable carrier.
[0037] In the polymer-modified Factor IX glycoconjugates of the
invention, essentially each of the amino acid residues to which the
polymer is bound has the same structure. For example, if one
peptide includes an asparagine linked glycosyl residue, 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. In
other embodiments, this is true of a glycosyl residue linked to a
threonine or a serine.
[0038] 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
[0039] 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.
[0040] 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 (a' and a'' are independently selected from 0 and 1-at
least one of a' and a'' is 1); The figure is exemplary in that any
glycosylated Factor IX molecule may comprise any mixture of mono-,
bi- tri-, or tetra-antennary N-linked glycosyl residues and any one
or more of the branches may further comprise a modified sialic acid
moiety of the invention. Moreover, the figure illustrates that the
modified glycan can be positioned at any one or more N- or O-linked
glycosylation site without limitation.
[0041] FIG. 3 is a plot comparing the in vivo residence lifetimes
of unglycosylated Factor IX and enzymatically glycoPEGylated Factor
IX.
[0042] FIG. 4 is a table comparing the activities of the species
shown in FIG. 3.
[0043] FIG. 5 is the amino acid sequence of Factor IX.
[0044] FIG. 6 is a graphic presentation of the pharmacokinetic
properties of various glycoPEGylated Factor IX molecules compared
to a non-pegylated Factor IX. LS refers to "low substitution" (the
peptide is glycoPEGylated using ST3Gal3 without desialylation). HS
refers to high substitution (the peptide is glycoPEGylated using
ST3Gal3, following desialylation). Unmodified Gal residues are
optionally capped with Sia.
[0045] FIG. 7 is a synthetic scheme for producing an exemplary
PEG-glycosyl linking group precursor (modified sugar) of us in
preparing the conjugates of the invention.
[0046] FIG. 8 is a table of sialyl transferases of use to transfer
onto an acceptor a modified sialic acid moiety, such as those set
forth herein and unmodified sialic acid moieties.
[0047] FIG. 9 shows an exemplary O-linked glycan structure on a
Factor IX glycoconjugate of the invention. Each index n is
independently selected.
[0048] FIG. 10 shows an exemplary N-linked glycan structure on a
mutant Factor IX glycoconjugate of the invention expressed in
insect cells (and remodeled and glycopegylated) in which the mutant
includes one or more N-linked glycosylation sites.
[0049] FIG. 11 shows an exemplary N-linked glycan structure on a
mutant Factor IX glycoconjugate of the invention expressed in
mammalian cells (and glycopegylated) in which the mutant includes
one or more N-linked glycosylation sites: A) N-linked glycans of
Factor IX expressed in CHO; B) N-linked glycans of CHO-derived
Factor IX glycoPEGylated with CST-II or .alpha.2,8
sialyltransferase; C)N-linked glycans of CHO-derived Factor IX
glycoPEGylated with CST-II and/or ST3Gal3. A glycine linker can be
interposed between the linear and/or branched PEG species such as
discussed herein.
[0050] FIG. 12 illustrates exemplary modified sialic acid
nucleotides useful in the practice of the invention. A. Structure
of exemplary branched (e.g., 30 kDa, 40 kDa) CMP-sialic acid-PEG
sugar nucleotides. B. Structure of linear CMP-sialic acid-PEG
(e.g., 10 kDa).
DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED
EMBODIMENTS
Abbreviations
[0051] PEG, poly(ethylene glycol); PPG, poly(propylene glycol);
Ara, arabinosyl; Fru, fructosyl; Fuc, fucosyl; Gal, galactosyl;
GalNAc, N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc,
N-acetylglucosaminyl; Man, mannosyl; ManAc, mannosaminyl acetate;
Xyl, xylosyl; NeuAc (N-acetylneuraminyl), Sia (sialyl); M6P,
mannose-6-phosphate.
DEFINITIONS
[0052] 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 performed according to conventional methods in the
art and various general references (see generally, 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.
[0053] 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 (.alpha. or
.beta.), 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., GlcNAc).
Each saccharide is preferably a pyranose. For a review of standard
glycobiology nomenclature, see, Essentials of Glycobiology Varki et
al. eds. CSHL Press (1999).
[0054] 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.
[0055] 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 Neu5Ac, NeuAc, or NANA). A second member
of the family is N-glycolyl-neuraminic acid (Neu5Gc 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 (KDN) (Nadano
et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J.
Biol. Chem. 265: 21811-21819 (1990)). Also included are
9-substituted sialic acids such as a 9-O--C.sub.1-C.sub.6
acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,
9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. 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 Oct.
1, 1992.
[0056] "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, .beta.-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 L-isomer. The L-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 peptides 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).
[0057] The term "peptide conjugate," refers to species of the
invention in which a peptide is conjugated with a modified sugar as
set forth herein.
[0058] 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, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. 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.
[0059] 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 enzyme substrates including, but not limited to sugar
nucleotides (mono-, di-, and 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.
[0060] 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 poly(ethylene glycol). Poly(ethylene
imine) is an exemplary polyamine, and poly(acrylic) acid is a
representative poly(carboxylic acid).
[0061] The polymer backbone of the water-soluble polymer can be
poly(ethylene 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
poly(ethylene glycol) is intended to be inclusive and not exclusive
in this respect. The term PEG includes poly(ethylene 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.
[0062] 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
poly(ethylene glycol) can be represented in general form as
R(-PEG-OH).sub.m in which R represents the core moiety, such as
glycerol or pentaerythritol, and m represents the number of arms.
Multi-armed PEG 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.
[0063] 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 poly(propylene
glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol
and the like, poly(oxyethylated polyol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide),
poly(.alpha.-hydroxy acid), poly(vinyl 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.
[0064] 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.
[0065] The term "half-life" or "t1/2", 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) and/or 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 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
known 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).
[0066] 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., a Factor
IX peptide of the present invention. A subgenus of
"glycoconjugation" is "glycol-PEGylation," in which the modifying
group of the modified sugar is poly(ethylene glycol), and alkyl
derivative (e.g., m-PEG) or reactive derivative (e.g.,
H.sub.2N-PEG, HOOC-PEG) thereof.
[0067] 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.
[0068] 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 linking 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.fwdarw.Schiff base formation.fwdarw.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 unit(s) or removal of one
or more glycosyl unit from a parent saccharide structure.
[0069] 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 known to those of skill in the
art. Exemplary targeting moieties include antibodies, antibody
fragments, transferrin, HS-glycoprotein, coagulation factors, serum
proteins, .beta.-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO and the
like.
[0070] 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-.alpha.,
-.beta., -.gamma.), 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.
Tumor Necrosis Factor Receptor ((TNFR)/Fc domain fusion
protein)).
[0071] 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 oil/water
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.
[0072] As used herein, "administering," means oral administration,
administration as a suppository, topical contact, intravenous,
intraperitoneal, intramuscular, intralesional, intranasal or
subcutaneous administration, or the implantation of a slow-release
device e.g., a mini-osmotic pump, to the subject. Administration 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.
[0073] 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.
[0074] 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).
[0075] The term "effective amount" or "an amount effective to" or a
"therapeutically effective amount" or any grammatically equivalent
term means the amount that, when administered to an animal for
treating a disease, is sufficient to effect treatment for that
disease.
[0076] 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%.
[0077] 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.
[0078] 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).
[0079] "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 of the invention, which are
at least about 80%, preferably at least about 90% and more
preferably at least about 95% homogenous.
[0080] "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%.
[0081] 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 purity 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.
[0082] "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 .alpha.1,2
fucosyltransferase, a substantially uniform fucosylation pattern
exists if substantially all (as defined below) of the
Gal.beta.1,4-GlcNAc-R and sialylated analogues thereof are
fucosylated in a peptide conjugate of the invention. It will be
understood by one of skill in the art, that the starting material
may contain glycosylated acceptor moieties (e.g., fucosylated
Gal.beta.1,4-GlcNAc-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 glycosylated in the starting material.
[0083] 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.
[0084] 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., --CH.sub.2O--
is intended to also recite --OCH.sub.2--.
[0085] 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. C.sub.1-C.sub.10 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 higher homologs and isomers. 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".
[0086] 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
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, 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.
[0087] 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.
[0088] 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, --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2-S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.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, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. 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).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--.
[0089] 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.
[0090] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" is mean to
include, but not be limited to, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0091] 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 linked 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 atom(s) 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]furanyl, benzo[b]thienyl, 2,3-dihydrobenzo[1,4]dioxin-6-yl,
benzo[1,3]dioxol-5-yl and 6-quinolyl. Substituents for each of the
above noted aryl and heteroaryl ring systems are selected from the
group of acceptable substituents described below.
[0092] 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 term
"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).
[0093] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") is meant to include both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0094] 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', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR',
-halogen, --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''')=NR'''',
--NR--C(NR'R'')=NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2 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 thioalkoxy 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., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0095] 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', .dbd.O, .dbd.NR',
.dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R''R''', --OC(O)R',
--C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR'--C(O)NR''R''', --NR''C(O).sub.2R',
--NR--C(NR'R''R''').dbd.NR'''', --NR--C(NR'R'').dbd.NR''',
--S(O)R', --S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN
and --NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)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.
[0096] 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').sub.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-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CRR'--, --O--, --NR--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.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').sub.s--X--(CR''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituents R, R', R'' and R''' are preferably independently
selected from hydrogen or substituted or unsubstituted
(C.sub.1-C.sub.6)alkyl.
[0097] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
Introduction
[0098] As described above, Factor IX is vital in the blood
coagulation cascade. The structure and sequence of Factor IX is
provided in FIG. 1 and FIG. 5. A deficiency of Factor IX in the
body characterizes a type of hemophilia (type B). Treatment of this
disease is usually limited to intravenous transfusion of human
plasma protein concentrates of Factor IX. However, in addition to
the practical disadvantages of time and expense, transfusion of
blood concentrates involves the risk of transmission of viral
hepatitis, acquired immune deficiency syndrome or thromboembolic
diseases to the recipient.
[0099] While Factor IX is an important and useful compound for
therapeutic applications, present methods for the production of
Factor IX from recombinant cells (U.S. Pat. No. 4,770,999) result
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.
[0100] 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.
[0101] The conjugates of the invention are formed by the enzymatic
attachment of a modified sugar to the glycosylated or
unglycosylated peptide. A glycosylation site and/or a glycosyl
residue provides a locus for conjugating a sugas bearing a
modifying group to the peptide, e.g., by glycoconjugation. An
exemplary modifying group is a water-soluble polymer, such as
poly(ethylene glycol), e.g., methoxy-poly(ethylene glycol).
Modification of the Factor IX peptides, e.g., with a water-soluble
peptide 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.
[0102] The methods of the invention make it possible to assemble
peptides and glycopeptides that have 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.
[0103] 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, 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.
The Conjugates
[0104] In a first aspect, the present invention provides a
conjugate between a selected modifying group and an Factor IX
peptide.
[0105] The link between the peptide and the modifying moiety
includes a glycosyl linking group interposed between the peptide
and the selected moiety. As discussed herein, the selected
modifying 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, or a glycosyl residue attached
thereto. 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.
[0106] 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.
[0107] The conjugates of the invention will typically correspond to
the general structure:
##STR00009##
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 wide array of linking
groups, infra. Alternatively, the linker may be a single bond or a
"zero order linker."
[0108] 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.
[0109] An exemplary water-soluble polymer is poly(ethylene glycol),
e.g., methoxy-poly(ethylene glycol). The poly(ethylene glycol) used
in the present invention is not restricted to any particular form
or molecular weight range. For unbranched poly(ethylene glycol)
molecules the molecular weight is preferably between 500 and
100,000. A molecular weight of 2000-60,000 is preferably used and
preferably of from about 5,000 to about 30,000.
[0110] In another embodiment the poly(ethylene 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 02/09766;
Kodera Y., Bioconjugate Chemistry 5: 283-288 (1994); and Yamasaki
et al., Agric. Biol. Chem., 52: 2125-2127, 1998. In a preferred
embodiment the molecular weight of each poly(ethylene glycol) of
the branched PEG is less than or equal to 40,000 daltons.
[0111] 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 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 a
glycosyl linking group, e.g., 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
glycosyl residue of the peptide, and each glycosyl residue of the
peptide to which the glycosyl linking group is attached has the
same structure.
[0112] 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.
[0113] 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. When the modifying moiety
is attached to a sialic acid, it is generally preferred that the
modifying moiety is substantially non-fluorescent.
[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:
TABLE-US-00001 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQ
CESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCK
NSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVF
PDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNG
KVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNV
IRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFL
KFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFC
AGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTK
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. Pat. Nos. 4,770,999, 5,521,070 in
which a tyrosine is replaced by an alanine in the first position,
U.S. Pat. No. 6,037,452, in which Factor XI is linked to an
alkylene oxide group, and U.S. Pat. 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 are 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 thromboplastin time
assay as described in, for example, Biggs (1972, Human Blood
Coagulation Homeostasis 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 (Amer. J. Clin.
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 linking
group. The glycosyl linking group is covalently attached to either
an amino acid residue or a glycosyl residue of the 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.
[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.
[0120] Preferably, neither the amino nor the carboxy terminus of
the Factor IX peptide is derivatized with a polymeric modifying
moiety.
[0121] The peptides of the invention include at least one N-linked
or O-linked glycosylation site, which is glycosylated with a
glycosyl residue that includes a polymeric modifying moiety, e.g.,
a PEG moiety. In an exemplary embodiment, the PEG is covalently
attached to the 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 peptide. Alternatively,
the glycosyl linking group is attached to one or more glycosyl
units of a glycopeptide. The invention also provides conjugates in
which a glycosyl linking group is attached to both an amino acid
residue and a glycosyl residue.
[0122] 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.
[0123] In an exemplary embodiment, the invention utilizes a
modified sugar amine that has the formula:
##STR00010##
in which J is a glycosyl moiety (e.g., a nucleotide sugar), L is a
bond or a linker and R.sup.1 is the modifying group, e.g., a
polymeric modifying moiety. Exemplary bonds are those that are
formed between an NH.sub.2 moiety on the glycosyl moiety and a
group of complementary reactivity on the modifying group. For
example, when R.sup.1 includes a carboxylic acid moiety, this
moiety may be activated and coupled with the NH.sub.2 moiety on the
glycosyl residue affording a bond having the structure
NHC(O)R.sup.1. J is preferably a glycosyl moiety that is "intact",
not having been degraded by exposure to conditions that cleave the
pyranose or furanose structure, e.g. oxidative conditions, e.g.,
sodium periodate.
[0124] 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 (R.sup.1). Other
exemplary 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 corresponding amide or urethane by reaction with
an activated carboxylic acid or carbonate of the modifying
group.
[0125] Another exemplary linker is a PEG moiety, e.g., a PEG moiety
that is functionalized with an amino acid residue. The PEG linker
is conjugated to the glycosyl group through the amino acid residue
at one PEG terminus and bound to R.sup.1 through the other PEG
terminus. Alternatively, the amino acid residue is bound to R.sup.1
and the PEG terminus, which is not bound to the amino acid, is
bound to the glycosyl group.
[0126] An exemplary species of NH-L-R.sup.1 has the formula:
--NH{C(O)(CH.sub.2).sub.nNH}.sub.s{C(O)(CH.sub.2).sub.b(OCH.sub.2CH.sub.2-
).sub.c--O--(CH.sub.2).sub.dNH}.sub.tR.sup.1, 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 an --NH
moiety is replaced by another group, for example, --S, --O or
--CH.sub.2. As those of skill will appreciate one or more of the
bracketed moieties corresponding to indices s and t can be replaced
with a substituted or unsubstituted alkyl or heteroalkyl
moiety.
[0127] More particularly, the invention utilizes compounds in which
NH-L-R.sup.1 is:
NHC(O)(CH.sub.2).sub.aNHC(O)(CH.sub.2).sub.b(OCH.sub.2CH.sub.2).sub.c--O--
-(CH.sub.2).sub.dNHR.sup.1,
NHC(O)(CH.sub.2).sub.b(OCH.sub.2CH.sub.2).sub.c--O--(CH.sub.2).sub.dNHR.s-
up.1,
NHC(O)O(CH.sub.2).sub.b(OCH.sub.2CH.sub.2).sub.c--O--(CH.sub.2).sub.-
dNHR.sup.1,
NH(CH.sub.2).sub.aNHC(O)(CH.sub.2).sub.b(OCH.sub.2CH.sub.2).sub.c--O--(CH-
.sub.2).sub.dNHR.sup.1, NHC(O)(CH.sub.2).sub.aNHR.sup.1,
NH(CH.sub.2).sub.aNHR.sup.1, and NHR.sup.1. 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 about 2500.
[0128] In an exemplary embodiment, c is selected such that the PEG
moiety is approximately 1 kD, 5 kD, 10, kD, 15 kD, 20 kD or 30
kD.
[0129] In the discussion that follows, the invention is illustrated
by reference to the use of selected derivatives of furanose and
pyranose. 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.
[0130] In an exemplary embodiment, the invention provides a
glycopeptide that is conjugated to a polymeric modifying moiety
through an intact glycosyl linking group having a formula that is
selected from:
##STR00011##
In Formulae I R.sup.2 is H, CH.sub.2OR.sup.7, COOR.sup.7 or
OR.sup.7, in which R.sup.7 represents H, substituted or
unsubstituted alkyl or substituted or unsubstituted heteroalkyl.
When COOR.sup.7 is a carboxylic acid or carboxylate, both forms are
represented by the designation of the single structure COO.sup.- or
COOH. In Formulae I and II, the symbols R.sup.3, R.sup.4, R.sup.5,
R.sup.6 and R.sup.6' independently represent H, substituted or
unsubstituted alkyl, OR.sup.8, NHC(O)R.sup.9. The index d is 0 or
1. R.sup.8 and R.sup.9 are independently selected from H,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, sialic acid or polysialic acid. At least one of
R.sup.3, R.sup.4, R.sup.5, R.sup.6 or R.sup.6' includes the
polymeric modifying moiety e.g., PEG, linked through a bond or a
linking group. In an exemplary embodiment, R.sup.6 and R.sup.6,
together with the carbon to which they are attached are components
of the pyruvyl side chain of sialic acid. In a further exemplary
embodiment, this side chain is functionalized with the polymeric
modifying moiety. In another exemplary embodiment, R.sup.6 and
R.sup.6', together with the carbon to which they are attached are
components of the side chain of sialic acid and the polymeric
modifying moiety is a component of R.sup.5.
[0131] In a further exemplary embodiment, the polymeric modifying
moiety is bound to the sugar core, generally through a heteroatom,
e.g, nitrogen, on the core through a linker, L, as shown below:
##STR00012##
R.sup.1 is the polymeric moiety and L is selected from a bond and a
linking group. The index w represents an integer selected from 1-6,
preferably 1-3 and more preferably 1-2. Exemplary linking groups
include substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl moieties and sialic acid. An exemplary
component of the linker is an acyl moiety.
[0132] An exemplary compound according to the invention has a
structure according to Formulae I or II, in which at least one of
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 or R.sup.6' has the
formula:
##STR00013##
[0133] In another example according to this embodiment at least one
of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 or R.sup.6' has the
formula:
##STR00014##
in which s is an integer from 0 to 20 and R.sup.1 is a linear
polymeric modifying moiety.
[0134] In an exemplary embodiment, the polymeric modifying
moiety-linker construct is a branched structure that includes two
or more polymeric chains attached to central moiety. In this
embodiment, the construct has the formula:
##STR00015##
in which R.sup.1 and L are as discussed above and w' is an integer
from 2 to 6, preferably from 2 to 4 and more preferably from 2 to
3.
[0135] When L is a bond it is formed between a reactive functional
group on a precursor of R.sup.1 and a reactive functional group of
complementary reactivity on the saccharyl core. When L is a
non-zero order linker, a precursor of L can be in place on the
glycosyl moiety prior to reaction with the R.sup.1 precursor.
Alternatively, the precursors of R.sup.1 and L can be incorporated
into a preformed cassette that is subsequently attached to the
glycosyl moiety. As set forth herein, the selection and preparation
of precursors with appropriate reactive functional groups is within
the ability of those skilled in the art. Moreover, coupling the
precursors proceeds by chemistry that is well understood in the
art.
[0136] In an exemplary embodiment, L is a linking group that is
formed from an amino acid, or small peptide (e.g., 1-4 amino acid
residues) providing a modified sugar in which the polymeric
modifying moiety is attached through a substituted alkyl linker.
Exemplary linkers include glycine, lysine, serine and cysteine. The
PEG moiety can be attached to the amine moiety of the linker
through an amide or urethane bond. The PEG is linked to the sulfur
or oxygen atoms of cysteine and serine through thioether or ether
bonds, respectively.
[0137] In an exemplary embodiment, R.sup.5 includes the polymeric
modifying moiety. In another exemplary embodiment, R.sup.5 includes
both the polymeric modifying moiety and a linker, L, joining the
modifying moiety to the remainder of the molecule. As discussed
above, L can be a linear or branched structure. Similarly, the
polymeric modifying can be branched or linear.
[0138] In one embodiment, the present invention provides an Factor
IX peptide comprising the moiety:
##STR00016##
wherein D is a member selected from --OH and R.sup.1-L-HN--; G is a
member selected from H and R.sup.1-L- and
--C(O)(C.sub.1-C.sub.6)alkyl; R.sup.1 is a moiety comprising a
straight-chain or branched poly(ethylene glycol) residue; and L is
a linker, e.g., a bond ("zero order"), substituted or unsubstituted
alkyl and substituted or unsubstituted heteroalkyl. In exemplary
embodiments, when D is OH, G is R.sup.1-L-, and when G is
--C(O)(C.sub.1-C.sub.6)alkyl, D is R.sup.1-L-NH--.
[0139] 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 peptide 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
(e.g., oxidase) processes. 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:
##STR00017##
[0140] In the formulae above, R.sup.1 and L are as described above.
Further detail about the structure of exemplary R.sup.1 groups is
provided below.
[0141] 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 formula:
##STR00018##
in which the radicals are as discussed above. Such saccharyl
moieties include, without limitation, glucose, glucosamine,
N-acetyl-glucosamine, galactose, galactosamine,
N-acetyl-galactosamine, mannose, mannosamine, N-acetyl-mannosamine,
and the like.
[0142] Due to the versatility of the methods available for
modifying glycosyl residues on a therapeutic peptide such as Factor
IX, the glycosyl structures on the peptide conjugates of the
invention can have substantially any structure. Moreover, the
glycans can be O-linked or N-linked. As exemplified in the
discussion below, each of the pyranose and furanose derivatives
discussed above can be a component of a glycosyl moiety of a
peptide.
[0143] The invention provides a modified Factor IX peptide that
includes a glycosyl group having the formula:
##STR00019##
[0144] In other embodiments, the group has the formula:
##STR00020##
[0145] In a still further exemplary embodiment, the group has the
formula:
##STR00021##
[0146] In yet another embodiment, the group has the formula:
##STR00022##
in which the index p represents and integer from 1 to 10; and c is
either 0 or 1.
[0147] In an exemplary embodiment according to each of the formulae
set forth above, the PEG-glycosyl linking group is attached at
Serine 61 (Ser 61) of Factor IX.
[0148] In an exemplary embodiment, a glycoPEGylated Factor IX
peptide of the invention includes at least one N-linked glycosyl
residue selected from the glycosyl residues set forth below:
##STR00023##
[0149] In the formulae above, the index t is 0 or 1 and the index p
is an integer from 1 to 10. The symbol R.sup.15 represents H, OH
(e.g., Gal-OH), a sialyl moiety, a polymer modified sialyl moiety
(i.e., glycosyl linking group-polymeric modifying moiety
(Sia-L-R.sup.1)) or a sialyl moiety to which is bound a polymer
modified sialyl moiety (e.g., Sia-Sia-L-R.sup.1) ("Sia-Sia.sup.p").
Exemplary polymer modified saccharyl moieties have a structure
according to Formulae I and II. An exemplary Factor IX peptide of
the invention will include at least one glycan having a R.sup.15'
that includes a structure according to Formulae I or II. The
oxygen, with the open valence, of Formulae I and II is preferably
attached through a glycosidic linkage to a carbon of a Gal or
GalNAc moiety. In a further exemplary embodiment, the oxygen is
attached to the carbon at position 3 of a galactose residue. In an
exemplary embodiment, the modified sialic acid is linked
.alpha.2,3-to the galactose residue. In another exemplary
embodiment, the sialic acid is linked .alpha.2,6-to the galactose
residue.
[0150] In another exemplary embodiment, the invention provides an
Factor IX peptide conjugate that includes a glycosyl linking group,
such as those set forth above, that is covalently attached to an
amino acid residue of the peptide. In one embodiment according to
this motif, the glycosyl linking moiety is linked to a galactose
residue through a Sia residue:
##STR00024##
[0151] An exemplary species according to this motif is prepared by
conjugating Sia-L-R.sup.1 to a terminal sialic acid of a glycan
using an enzyme that forms Sia-Sia bonds, e.g., CST-II, ST8Sia-II,
ST8Sia-III and ST8Sia-IV.
[0152] In another exemplary embodiment, the glycans have a formula
that is selected from the group:
##STR00025##
and combinations thereof.
[0153] The glycans of this group generally correspond to those
found on an Factor IX peptide that is produced by insect (e.g.,
Sf-9) cells, following remodeling according to the methods set
forth herein. For example insect-derived Factor IX that is
expressed with a tri-mannosyl core is subsequently contacted with a
GlcNAc donor and a GlcNAc transferase and a Gal donor and a Gal
transferase. Appending GlcNAc and Gal to the tri-mannosyl core is
accomplished in either two steps or a single step. A modified
sialic acid is added to at least one branch of the glycosyl moiety
as discussed herein. Those Gal moieties that are not functionalized
with the modified sialic acid are optionally "capped" by reaction
with a sialic acid donor in the presence of a sialyl
transferase.
[0154] In an exemplary embodiment, the glycosyl linking group is
attached to a member selected from Asn 157, Asn 167 and
combinations thereof.
[0155] In an exemplary embodiment, at least 60% of terminal Gal
moieties in a population of peptides is capped with sialic acid,
preferably at least 70%, more preferably, at least 80%, still more
preferably at least 90% and even more preferably at least 95%, 96%,
97%, 98% or 99% are capped with sialic acid.
[0156] In each of the formulae above, R.sup.15/R.sup.15' is as
discussed above. Moreover, an exemplary modified Factor IX peptide
of the invention will include at least one glycan with an
R.sup.15/R.sup.15' moiety having a structure according to Formulae
I or II.
[0157] In an exemplary embodiment, the glycosyl linking moiety has
the formula:
##STR00026##
in which b is 0 or 1. The index s represents and integer from 1 to
10; and f represents and integer from 1 to 2500. Generally
preferred is the use of a PEG moiety that has a molecular weight of
about 20 kDa. Also preferred is the attachment of the glycosyl
linking group a member selected from Ser 61, Ser 63, Thr 159, Thr
169, Thr 172 and combinations thereof.
[0158] In another exemplary embodiment, the Factor IX is derived
from insect cells, remodeled by adding GlcNAc and Gal to the
mannose core and glycopegylated using a sialic acid bearing a
linear PEG moiety, affording an Factor IX peptide that comprises at
least one moiety having the formula:
##STR00027##
in which s represents and integer from 1 to 10; and f represents
and integer from 1 to 2500.
[0159] As discussed herein, the PEG of use in the conjugates of the
invention can be linear or branched. An exemplary precursor of use
to form the branched conjugates according to this embodiment of the
invention has the formula:
##STR00028##
[0160] The branched polymer species according to this formula are
essentially pure water-soluble polymers. X.sup.3' is a moiety that
includes an ionizable, e.g., OH, COOH, H.sub.2PO.sub.4, HSO.sub.3,
HPO.sub.3, and salts thereof, etc.) or other reactive functional
group, e.g., infra. C is carbon. X.sup.5 is preferably a
non-reactive group (e.g., H, unsubstituted alkyl, unsubstituted
heteroalkyl), and can be a polymeric arm. R.sup.16 and R.sup.17 are
independently selected polymeric arms, e.g., nonpeptidic,
nonreactive polymeric arms (e.g., PEG)). X.sup.2 and X.sup.4 are
linkage fragments that are preferably essentially non-reactive
under physiological conditions, which may be the same or different.
An exemplary linker includes neither aromatic nor ester moieties.
Alternatively, these linkages can include one or more moiety that
is designed to degrade under physiologically relevant conditions,
e.g., esters, disulfides, etc. X.sup.2 and X.sup.4 join polymeric
arms R.sup.16 and R.sup.17 to C. When X.sup.3' is reacted with a
reactive functional group of complementary reactivity on a linker,
sugar or linker-sugar cassette, X.sup.3' is converted to a
component of linkage fragment X.sup.3
[0161] Exemplary linkage fragments for X.sup.2, X.sup.3 and X.sup.4
are independently selected and include S, SC(O)NH, HNC(O)S, SC(O)O,
O, NH, NHC(O), (O)CNH and NHC(O)O, and OC(O)NH, CH.sub.2S,
CH.sub.2O, CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2S, (CH.sub.2).sub.oO,
(CH.sub.2).sub.oS or (CH.sub.2).sub.oY'-PEG wherein, Y' is S, NH,
NHC(O), C(O)NH, NHC(O)O, OC(O)NH, or O and o is an integer from 1
to 50. In an exemplary embodiment, the linkage fragments X.sup.2
and X.sup.4 are different linkage fragments.
[0162] In an exemplary embodiment, the precursor (III), or an
activated derivative thereof, is reacted with, and thereby bound to
a sugar, an activated sugar or a sugar nucleotide through a
reaction between X.sup.3' and a group of complementary reactivity
on the sugar moiety, e.g., an amine. Alternatively, X.sup.3' reacts
with a reactive functional group on a precursor to linker, L. One
or more of R.sup.2, R.sup.3, R.sup.4, R.sup.5 R.sup.6 or R.sup.6'
of Formulae I and II can include the branched polymeric modifying
moiety, or this moiety bound through L.
[0163] In an exemplary embodiment, the moiety:
##STR00029##
is the linker arm, L. In this embodiment, an exemplary linker is
derived from a natural or unnatural amino acid, amino acid analogue
or amino acid mimetic, or a small peptide formed from one or more
such species. For example, certain branched polymers found in the
compounds of the invention have the formula:
##STR00030##
[0164] X.sup.a is a linkage fragment that is formed by the reaction
of a reactive functional group, e.g., X.sup.3', on a precursor of
the branched polymeric modifying moiety and a reactive functional
group on the sugar moiety, or a precursor to a linker. For example,
when X.sup.3' is a carboxylic acid, it can be activated and bound
directly to an amine group pendent from an amino-saccharide (e.g.,
Sia, GalNH.sub.2, GlcNH.sub.2, ManNH.sub.2, etc.), forming an
X.sup.a that is an amide. Additional exemplary reactive functional
groups and activated precursors are described hereinbelow. The
index c represents an integer from 1 to 10. The other symbols have
the same identity as those discussed above.
[0165] In another exemplary embodiment, X.sup.a is a linking moiety
formed with another linker:
##STR00031##
in which X.sup.b is a second linkage fragment and is independently
selected from those groups set forth for X.sup.a, and, similar to
L, L.sup.1 is a bond, substituted or unsubstituted alkyl or
substituted or unsubstituted heteroalkyl.
[0166] Exemplary species for X.sup.a and X.sup.b include S,
SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), C(O)NH and NHC(O)O, and
OC(O)NH.
[0167] In another exemplary embodiment, X.sup.4 is a peptide bond
to R.sup.17, which is an amino acid, di-peptide (e.g., Lys-Lys) or
tri-peptide (E.G., Lys-Lys-Lys) in which the alpha-amine
moiety(ies) and/or side chain heteroatom(s) are modified with a
polymeric modifying moiety.
[0168] In a further exemplary embodiment, the conjugates of the
invention include a moiety, e.g., an R.sup.15/R.sup.15' moiety that
has a formula that is selected from:
##STR00032##
in which the identity of the radicals represented by the various
symbols is the same as that discussed hereinabove. L.sup.a is a
bond or a linker as discussed above for L and L.sup.1, e.g.,
substituted or unsubstituted alkyl or substituted or unsubstituted
heteroalkyl moiety. In an exemplary embodiment, L.sup.a is a moiety
of the side chain of sialic acid that is functionalized with the
polymeric modifying moiety as shown. Exemplary L.sup.a moieties
include substituted or unsubstituted alkyl chains that include one
or more OH or NH.sub.2.
[0169] In yet another exemplary embodiment, the invention provides
conjugates having a moiety, e.g., an R.sup.15/R.sup.15' moiety with
formula:
##STR00033##
The identity of the radicals represented by the various symbols is
the same as that discussed hereinabove. As those of skill will
appreciate, the linker arm in Formulae VI and VII is equally
applicable to other modified sugars set forth herein. In exemplary
embodiment, the species of Formulae VI and VII are the R.sup.15
moieties attached to the glycan structures set forth herein.
[0170] In yet another exemplary embodiment, the Factor IX peptide
includes an R.sup.15' moiety with the formula:
##STR00034##
in which the identities of the radicals are as discussed above. An
exemplary species for L.sup.a is
--(CH.sub.2).sub.jC(O)NH(CH.sub.2).sub.hC(O)NH--, in which h and j
are independently selected integers from 0 to 10. A further
exemplary species is --C(O)NH--.
[0171] The embodiments of the invention set forth above are further
exemplified by reference to species in which the polymer is a
water-soluble polymer, particularly poly(ethylene glycol) ("PEG"),
e.g., methoxy-poly(ethylene glycol). Those of skill will appreciate
that the focus in the sections that follow is for clarity of
illustration and the various motifs set forth using PEG as an
exemplary polymer are equally applicable to species in which a
polymer other than PEG is utilized.
[0172] PEG of any molecular weight, e.g. 1 kDa, 2 kDa, 5 kDa, 10
kDa, 15 kDa, 20 kDa, 30 kDa and 40 kDa is of use in the present
invention.
[0173] In an exemplary embodiment, the R.sup.15 moiety has a
formula that is a member selected from the group:
##STR00035##
In each of the structures above, the linker fragment
--NH(CH.sub.2).sub.a-- can be present or absent. In other exemplary
embodiments, the conjugate includes an R.sup.15 moiety selected
from the group:
##STR00036##
[0174] In each of the formulae above, the indices e and f are
independently selected from the integers from 1 to 2500. In further
exemplary embodiments, e and f are selected to provide a PEG moiety
that is about 1 kD, 2 kD, 10 kD, 15 kD, 20 kD, 30 kD or 40 kD. The
symbol Q represents substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.6 alkyl, e.g., methyl), substituted or unsubstituted
heteroalkyl or H.
[0175] Other branched polymers have structures based on di-lysine
(Lys-Lys) peptides, e.g.:
##STR00037##
and tri-lysine peptides (Lys-Lys-Lys), e.g.:
##STR00038##
In each of the figures above, e, f, f' and f'' represent integers
independently selected from 1 to 2500. The indices q, q' and q''
represent integers independently selected from 1 to 20.
[0176] In another exemplary embodiment, the Factor IX peptide
comprises a glycosyl moiety selected from the formulae:
##STR00039##
in which L.sup.a is a bond or a linker as described herein; the
index t represents 0 or 1; and the index c represents 0 or 1. Each
of these groups can be included as components of the mono-, bi-,
tri- and tetra-antennary saccharide structures set forth above.
[0177] In yet another embodiment, the conjugates of the invention
include a modified glycosyl residue that includes the substructure
selected from:
##STR00040##
in which the index a and the linker L.sup.a are as discussed above.
The index p is an integer from 1 to 10. The index c represents 0 or
1. Each of these groups can be included as components of the mono-,
bi-, tri- and tetra-antennary saccharide structures set forth
above.
[0178] 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, GalNAc, Glc, GlcNAc, Fuc, Xyl, Man, and the like. A
representative modified sugar according to this embodiment has the
formula:
##STR00041##
in which R.sup.11-R.sup.14 are members independently selected from
H, OH, C(O)CH.sub.3, NH, and NH C(O)CH.sub.3. R.sup.10 is a link to
another glycosyl residue (--O-glycosyl) or to an amino acid of the
Factor IX peptide (--NH-(Factor IX)). R.sup.14 is OR.sup.1,
NHR.sup.1 or NH-L-R.sup.1. R.sup.1 and NH-L-R.sup.1 are as
described above.
[0179] Selected conjugates according to this motif 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:
##STR00042##
[0180] As discussed above, the invention provides saccharides
bearing a modifying group, activated analogues of these species and
conjugates formed between species such as peptides and lipids and a
modified saccharide of the invention.
Modified Sugars
[0181] The present invention uses modified sugars and modified
sugar nucleotides to form conjugates of the modified sugars. In
modified sugar compounds of use in 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 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.
[0182] Any sugar can be utilized as the sugar core of the glycosyl
linking group 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.
[0183] Exemplary modified sugars are modified with water-soluble or
water-insoluble polymers. Examples of useful polymer are further
exemplified below.
Water-Soluble Polymers
[0184] 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.
[0185] 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)).
[0186] 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."
[0187] The present invention is further illustrated by reference to
a poly(ethylene glycol) conjugate. Several reviews and monographs
on the functionalization and conjugation of PEG are available. See,
for example, Harris, Macronol. Chem. Phys. C25: 325-373 (1985);
Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et al.,
Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,
Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304
(1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); and Bhadra,
et al., Pharmazie, 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. Pat. 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).
[0188] U.S. Pat. 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.
[0189] WO 99/45964 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 poly(ethylene glycols) are described in WO 96/21469, U.S.
Pat. 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 poly(ethylene glycol) and the
biologically active species. U.S. Pat. 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.
[0190] Conjugates that include degradable PEG linkages are
described in WO 99/34833; and WO 99/14259, as well as in U.S. Pat.
No. 6,348,558. Such degradable linkages are applicable in the
present invention.
[0191] 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.
[0192] The modified sugars are prepared by reacting the glycosyl
core (or a linker on the core) with a polymeric modifying moiety
(or a linker on the polymeric modifying moiety). The discussion
that follows provides examples of selected polymeric modifying
moieties of use in the invention. For example, representative
polymeric modifying moieties include structures that are based on
side chain-containing amino acids, e.g., serine, cysteine, lysine,
and small peptides, e.g., lys-lys. Exemplary structures
include:
##STR00043##
Those of skill will appreciate that the free amine in the di-lysine
structures can also be pegylated through an amide or urethane bond
with a PEG moiety.
[0193] 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:
##STR00044##
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.
[0194] 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
.alpha.-amine shown as unmodified in the structure above.
Similarly, the use of a tri-lysine functionalized with three or
four polymeric subunits labeled with the polymeric modifying moiety
in a desired manner is within the scope of the invention.
[0195] The polymeric modifying moieties can be activated for
reaction with the glycosyl core. Exemplary structures of activated
species (e.g., carbonates and active esters) include:
##STR00045##
[0196] Other activating, or leaving groups, appropriate for
activating linear and branched PEGs of use in preparing the
compounds set forth herein include, but are not limited to the
species:
##STR00046##
PEG molecules that are activated with these and other species and
methods of making the activated PEGs are set forth in WO
04/083259.
[0197] Those of skill in the art will appreciate that one or more
of the m-PEG arms of the branched polymers shown above can be
replaced by a PEG moiety with a different terminus, e.g., OH, COOH,
NH.sub.2, C.sub.2-C.sub.10-alkyl, etc. Moreover, the structures
above are readily modified by inserting alkyl linkers (or removing
carbon atoms) between the .alpha.-carbon atom and the functional
group of the amino acid side chain. Thus, "homo" derivatives and
higher homologues, as well as lower homologues are within the scope
of cores for branched PEGs of use in the present invention.
[0198] The branched PEG species set forth herein are readily
prepared by methods such as that set forth in the scheme below:
##STR00047##
in which X.sup.d is O or S and r is an integer from 1 to 5. The
indices e and f are independently selected integers from 1 to 2500.
In an exemplary embodiment, one or both of these indices are
selected such that the polymer is about 10 kD, 15 kD or 20 kD in
molecular weight.
[0199] 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
X.sup.d. The mono-functionalize 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 in situ using a dehydrating agent such as
dicyclohexylcarbodiimide, carbonyldiimidazole, etc.
[0200] In other exemplary embodiments, the urea moiety is replaced
by a group such as a amide.
[0201] In an illustrative embodiment, the modified sugar is sialic
acid and selected modified sugar compounds of use in the invention
have the formulae:
##STR00048##
The indices a, b and d are integers from 0 to 20. The index c is an
integer from 1 to 2500. The structures set forth above can be
components of R.sup.15.
[0202] 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:
##STR00049##
The structures set forth above can be components of
R.sup.15/R.sup.15'.
[0203] 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-acyl derivatives,
and the like.
[0204] Although the present invention is exemplified in the
preceding sections by reference to PEG, as those of skill will
appreciate, an array of polymeric modifying moieties is of use in
the compounds and methods set forth herein.
[0205] In selected embodiments, R.sup.1 or L-R.sup.1 is a branched
PEG, for example, one of the species set forth above. Illustrative
modified sugars according to this embodiment include:
##STR00050##
in which X.sup.4 is a bond or O. In each of the structures above,
the alkylamine linker --(CH.sub.2)aNH- can be present or absent.
The structures set forth above can be components of
R.sup.15/R.sup.15'.
[0206] As discussed herein, the polymer-modified sialic acids of
use in the invention may also be linear structures. Thus, the
invention provides for conjugates that include a sialic acid moiety
derived from a structure such as:
##STR00051##
in which q and e are as discussed above.
Water-Insoluble Polymers
[0207] 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. See,
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.
[0208] The motifs forth above for R.sup.1, L-R.sup.1, R.sup.15,
R.sup.15' and other radicals are equally applicable to
water-insoluble polymers, which may be incorporated into the linear
and branched structures without limitation utilizing chemistry
readily accessible to those of skill in the art.
[0209] Representative water-insoluble polymers include, but are not
limited to, polyphosphazines, poly(vinyl alcohols), polyamides,
polycarbonates, polyalkylenes, polyacrylamides, polyalkylene
glycols, polyalkylene oxides, polyalkylene terephthalates,
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), poly(methyl acrylate),
poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl
acrylate) polyethylene, polypropylene, poly(ethylene glycol),
poly(ethylene oxide), poly (ethylene terephthalate), poly(vinyl
acetate), polyvinyl chloride, polystyrene, polyvinyl pyrrolidone,
pluronics and polyvinylphenol and copolymers thereof.
[0210] Synthetically modified natural polymers of use in conjugates
of the invention include, but are 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.
[0211] 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,
Wis.), Fluka (Ronkonkoma, N.Y.), and BioRad (Richmond, Calif.), or
else synthesized from monomers obtained from these suppliers using
standard techniques.
[0212] Representative biodegradable polymers of use in the
conjugates of the invention include, but are not limited to,
polylactides, polyglycolides and copolymers thereof, poly(ethylene
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.
[0213] 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 crosslinkable functional groups per polymer
chain.
[0214] 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-soluble, the polymer molecule, as a whole, does not to any
substantial measure dissolve in water.
[0215] For purposes of the present invention, the term
"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.
[0216] 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 region, and the hydrophilic region
are selected to ensure that useful bioresorbable compositions
remain water-insoluble.
[0217] Exemplary resorbable polymers include, for example,
synthetically produced resorbable block copolymers of
poly(.alpha.-hydroxy-carboxylic acid)/poly(oxyalkylene, (see, Cohn
et al., U.S. Pat. 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., J.
Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J.
Biomed. Mater. Res. 22: 993-1009 (1988).
[0218] 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
biosresorbable 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.
[0219] 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 excretable and/or
metabolizable fragment.
[0220] Higher order copolymers can also be used in the present
invention. For example, Casey et al., U.S. Pat. No. 4,438,253,
which issued on Mar. 20, 1984, discloses tri-block 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.
[0221] Other polymers based on lactic and/or glycolic acids can
also be utilized. For example, Spinu, U.S. Pat. No. 5,202,413,
which issued on Apr. 13, 1993, discloses biodegradable multi-block
copolymers having sequentially ordered blocks of polylactide and/or
polyglycolide produced by ring-opening polymerization of lactide
and/or 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.
[0222] 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.
[0223] 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, poly(vinyl pyrrolidine), poly(vinyl
alcohol), poly(alkyl oxazolines), polysaccharides, carbohydrates,
peptides, proteins and copolymers and mixtures thereof.
Furthermore, the hydrophilic region can also be, for example, a
poly(alkylene) oxide. Such poly(alkylene) oxides can include, for
example, poly(ethylene) oxide, poly(propylene) oxide and mixtures
and copolymers thereof.
[0224] 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 and bioresorbable. Moreover, hydrogel
compositions can include subunits that exhibit one or more of these
properties.
[0225] 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. Pat. Nos. 5,410,016, which issued on Apr. 25,
1995 and 5,529,914, which issued on Jun. 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 poly(ethylene glycol);
whereas, the hydrolytically labile extensions can be a
poly(.alpha.-hydroxy acid), such as polyglycolic acid or polylactic
acid. See, Sawhney et al., Macromolecules 26: 581-587 (1993).
[0226] 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.
[0227] 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. Pat. No.
4,522,811. For example, liposome formulations may be prepared by
dissolving appropriate lipid(s) (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.
[0228] 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 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,
is of use in the present invention.
[0229] 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.
The Methods
[0230] 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.
[0231] In exemplary embodiments, the conjugate is formed between a
polymeric modifying moiety and a glycosylated or non-glycosylated
peptide. The polymer is conjugated to the peptide via a glycosyl
linking group, which is interposed between, and covalently linked
to both the peptide (or glycosyl residue) 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. The reaction is conducted under conditions
appropriate to form a covalent bond between the modified sugar and
the peptide. The sugar moiety of the modified sugar is preferably
selected from nucleotide sugars.
[0232] In an exemplary embodiment, the modified sugar, such as
those set forth above, is 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-mannose, GDP-fucose, CMP-sialic acid, or
CMP-NeuAc. In an exemplary embodiment, the nucleotide phosphate is
attached to C-1.
[0233] Thus, in an illustrative embodiment in which the glycosyl
moiety is sialic acid, the method of the invention utilizes
compounds having the formulae:
##STR00052##
in which L-R.sup.1 is as discussed above, and L.sup.1-R.sup.1
represents a linker bound to the modifying group. As with L,
exemplary linker species according to L.sup.1 include a bond, alkyl
or heteroalkyl moieties.
[0234] Moreover, as discussed above, the present invention provides
for the use of 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 of use to
prepare conjugates within the scope of the present invention:
##STR00053##
in which X.sup.4 is O or a bond.
[0235] The invention also provides for the use of sugar nucleotides
modified with L-R.sup.1 at the 6-carbon position. Exemplary species
according to this embodiment include:
##STR00054##
in which the R groups, and L, represent moieties as discussed
above. The index "y" is 0, 1 or 2. In an exemplary embodiment, L is
a bond between NH and R.sup.1. The base is a nucleic acid base.
[0236] Exemplary nucleotide sugars of use in the invention in which
the carbon at the 6-position is modified include species having the
stereochemistry of GDP mannose, e.g.:
##STR00055##
in which X.sup.5 is a bond or O. The index i represents 0 or 1. The
index a represents an integer from 1 to 20. The indices e and f
independently represent integers from 1 to 2500. Q, as discussed
above, is H or substituted or unsubstituted C.sub.1-C.sub.6 alkyl.
As those of skill will appreciate, the serine derivative, in which
S is replaced with 0 also falls within this general motif.
[0237] In a still further exemplary embodiment, the invention
provides a conjugate in which the modified sugar is based on the
stereochemistry of UDP galactose. An exemplary nucleotide sugar of
use in this invention has the structure:
##STR00056##
[0238] In another exemplary embodiment, the nucleotide sugar is
based on the stereochemistry of glucose. Exemplary species
according to this embodiment have the formulae:
##STR00057##
[0239] 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 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., MODIFICATION OF PROTEINS;
Advances in Chemistry Series, Vol. 198, American Chemical Society,
Washington, D.C., 1982.
[0240] Useful reactive functional groups pendent from a sugar
nucleus or modifying group include, but are not limited to: [0241]
(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; [0242] (b)
hydroxyl groups, which can be converted to, e.g., esters, ethers,
aldehydes, etc. [0243] (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
alkoxide ion, thereby resulting in the covalent attachment of a new
group at the functional group of the halogen atom; [0244] (d)
dienophile groups, which are capable of participating in
Diels-Alder reactions such as, for example, maleimido groups;
[0245] (e) aldehyde or ketone 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; [0246] (f) sulfonyl halide groups for subsequent reaction
with amines, for example, to form sulfonamides; [0247] (g) thiol
groups, which can be, for example, converted to disulfides or
reacted with acyl halides; [0248] (h) amine or sulfhydryl groups,
which can be, for example, acylated, alkylated or oxidized; [0249]
(i) alkenes, which can undergo, for example, cycloadditions,
acylation, Michael addition, etc; and [0250] (j) epoxides, which
can react with, for example, amines and hydroxyl compounds.
[0251] 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.
[0252] 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 construed 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, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and
Schafer et al., J. Org. Chem. 65: 24 (2000)).
[0253] In an exemplary embodiment, the modified sugar is based upon
a 6-amino-N-acetyl-glycosyl moiety. As shown in FIG. 5 for
N-acetylgalactosamine, the 6-amino-sugar moiety is readily prepared
by standard methods.
[0254] In the scheme above, the index n represents an integer from
1 to 2500. In an exemplary embodiment, this index is selected such
that the polymer is about 10 kD, 15 kD or 20 kD in molecular
weight. 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.
[0255] The acceptor peptide is typically synthesized de novo, or
recombinantly expressed in a prokaryotic cell (e.g., bacterial
cell, such as E. coli) or in a eukaryotic cell such 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 one or more N- or O-linked
glycosylation sites to the peptide sequence.
[0256] The method of the invention also provides for modification
of incompletely glycosylated peptides that are produced
recombinantly. Many recombinantly produced glycoproteins are
incompletely glycosylated, exposing carbohydrate residues that may
have undesirable properties, e.g., immunogenicity, recognition by
the RES. 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.
[0257] Those of skill will appreciate that the invention can be
practiced using substantially any peptide or glycopeptide from any
source. Exemplary peptides with which the invention can be
practiced are set forth in WO 03/031464, and the references set
forth therein.
[0258] 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, GlcNAc, 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.
[0259] Moreover, in addition to peptides, the methods of the
present invention can be practiced with other biological structures
(e.g., glycolipids, lipids, sphingoids, ceramides, whole cells, and
the like, containing a glycosylation site).
[0260] 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 mutation(s) are
preferably made using methods known in the art.
[0261] 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, Proc. Natl. Acad. Sci.
USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994); and
U.S. Pat. Nos. 5,605,793, 5,837,458, 5,830,721 and 5,811,238.
[0262] Exemplary peptides with which the present invention can be
practiced, methods of adding or removing glycosylation sites, and
adding or removing glycosyl structures or substructures are
described in detail in WO03/031464 and related U.S. and PCT
applications.
[0263] The present invention also takes advantage of adding to (or
removing from) a peptide one or more selected glycosyl residues,
after which a modified sugar is conjugated to at least one of the
selected glycosyl residues of the peptide. The present embodiment
is useful, for example, when it is desired to conjugate the
modified sugar to a selected glycosyl residue that is either not
present on a 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.
[0264] Addition or removal of any carbohydrate moieties present on
the glycopeptide is accomplished either chemically or
enzymatically. An exemplary chemical deglycosylation is brought
about by exposure of the polypeptide variant to the compound
trifluoromethanesulfonic acid, or an equivalent compound. This
treatment results in the cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while
leaving the peptide intact. Chemical deglycosylation is described
by Hakimuddin et al., Arch. Biochem. Biophys. 259: 52 (1987) and by
Edge et al., Anal Biochem. 118: 131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptide variants can be achieved by
the use of a variety of endo- and exo-glycosidases as described by
Thotakura et al., Meth. Enzymol. 138: 350 (1987).
[0265] In an exemplary embodiment, the peptide is essentially
completely desialylated with neuraminidase prior to performing
glycoconjugation or remodeling steps on the peptide. Following the
glycoconjugation or remodeling, the peptide is optionally
re-sialylated using a sialyltransferase. In an exemplary
embodiment, the re-sialylation occurs at essentially each (e.g.,
>80%, preferably greater than 85%, greater than 90%, preferably
greater than 95% and more preferably greater than 96%, 97%, 98% or
99%) terminal saccharyl acceptor in a population of sialyl
acceptors. In a preferred embodiment, the saccharide has a
substantially uniform sialylation pattern (i.e., substantially
uniform glycosylation pattern).
[0266] Chemical addition of glycosyl moieties is carried out by any
art-recognized method. Enzymatic addition of sugar moieties is
preferably achieved using a modification of the methods set forth
herein, substituting native glycosyl units for the modified sugars
used in the invention. Other methods of adding sugar moieties are
disclosed in U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and
5,922,577.
[0267] 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 sulfhydryl 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.
BIOCHEM., pp. 259-306 (1981).
[0268] In one embodiment, the invention provides a method for
linking two or more peptides through a linking group. The linking
group is of any useful structure and may be selected from straight-
and branched-chain structures. Preferably, each terminus of the
linker, which is attached to a peptide, includes a modified sugar
(i.e., a nascent intact glycosyl linking group).
[0269] In an exemplary method of the invention, two peptides are
linked together via a linker moiety that includes a polymeric
(e.g., PEG linker). The construct conforms to the general structure
set forth in the cartoon above. As described herein, the construct
of the invention includes two intact glycosyl linking groups (i.e.,
s+t=1). The focus on a PEG linker that includes two glycosyl groups
is for purposes of clarity and should not be interpreted as
limiting the identity of linker arms of use in this embodiment of
the invention.
[0270] Thus, a PEG moiety is functionalized at a first terminus
with a first glycosyl unit and at a second terminus with a second
glycosyl unit. The first and second glycosyl units are preferably
substrates for different transferases, allowing orthogonal
attachment of the first and second peptides to the first and second
glycosyl units, respectively. In practice, the
(glycosyl).sup.1-PEG-(glycosyl).sup.2 linker is contacted with the
first peptide and a first transferase for which the first glycosyl
unit is a substrate, thereby forming
(peptide).sup.1-(glycosyl).sup.1-PEG-(glycosyl).sup.2. Transferase
and/or unreacted peptide is then optionally removed from the
reaction mixture. The second peptide and a second transferase for
which the second glycosyl unit is a substrate are added to the
(peptide).sup.1-(glycosyl).sup.1-PEG-(glycosyl).sup.2 conjugate,
forming
(peptide).sup.1-(glycosyl).sup.1-PEG-(glycosyl).sup.2-(peptide).sup.2;
at least one of the glycosyl residues is either directly or
indirectly O-linked. Those of skill in the art will appreciate that
the method outlined above is also applicable to forming conjugates
between more than two peptides by, for example, the use of a
branched PEG, dendrimer, poly(amino acid), polysaccharide or the
like.
[0271] 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.
[0272] In Scheme 1, 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 corresponding protected
amino acid amide adduct. The adduct is treated with an aldolase to
form .alpha.-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 attachment 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.
##STR00058##
Conjugation of Modified Sugars to Peptides
[0273] 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), enzyme(s) and acceptor peptide(s) 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.
[0274] A number of methods of using glycosyltransferases to
synthesize desired oligosaccharide structures are known and are
generally applicable to the instant invention. 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, commonly owned U.S. Pat. Nos. 6,399,336, and 6,440,703,
and commonly owned published PCT applications, WO 03/031464, WO
04/033651, WO 04/099231, which are incorporated herein by
reference.
[0275] 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.
[0276] 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.
[0277] 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.
[0278] 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 GlcNAc 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-peptide moiety. In further embodiments, the GlcNAc residue on
the glycosyl donor molecule is modified. For example, the GlcNAc
residue may comprise a 1,2 oxazoline moiety.
[0279] 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.
[0280] 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.degree. C. to about 55.degree. C., and more preferably about
20.degree. C. to about 37.degree. C. In another exemplary
embodiment, one or more components of the present method are
conducted at an elevated temperature using a thermophilic
enzyme.
[0281] 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 h, with recoverable amounts usually being obtained
within 24 h 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.
[0282] The present invention also provides for the industrial-scale
production of modified peptides. As used herein, an industrial
scale generally produces at least one gram of finished, purified
conjugate.
[0283] 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 glycosyl unit with agents other than PEG including other PEG
moieties, therapeutic moieties, and biomolecules.
[0284] 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 masked 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.
[0285] In an exemplary embodiment, an acceptor for a
sialyltransferase is present on the peptide to be modified either
as a naturally occurring structure or it is placed there
recombinantly, enzymatically or chemically. Suitable acceptors,
include, for example, galactosyl acceptors such as
Gal.beta.1,4GlcNAc, Gal.beta.1,4GalNAc, Gal.beta.1,3GalNAc,
lacto-N-tetraose, Gal.beta.1,3GlcNAc, Gal.beta.1,3Ara,
Gal.beta.1,6GlcNAc, Gal.beta.1,4Glc (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)). Exemplary sialyltransferases
are set forth herein.
[0286] 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 GalNAc residue is added by the action of a
GalNAc transferase.
[0287] 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 GlcNAc. The method includes
incubating the peptide to be modified with a reaction mixture that
contains a suitable amount of a galactosyltransferase (e.g.,
Gal.beta.1,3 or Gal.beta.1,4), 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 residue is
added. Other methods of assembling a selected saccharide acceptor
will be apparent to those of skill in the art.
[0288] 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. Pat. No. 5,716,812) are
useful for the attaching and trimming reactions. In another
embodiment of this method, the sialic acid moieties of the peptide
are essentially completely removed (e.g., at least 90, at least 95
or at least 99%), exposing an acceptor for a modified sialic
acid.
[0289] 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.
[0290] 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 GlcNAc
moiety conjugated to the PEG moiety. The modified GlcNAc is
attached to one or both of the terminal mannose residues of the
biantennary structure. Alternatively, an unmodified GlcNAc can be
added to one or both of the termini of the branched species.
[0291] 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 GlcNAc residue added onto the terminal mannose
residues. Alternatively, an unmodified Gal can be added to one or
both terminal GlcNAc residues.
[0292] In yet a further example, a PEG moiety is added onto a Gal
residue using a modified sialic acid such as those discussed
above.
[0293] In another exemplary embodiment, a high mannose structure is
"trimmed back" to the mannose from which the biantennary structure
branches. In one example, a PEG moiety is added via a GlcNAc
modified with the polymer. Alternatively, an unmodified GlcNAc is
added to the mannose, followed by a Gal with an attached PEG
moiety. In yet another embodiment, unmodified GlcNAc and Gal
residues are sequentially added to the mannose, followed by a
sialic acid moiety modified with a PEG moiety.
[0294] A high mannose structure can also be trimmed back to the
elementary tri-mannosyl core.
[0295] In a further exemplary embodiment, high mannose is "trimmed
back" to the GlcNAc to which the first mannose is attached. The
GlcNAc is conjugated to a Gal residue bearing a PEG moiety.
Alternatively, an unmodified Gal is added to the GlcNAc, followed
by the addition of a sialic acid modified with a water-soluble
sugar. In yet a further example, the terminal GlcNAc is conjugated
with Gal and the GlcNAc is subsequently fucosylated with a modified
fucose bearing a PEG moiety.
[0296] High mannose may also be trimmed back to the first GlcNAc
attached to the Asn of the peptide. In one example, the GlcNAc of
the GlcNAc-(Fuc).sub.a residue is conjugated with ha GlcNAc bearing
a water soluble polymer. In another example, the GlcNAc of the
GlcNAc-(Fuc)a residue is modified with Gal, which bears a water
soluble polymer. In a still further embodiment, the GlcNAc is
modified with Gal, followed by conjugation to the Gal of a sialic
acid modified with a PEG moiety.
[0297] Other exemplary embodiments are set forth in commonly owned
U.S. Patent application Publications: 20040132640; 20040063911;
20040137557; U.S. patent application Ser. Nos. 10/369,979;
10/410,913; 10/360,770; 10/410,945 and PCT/US02/32263 each of which
is incorporated herein by reference.
[0298] 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 intermediate between the peptide core and the
terminus of the carbohydrate.
[0299] In an exemplary embodiment, an existing sialic acid is
removed from a 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.
[0300] In another exemplary embodiment, an enzyme that transfers
sialic acid onto sialic acid is utilized. This method can be
practiced without treating a sialylated glycan with a sialidase to
expose glycan residues beneath the sialic acid. An exemplary
polymer-modified sialic acid is a sialic acid modified with
poly(ethylene glycol). Other exemplary enzymes that add sialic acid
and modified sialic acid moieties onto glycans that include a
sialic acid residue or exchange an existing sialic acid residue on
a glycan for these species include ST3Gal3, CST-II, ST8Sia-II,
ST8Sia-III and ST8Sia-IV.
[0301] In yet a further approach, 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.
[0302] Any modified sugar can be used with its appropriate
glycosyltransferase, depending on the terminal sugars of the
oligosaccharide side chains of the glycopeptide. 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).
[0303] In a further exemplary embodiment, UDP-galactose-PEG is
reacted with .beta.1,4-galactosyltransferase, thereby transferring
the modified galactose to the appropriate terminal
N-acetylglucosamine structure. The terminal GlcNAc 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 and/or glycosyltransferase, as required.
[0304] In another exemplary embodiment, a GlcNAc transferase, such
as GNT1-5, is utilized to transfer PEGylated-GlcNAc 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
GlcNAc as a GlcNAc-linked-Asn on the glycopeptide. UDP-Gal-PEG and
the appropriate galactosyltransferase is used to introduce the
PEG-galactose functionality onto the exposed GlcNAc.
[0305] 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 backbone. Exemplary
glycosyltransferases useful in practicing the present invention
include, but are not limited to, GalNAc transferases (GalNAc
T1-14), GlcNAc 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.
[0306] 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.
[0307] Enzymes and reaction conditions for preparing the conjugates
of the present invention are discussed in detail in the parent of
the instant application as well as co-owned published PCT patent
applications WO 03/031464, WO 04/033651, WO 04/099231.
[0308] In a selected embodiment, a Factor IX peptide, expressed in
insect cells, is remodeled such that glycans on the remodeled
glycopeptide include a GlcNAc-Gal glycosyl residue. The addition of
GlcNAc and Gal can occur as separate reactions or as a single
reaction in a single vessel. In this example, GlcNAc-transferase I
and Gal-transferase I are used. The modified sialyl moiety is added
using ST3Gal-III.
[0309] In another embodiment, the addition of GlcNAc, Gal and
modified Sia can also occur in a single reaction vessel, using the
enzymes set forth above. Each of the enzymatic remodeling and
glycoPEGylation steps are carried out individually.
[0310] When the peptide is expressed in mammalian cells, different
methods are of use. In one embodiment, the peptide is conjugated
without need for remodeling prior to conjugation by contacting the
peptide with a sialyltransferase that transfers the modified sialic
acid directly onto a sialic acid on the peptide forming
Sia-Sia-L-R.sup.1, or exchanges a sialic acid on the peptide for
the modified sialic acid, forming Sia-L-R.sup.1. An exemplary
enzyme of use in this method is CST-II. Other enzymes that add
sialic acid to sialic acid are known to those of skill in the art
and examples of such enzymes are set forth the figures appended
hereto.
[0311] In yet another method of preparing the conjugates of the
invention, the peptide expressed in a mammalian system is
desialylated using a sialidase. The exposed Gal residue is
sialylated with a modified sialic acid using a sialyltransferase
specific for O-linked glycans, providing an Factor IX peptide with
an O-linked modified glycan. The desialylated, modified Factor IX
peptide is optionally partially or fully re-sialylated by using a
sialyltransferase such as ST3GalIII. Using ST3GalIII with the
desialylated peptide, both O and N-linked sites are glycoPEGylated.
Using ST3GalI, one or more of the O-linked sites (e.g., Thr159,
Thr169, Thr172 of FIG. 1) are essentially selectively
glycoPEGylated. The use of CST-II provides a route to essentially
selectively glycoPEGylate one or more N-linked site (e.g., Asn 157,
Asn 167 of FIG. 1).
[0312] In an exemplary embodiment, the Factor IX of the invention
includes at least one linear 2 kDa PEG moiety covalently attached
thereto through an intact glycosyl linking group. A presently
preferred Factor IX peptide conjugate of the invention includes up
to 9 2 kDa PEG moieties attached to both N- and O-linked sites
through an intact glycosyl linking group, more preferably from 5-9
PEG moieties. In another exemplary embodiment, the Factor IX of the
invention includes at least one linear 30 kDa PEG moiety covalently
attached thereto through an intact glycosyl linking group. In a
presently preferred embodiment, the Factor IX peptide conjugate of
the invention includes from 1 to 3 PEG moieties.
[0313] In another aspect, the invention provides a method of making
a PEGylated Factor IX of the invention. The method includes: (a)
contacting a substrate Factor IX peptide comprising a glycosyl
group selected from:
##STR00059##
with a PEG-sialic acid donor having the formula:
##STR00060##
and an enzyme that transfers PEG-sialic acid from said donor onto a
member selected from the GalNAc, Gal and the Sia of said glycosyl
group, under conditions appropriate for said transfer. An exemplary
modified sialic acid donor is CMP-sialic acid modified, through a
linker moiety, with a polymer, e.g., a straight chain or branched
poly(ethylene glycol) moiety. As discussed herein, the peptide is
optionally glycosylated with GalNAc and/or Gal and/or Sia
("Remodeled") prior to attaching the modified sugar. The remodeling
steps can occur in sequence in the same vessel without purification
of the glycosylated peptide between steps. Alternatively, following
one or more remodeling step, the glycosylated peptide can be
purified prior to submitting it to the next glycosylation or
glycPEGylation step.
[0314] As illustrated in the examples and discussed further below,
placement of an acceptor moiety for the PEG-sugar is accomplished
in any desired number of steps. For example, in one embodiment, the
addition of GalNAc to the peptide can be followed by a second step
in which the PEG-sugar is conjugated to the GalNAc in the same
reaction vessel. Alternatively, these two steps can be carried out
in a single vessel approximately simultaneously.
[0315] In an exemplary embodiment, the PEG-sialic acid donor has
the formula:
##STR00061##
[0316] In another exemplary embodiment, the PEG-sialic acid donor
has the formula:
##STR00062##
[0317] In a further exemplary embodiment, the Factor IX peptide is
expressed in an appropriate expression system prior to being
glycopegylated or remodeled. Exemplary expression systems include
Sf-9/baculovirus and Chinese Hamster Ovary (CHO) cells.
Purification of Factor IX Conjugates
[0318] The products produced by the above processes can be used
without purification. However, it is usually preferred to recover
the product and one or more of the intermediates, e.g., nucleotide
sugars, branched and linear PEG species, modified sugars and
modified nucleotide sugars. 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 membranes have molecular
weight cutoff of about 3000 to about 10,000 can be used to remove
proteins such as glycosyl transferases.
[0319] If the peptide is produced intracellularly, as a first step,
the particulate debris, either host cells or lysed fragments, is
removed. Following glycoPEGylation, the PEGylated peptide is
purified by art-recognized methods, 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 (DEAE) 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.
[0320] 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.
[0321] A protease inhibitor, e.g., methylsulfonylfluoride (PMSF)
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics or preservatives may be included to
prevent the growth of adventitious contaminants.
[0322] Within another embodiment, supernatants from systems which
produce the modified glycopeptide of the invention are first
concentrated using a commercially available protein 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 acrylamide, agarose, dextran, cellulose, or other
types commonly employed in protein purification. Alternatively, a
cation-exchange step may be employed. Suitable cation exchangers
include various insoluble matrices comprising sulfopropyl or
carboxymethyl groups. Sulfopropyl groups are particularly
preferred.
[0323] Other methods of use in purification include size exclusion
chromatography (SEC), hydroxyapatite chromatography, hydrophobic
interaction chromatography and chromatography on Blue Sepharose.
These and other useful methods are illustrated in co-assigned U.S.
Provisional Patent No. (Attorney Docket No. 40853-01-5168-PI, filed
May 6, 2005).
[0324] 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 further purify a polypeptide conjugate
composition. Some or all of the foregoing purification steps, in
various combinations, can also be employed to provide a homogeneous
or essentially homogeneous modified glycoprotein.
[0325] 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, RP-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
[0326] 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 biomolecule and a glycosylated or non-glycosylated peptide. The
polymer, therapeutic moiety or biomolecule is conjugated to the
peptide via an intact glycosyl linking group interposed between and
covalently linked to both the peptide and the polymer, therapeutic
moiety or biomolecule.
[0327] 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 Pharmaceutical 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).
[0328] 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. Pat. Nos. 4,897,268 and 5,075,109.
[0329] Commonly, the pharmaceutical compositions are administered
parenterally, e.g., intravenously. Thus, the invention provides
compositions for parenteral administration that include 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.
[0330] 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 11, more preferably from 5 to 9 and
most preferably from 7 and 8.
[0331] 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., Ann. Rev. Biophys. Bioeng. 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 known in the art
(see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044).
[0332] Standard methods for coupling targeting agents to liposomes
can be used. These methods generally involve incorporation into
liposomes of lipid components, such as phosphatidylethanolamine,
which can be activated for attachment of targeting agents, or
derivatized lipophilic compounds, such as lipid-derivatized
glycopeptides of the invention.
[0333] 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.
[0334] 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 .sup.125I, .sup.14C, or
tritium.
[0335] 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 formulation 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 1000 .mu.g/kg body weight of the active
material. Preferable doses for treatment of coagulation disorders
are about 50 to about 3000 .mu.g/kg three times a week. More
preferably, about 500 to about 2000 .mu.g/kg three times a week.
More preferrably, about 750 to about 1500 .mu.g/kg three times a
week, and more preferrably about 1000 .mu.g/kg three times a week.
Because the present invention provides a Factor IX with an enhanced
in vivo residence time, the stated dosages are optionally lowered
when a composition of the invention is administered.
[0336] The following examples are provided to illustrate the
conjugates, and methods and of the present invention, but not to
limit the claimed invention.
[0337] Preparative methods for species of use in preparing the
compositions of the invention are generally set forth in various
patent publications, e.g., US 20040137557; WO 04/083258; and WO
04/033651. The following examples are provided to illustrate the
conjugates, and methods and of the present invention, but not to
limit the claimed invention.
[0338] 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 Cysteine-PEG.sub.2 (2)
##STR00063##
[0339] 1.1 Synthesis of (1)
[0340] 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 stirred 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 kilodalton 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
white solid (1). Structural data for the compound were as follows:
.sup.1H-NMR (500 MHz; D.sub.2O) .delta. 2.83 (t, 2H,
O--C--CH.sub.2--S), 3.05 (q, 1H, S--CHH--CHN), 3.18 (q, 1H, (q, 1H,
S--CHH--CHN), 3.38 (s, 3H, CH.sub.3O), 3.7 (t, OCH.sub.2CH.sub.2O),
3.95 (q, 1H, CHN). The purity of the product was confirmed by SDS
PAGE.
1.2 Synthesis of (2)
[0341] Triethylamine (.about.0.5 mL) was added dropwise to a
solution of 1 (440 mg, 22 .mu.mol) dissolved in anhydrous
CH.sub.2Cl.sub.2 (30 mL) until the solution was basic. A solution
of 20 kilodalton mPEG-O-p-nitrophenyl carbonate (660 mg, 33
.mu.mol) and N-hydroxysuccinimide (3.6 mg, 30.8 .mu.mol) in
CH.sub.2Cl.sub.2 (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 7.0. 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 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 575 mg (70%) of a white solid (2).
Structural data for the compound were as follows: .sup.1H-NMR (500
MHz; D.sub.2O) .delta. 2.83 (t, 2H, O--C--CH.sub.2--S), 2.95 (t,
2H, O--C--CH.sub.2--S), 3.12 (q, 1H, S--CHH--CHN), 3.39 (s, 3H
CH.sub.3O), 3.71 (t, OCH.sub.2CH.sub.2O). The purity of the product
was confirmed by SDS PAGE.
Example 2
GlycoPEGylation of Factor IX Produced in CHO Cells
[0342] This example sets forth the preparation of asialoFactor IX
and its sialylation with CMP-sialic acid-PEG.
2. 1 Desialylation of rFactor IX
[0343] 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 H.sub.2O. This solution was transferred to a Centricon
Plus 20, PL-10 centrifugal filter with another 6 mL USP H.sub.2O.
The solution was concentrated to 2 mL and then diluted with 15 mL
50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl.sub.2, 0.05%
NaN.sub.3 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
.alpha.2-3,6,8-Neuraminidase-agarose conjugate (Vibrio cholerae,
Calbiochem, 450 .mu.L). The reaction mixture was rotated gently for
26.5 hours at 32.degree. 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 NaCl, 0.05% NaN.sub.3. 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 .about.2 mL in a Centricon Plus 20 PL-10
centrifugal filter. The solution was twice diluted with 15 mL of 50
mM Tris-HCl pH 7.4, 0.15 M NaCl, 0.05% NaN.sub.3 and reconcentrated
to 2 mL. The final desialyated rFactor IX solution was diluted to 3
mL final volume (.about.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 .mu.L (15 .mu.g) samples first diluted with 10 .mu.L Tris
buffer and mixed with 12 .mu.L sample loading buffer. Gels were
loaded, run and fixed using standard procedures. Gels were stained
with Colloidal Blue Stain (FIG. 154), showing a band for
desialylated Factor IX.
Example 3
Preparation of PEG (1 kDa and 10 kDa)-SA-Factor IX
[0344] 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-HCl pH 7.4, 0.15 M
NaCl, 0.05% NaN.sub.3 and either CMP-SA-PEG-1k or 10k (7.25
.mu.mol) was added. The tubes were inverted gently to mix and 2.9 U
ST3Gal3 (326 .mu.L) was added (total volume 14.5 mL). The tubes
were inverted again and rotated gently for 65 hours at 32.degree.
C. The reactions were stopped by freezing at -20.degree. C. 10
.mu.g samples of the reactions were analyzed by SDS-PAGE. The
PEGylated proteins were purified on a Toso Haas Biosep G3000SW
(21.5.times.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 .mu.L (10 .mu.g)
of samples after dilution with 2 .mu.L of 50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 0.05% NaN.sub.3 buffer and mixing with 12 .mu.L sample
loading buffer and 1 .mu.L 0.5 M DTT and heated for 6 minutes at
85.degree. C. Gels were stained with Colloidal Blue Stain (FIG.
155) showing a band for PEG (1 kDa and 10 kDa)-SA-Factor IX.
Example 4
Direct Sialyl-GlycoPEGylation of Factor IX
[0345] This example sets forth the preparation of sialyl-PEGylation
of Factor IX without prior sialidase treatment.
4.1 Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(10 KDa)
[0346] 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% NaN.sub.3 and 0.01% polysorbate 80,
pH 5.0. The CMP-SA-PEG-(10 kDa) (27 mg, 2.5 .mu.mol) was then
dissolved in the solution and 1 U of ST3Gal3 was added. The
reaction was complete after gently mixing for 28 hours at
32.degree. C. The reaction was analyzed by SDS-PAGE as described by
Invitrogen. The product protein was purified on an Amersham
Superdex 200 (10.times.300 mm, 13 .mu.m) HPLC column with phosphate
buffered saline, pH 7.0 (PBS), 1 mL/min. R.sub.t=9.5 min.
Example 5
Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(20 kDa)
[0347] 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% NaN.sub.3 and 0.01% polysorbate 80,
pH 5.0. The CMP-SA-PEG-(20 kDa) (50 mg, 2.3 .mu.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.degree.
C. The reaction was analyzed by SDS-PAGE as described by
Invitrogen.
[0348] The product protein was purified on an Amersham Superdex 200
(10.times.300 mm, 13 .mu.m) HPLC column with phosphate buffered
saline, pH 7.0 (Fisher), 1 mL/min. R.sub.t=8.6 min.
Example 6
Sialic Acid Capping of GlycoPEGylated Factor IX
[0349] This examples sets forth the procedure for sialic acid
capping of sialyl-glycoPEGylated peptides. Here, Factor-IX is the
exemplary peptide.
6.1 Sialic Acid Capping of N-Linked and O-Linked Glycans of
Factor-IX-SA-PEG (10 kDa)
[0350] Purified r-Factor-IX-PEG (10 kDa) (2.4 mg) was concentrated
in a Centricon.RTM. Plus 20 PL-10 (Millipore Corp., Bedford, Mass.)
centrifugal filter and the buffer was changed to 50 mM Tris-HCl pH
7.2, 0.15 M NaCl, 0.05% NaN.sub.3 to a final volume of 1.85 mL. The
protein solution was diluted with 372 .mu.L of the same Tris buffer
and 7.4 mg CMP-SA (12 .mu.mol) was 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.degree. C.
[0351] A 10 .mu.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
.mu.L (10 .mu.g), were mixed with 12 .mu.L sample loading buffer
and 1 .mu.L 0.5 M DTT and heated for 6 minutes at 85.degree. C.
(FIG. 156, lane 4).
Example 7
Glycopegylated Factor IX Pharmacokinetic Study
[0352] 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.
TABLE-US-00002 ETP TEG Clot activity (relative (relative Compound
(% of plasma) specific activity specific 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-9-10K
11% 0.6 0.3 PEG-9-30K 14% 0.9 0.4
[0353] 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; 10K 100 U/kg; 30K 100
U/kg. All doses were based on measured clotting assay units.
[0354] The results are outlined in FIG. 6 and Table II.
TABLE-US-00003 TABLE II Dose Cmax AUC CL Compound (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-9-10K 100
0.350 2.80 35.7 PEG-9-30K 100 1.40 8.83 11.3
[0355] 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 administered at the highest dose.
Example 8
Preparation of Ls and Hs Glycopegylated Factor IX
[0356] GlycoPEGylated Factor IX with a low degree of substitution
with PEG were prepared from native Factor IX by an exchange
reaction catalyzed by ST3Gal-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.degree. C. For PEGylation with
CMP-SA-PEG 30 kD, 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.
[0357] 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.degree. C.
Desialylation was verified by SDS-PAGE, HPLC and MALDI glycan
analysis. Asialo Factor IX was purified on Q Sepharose FF to remove
the sialidase. The CaCl.sub.2 fraction was concentrated using an
Ultra15 concentrator and buffer exchanged into MES, pH 6.0 using a
PD10 column.
[0358] 2 kD and 10 kD PEGylation of asialo-Factor IX (0.5 mg/mL)
was carried out by incubation with ST3Gal-III (50 mU/mL) and
CMP-SA-PEG (0.5 mM) at 32.degree. C. for 16 h. For PEGylation with
CMPSA-PEG-30 kD, 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.degree. 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 9
Preparation of O-Glycopegylated Factor IX
[0359] O-glycan chains were introduced de novo into native Factor
IX (1 mg/mL) by incubation of the peptide with GalNAcT-II (25
mU/mL) and 1 mM UDP-GalNAc at 32.degree. C. After 4 h of
incubation, the PEGylation reaction was initiated by adding
CMPSA-PEG (2 Kd or 10 Kd at 0.5 mM or 30 kDd at 0.17 mM) and
ST6GalNAc-I (25 mU/mL) and incubating for an additional 20 h.
[0360] 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 are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
11415PRTHomo sapiens 1Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln
Gly Asn Leu Glu Arg1 5 10 15Glu Cys Met Glu Glu Lys Cys Ser Phe Glu
Glu Ala Arg Glu Val Phe 20 25 30Glu Asn Thr Glu Arg Thr Thr Glu Phe
Trp Lys Gln Tyr Val Asp Gly 35 40 45Asp Gln Cys Glu Ser Asn Pro Cys
Leu Asn Gly Gly Ser Cys Lys Asp 50 55 60Asp Ile Asn Ser Tyr Glu Cys
Trp Cys Pro Phe Gly Phe Glu Gly Lys65 70 75 80Asn Cys Glu Leu Asp
Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu 85 90 95Gln Phe Cys Lys
Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr 100 105 110Glu Gly
Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val 115 120
125Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr
130 135 140Arg Ala Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser
Thr Glu145 150 155 160Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser
Thr Gln Ser Phe Asn 165 170 175Asp Phe Thr Arg Val Val Gly Gly Glu
Asp Ala Lys Pro Gly Gln Phe 180 185 190Pro Trp Gln Val Val Leu Asn
Gly Lys Val Asp Ala Phe Cys Gly Gly 195 200 205Ser Ile Val Asn Glu
Lys Trp Ile Val Thr Ala Ala His Cys Val Glu 210 215 220Thr Gly Val
Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu225 230 235
240Thr Glu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His
245 250 255His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile
Ala Leu 260 265 270Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr
Val Thr Pro Ile 275 280 285Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile
Phe Leu Lys Phe Gly Ser 290 295 300Gly Tyr Val Ser Gly Trp Gly Arg
Val Phe His Lys Gly Arg Ser Ala305 310 315 320Leu Val Leu Gln Tyr
Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys 325 330 335Leu Arg Ser
Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly 340 345 350Phe
His Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro 355 360
365His Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser
370 375 380Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr
Thr Lys385 390 395 400Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys
Thr Lys Leu Thr 405 410 415
* * * * *