U.S. patent application number 11/580669 was filed with the patent office on 2007-05-10 for one pot desialylation and glycopegylation of therapeutic peptides.
This patent application is currently assigned to Neose Technologies, Inc.. Invention is credited to Robert J. Bayer, Shawn DeFrees, Matthew Kalo, Susann Taudte, Walter Scott Willett, David A. Zopf.
Application Number | 20070105755 11/580669 |
Document ID | / |
Family ID | 38004529 |
Filed Date | 2007-05-10 |
United States Patent
Application |
20070105755 |
Kind Code |
A1 |
DeFrees; Shawn ; et
al. |
May 10, 2007 |
One pot desialylation and glycopegylation of therapeutic
peptides
Abstract
The present invention provides conjugates between peptides and
PEG moieties. The conjugates are linked via an intact glycosyl
linking group that is interposed between and covalently attached to
the peptide and the modifying group. The conjugates are formed from
both glycosylated and unglycosylated peptides by the action of a
glycosyltransferase. The glycosyltransferase ligates a modified
sugar moiety onto either an amino acid or glycosyl residue on the
peptide. Also provided are pharmaceutical formulations including
the conjugates. Methods for preparing the conjugates are also
within the scope of the invention.
Inventors: |
DeFrees; Shawn; (North
Wales, PA) ; Zopf; David A.; (Wayne, PA) ;
Taudte; Susann; (Pennsburg, PA) ; Willett; Walter
Scott; (Doylestown, PA) ; Bayer; Robert J.;
(San Diego, CA) ; Kalo; Matthew; (San Diego,
CA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP (SF)
2 PALO ALTO SQUARE
3000 El Camino Real, Suite 700
PALO ALTO
CA
94306
US
|
Assignee: |
Neose Technologies, Inc.
102 Witmer Road
Horsham
PA
19044
|
Family ID: |
38004529 |
Appl. No.: |
11/580669 |
Filed: |
October 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US06/32649 |
Aug 21, 2006 |
|
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|
11580669 |
Oct 13, 2006 |
|
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60746868 |
May 9, 2006 |
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60733649 |
Nov 4, 2005 |
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60730607 |
Oct 26, 2005 |
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Current U.S.
Class: |
530/322 ;
435/68.1; 514/13.7; 514/20.9 |
Current CPC
Class: |
C12P 21/005 20130101;
A61K 38/4846 20130101 |
Class at
Publication: |
514/008 ;
435/068.1 |
International
Class: |
A61K 38/36 20060101
A61K038/36; C12P 21/06 20060101 C12P021/06 |
Claims
1. A method of altering glycosylation of a peptide, said method
comprising: (a) contacting said peptide with a sialidase, thereby
removing at least one sialic acid moiety from said peptide; (b)
contacting product from step (a) with at least one
glycosyltransferase and a sugar donor which is a substrate for said
at least one glycosyltransferase, thereby transferring at least one
sugar moiety from said donor a glycosyl moiety or amino acid of
said said product from step (a); (c) separating product from step
(b) from sialidase; and (d) contacting product from step (c) with a
sialic acid donor and a sialyltransferase, thereby forming a
peptide with altered glycosylation.
2. The method according to claim 1 wherein said peptide with
altered glycosylation comprises a modified sialyl residue having
the formula: ##STR150## wherein D is a member selected from --OH
and R.sup.1-L-HN--; G is a member selected from R.sup.1-L- and
--C(O)(C.sub.1-C.sub.6)alkyl-R.sup.1; R.sup.1 is a moiety
comprising a member selected from a straight-chain poly(ethylene
glycol) residue and branched poly(ethylene glycol) residue; and M
is a member selected from H, a metal and a single negative charge;
L is a linker which is a member selected from a bond, substituted
or unsubstituted alkyl and substituted or unsubstituted
heteroalkyl, such that 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-- said method
comprising: (a) contacting a peptide comprising the glycosyl
moiety: ##STR151## with a PEG-sialic acid donor moiety having the
formula: ##STR152## and an enzyme that transfers said PEG-sialic
acid onto the Gal of said glycosyl moiety, under conditions
appropriate for said transfer.
3. The method according to claim 2, wherein L-R.sup.1 has the
formula: ##STR153## wherein a is an integer selected from 0 to
20.
4. The method according to claim 2, wherein R.sup.1 has a structure
that is a member selected from: ##STR154## wherein e, f, m and n
are integers independently selected from 1 to 2500; and q is an
integer selected from 0 to 20.
5. The method according to claim 2, wherein R.sup.1 has a structure
that is a member selected from: ##STR155## wherein e, f and f' are
integers independently selected from 1 to 2500; and q and q' are
integers independently selected from 1 to 20.
6. The method according to claim 2, wherein R.sup.1 has a structure
that is a member selected from: ##STR156## wherein e and f are
integers independently selected from 1 to 2500.
7. The method according to claim 2, wherein said glycosyl linker
has the formula: ##STR157##
8. The method according to claim 2, wherein said peptide conjugate
comprises at least one of said glycosyl linker according to a
formula selected from: ##STR158## wherein AA is an amino acid
residue of said peptide conjugate and t is an integer selected from
0 and 1.
9. The method according to claim 2 wherein said peptide conjugate
comprises at least one of said glycosyl linker wherein each of said
glycosyl linker has a structure which is a member independently
selected from the following formulae: ##STR159## ##STR160## wherein
AA is an amino acid residue of said peptide conjugate and t is an
integer selected from 0 and 1.
10. The method of claim 2, wherein said peptide conjugate comprises
at least one of said glycosyl linker according to a formula
selected from: ##STR161## ##STR162## ##STR163## wherein AA is an
amino acid residue of said peptide conjugate and t is an integer
selected from 0 and 1 and wherein a member selected from 0 and 2 of
the sialyl moieties which do not comprise G are absent.
11. The method according to claim 2 wherein said peptide conjugate
comprises at least one said glycosyl linker according to a formula
selected from: ##STR164## ##STR165## ##STR166## ##STR167## wherein
AA is an amino acid residue of said peptide conjugate and t is an
integer selected from 0 and 1 and wherein a member selected from 0
and 2 of the sialyl moieties which do not comprise G are
absent.
12. The method according to claim 2, wherein said peptide has the
amino acid sequence of SEQ. ID. NO:1.
13. The method according to claim 2, wherein said glycosyl linker
is attached to said peptide through an amino acid residue selected
from serine and threonine.
14. The method according to claim 2, wherein said glycosyl linker
is attached to said peptide through an amino acid residue which is
an asparagine residue.
15. The method according to claim 14, wherein said asparagine
residue is a member selected from N152, N322 and combinations
thereof.
16. The method according to claim 2, wherein said peptide is a
bioactive peptide.
17. The method of claim 2, further comprising, prior to step (a):
(b) expressing said peptide in a suitable host.
18. The method of claim 17, wherein said host is a mammalian
expression system.
19. A method of treating a condition in a subject in need thereof,
said condition characterized by compromised clotting potency in
said subject, said method comprising the step of administering to
the subject an amount of the peptide conjugate produced according
to the method of claim 2, effective to ameliorate said condition in
said subject.
20. A method of enhancing clotting potency in a mammal, said method
comprising administering to said mammal an amount of the peptide
conjugate produced according to the method of claim 2.
Description
CROSS-REFERENCES TO OTHER APPLICATIONS
[0001] The present application is a continuation-in-part of
PCT/US06/32649, filed Aug. 21, 2006 and is related to U.S.
Provisional Patent Applications 60/746,868, filed May 9, 2006;
60/756,443, filed Jan. 5, 2006; 60/733,649, filed Nov. 4, 2005; and
60/730,607, filed Oct. 26, 2005, which are incorporated by
reference in their entirety for all purposes
BACKGROUND OF THE INVENTION
[0002] The attachment of synthetic polymers to the peptide backbone
in an attempt to improve the pharmacokinetic properties of
glycoprotein therapeutics is known in the art. 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.
[0003] 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).
[0004] In these non-specific methods, poly(ethylene glycol) 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.
[0005] 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).
[0006] In response to the need for improved therapeutic peptide,
the present invention provides a glycopegylated peptide that is
therapeutically active and which has pharmacokinetic parameters and
properties that are improved relative to an identical, or closely
analogous, peptide that is not glycopegylated. Furthermore, the
invention provides method for producing cost effectively and on an
industrial scale the improved peptides of the invention.
SUMMARY OF THE INVENTION
[0007] It has now been discovered that the controlled modification
of a peptide with one or more sugar moiety, either unmodified or
modified, e.g., with poly(ethylene glycol) moieties, affords a
novel peptide conjugate with pharmacokinetic properties that are
improved relative to the corresponding native unmodified, e.g.,
(un-pegylated), peptide. Furthermore, cost effective methods for
reliable and reproducible production of the peptide conjugates of
the invention have been discovered and developed.
[0008] Quite surprisingly, it has been discovered that a reaction
mixture that includes both a degradative enzyme (e.g., sialidase)
and a synthetic enzyme (e.g., sialyltransferase) can be used to
transfer a sugar residue (e.g., PEGylated sialic acid) onto a
peptide. Contrary to common wisdom, the degradative enzyme does not
significantly interfere with transfer of the sugar residue to the
peptide. Thus, the instant discovery provides for added convenience
in forming peptide conjugates, because the degradative and
synthetic enzymes can be utilized simultaneously without the need
to remove the degradative enzyme prior to performing the synthesis
step.
[0009] In a first aspect, the invention provides a method altering
glycosylation of a peptide. The method includes, (a) contacting the
peptide with a sialidase, thereby removing at least one sialic acid
moiety from the peptide. The peptide can be partially or wholly
desialylated as discussed above. In step (b), the product from step
(a) is contacted with at least one glycosyltransferase and at least
one sugar donor. The at least one sugar donor is a substrate for
the at least one glycosyltransferase. Preferably, step (b) results
in transfer of a sugar residue from the donor onto an amino acid or
glycosyl residue of the peptide. The sugar residue can be a
modified or unmodifed sugar residue. The conditions of step (b) are
maintained until a desired level of glycosylation of the peptide is
achieved, as discussed above. Following step (b), the sialidase
content of the reaction mixture is reduced and preferably,
essentially all of the sialidase is removed. In an exemplary
embodiment, the removal of sialidase is effected by a
chromatography (e.g., ion exchange, e.g., anion exchange) or
filtration step (e.g., nanofiltration). When the sialidase is
removed, the resulting glycosylated peptide (i.e., produced in step
(a)) is optionally contacted with a further at least one sugar
donor and at least one glycosyltransferase, transferring at least
one sugar residue onto an amino acid or glycosyl moiety of the
peptide. In a preferred embodiment, the sugar residue is sialic
acid. In a still further preferred embodiment, the sugar donor is
CMP-sialic acid. The glycosyltransferase is typically a
sialyltransferase.
[0010] An exemplary modified sugar donor includes a sugar residue
modified with a polymer (e.g., a water-soluble polymer, e.g., PEG),
a therapeutic agent, a diagnostic agent, a bioactive agent and/or a
peptide.
[0011] The process set forth above is of use for preparing
"glycopegylated" peptides in which a glycosyltransferase (or enzyme
having glycosyltransferase activity) transfers to an amino acid or
glycosyl residue of the peptide a sugar moiety modified with a PEG
moiety. As set forth herein, the process allows for control of the
degree of glycopegylation of the peptide by control of the speed of
the glycopegylation reaction and the time the reaction is allowed
to run. When the reaction is completed, exposed Gal residues on the
glycopegylated peptide can be "capped" using sialic acid.
[0012] In an exemplary embodiment, "glycopegylated" peptide
molecules of the invention are produced by the enzyme mediated
formation of a conjugate between a glycosylated or non-glycosylated
peptide and an enzymatically transferable saccharyl moiety that
includes a modifying group, such as a polymeric modifying group
such as poly(ethylene glycol), 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.
[0013] Thus, in one aspect, the present invention provides a
conjugate between a PEG moiety, e.g., PEG and a peptide that has an
in vivo activity similar or otherwise analogous to art-recognized
peptide. In the conjugate of the invention, the PEG moiety is
covalently attached to the peptide via an intact glycosyl linking
group. Exemplary intact glycosyl linking groups include sialic acid
moieties that are derivatized with PEG.
[0014] The polymeric modifying group can be attached at any
position of a glycosyl moiety of a peptide. Moreover, the polymeric
modifying group can be bound to a glycosyl residue at any position
in the amino acid sequence of a wild type or mutant peptide.
[0015] In an exemplary embodiment, the invention provides a peptide
that is conjugated through a glycosyl linking group to a polymeric
modifying group. Exemplary peptide conjugates include a glycosyl
linking group having a formula selected from: ##STR1##
[0016] In Formulae I and II, R.sup.2 is H, CH.sub.2OR.sup.7,
COOR.sup.7, COO.sup.- 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.8and
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 group 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 a sialyl moiety. In a further exemplary embodiment, this side
chain is functionalized with the polymeric modifying group.
[0017] In an exemplary embodiment, the polymeric modifying group 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: ##STR2## R.sup.1 is the polymeric modifying group 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.)
[0018] 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.
[0019] 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.
[0020] In an exemplary embodiment, the glycosyl linking group has a
structure according to Formula I and R.sup.5 includes the polymeric
modifying group. In another exemplary embodiment, R.sup.5 includes
both the polymeric modifying group and a linker, L, joining the
polymeric modifying group to the glycosyl core. L can be a linear
or branched structure. Similarly, the polymeric modifying group can
be branched or linear.
[0021] The polymeric modifying group 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.
[0022] The glycosyl core of the glycosyl linking groups of use in
the peptide conjugates are 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.
[0023] 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.
[0024] In one embodiment, the present invention provides a peptide
conjugate comprising the moiety: ##STR3## 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--.
[0025] In another aspect, the invention provides a peptide
conjugate comprising a peptide. The conjugate also comprises a
glycosyl linking group, wherein the glycosyl linking group is
attached to an amino acid residue of said peptide, and wherein said
glycosyl linking group comprises a sialyl linking group having a
formula which is a member selected from: ##STR4## are modifying
groups. R.sup.2 is a member selected from H, CH.sub.2OR.sup.7,
COOR.sup.7, COO.sup.- and OR.sup.7. R.sup.7 is a member selected
from H, substituted or unsubstituted alkyl and substituted or
unsubstituted heteroalkyl. R.sup.3 and R.sup.4 are members
independently selected from H, substituted or unsubstituted alkyl,
OR.sup.8, and NHC(O)R.sup.9. R.sup.8 and R.sup.9 are independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl and sialyl. L.sup.a is a linker selected
from a bond, substituted or unsubstituted alkyl and substituted or
unsubstituted heteroalkyl. X.sup.5, R.sup.16 and R.sup.17 are
independently selected from non-reactive group and polymeric arms
(e.g. PEG). X.sup.2 and X.sup.4 are independently selected linkage
fragments joining polymeric moieties R.sup.16 and R.sup.17 to C.
The index j is an integer selected from 1 to 15.
[0026] In another exemplary embodiment, the polymeric modifying
group has a structure according to the following formula: ##STR5##
in which the indices m and n are integers independently selected
from 0 to 5000. A.sup.1, A.sup.2, A.sup.3, A.sup.4, A.sup.5,
A.sup.6, A.sup.7, A.sup.8, A.sup.9, A.sup.10 and All are members
independently selected from H, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, --NA.sup.12A.sup.13, --OA.sup.12 and
--SiA.sup.12A.sup.13. A.sup.12 and A.sup.13 are members
independently selected from substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and
substituted or unsubstituted heteroaryl.
[0027] In an exemplary embodiment, the polymeric modifying group
has a structure according to the following formulae: ##STR6##
[0028] In another exemplary embodiment according to the formula
above, the polymeric modifying group has a structure according to
the following formula: ##STR7## In an exemplary embodiment, A.sup.1
and A.sup.2 are each members selected from --OH and
--OCH.sub.3.
[0029] Exemplary polymeric modifying groups according to this
embodiment include: ##STR8##
[0030] The invention provides a peptide conjugate comprising a
peptide. The conjugate also comprises a glycosyl linking group,
wherein the glycosyl linking group is attached to an amino acid
residue of the peptide, and wherein the glycosyl linking group
comprises a sialyl linking group having the formula: ##STR9## is a
modifying group. The index s is an integer selected from 1 to 20.
The index f is an integer selected from 1 to 2500. Q is a member
selected from H and substituted or unsubstituted C.sub.1-C.sub.6
alkyl.
[0031] In an exemplary embodiment, the invention provides a
modified sugar having the following formula: ##STR10##
[0032] The present invention provides methods of forming conjugates
of peptides. The methods include contacting a peptide with a
modified sugar donor that bears a modifying group covalently
attached to a sugar. The modified sugar moiety is transferred from
the donor onto an amino acid or glycosyl residue of the peptide by
the action of an enzyme. Representative enzymes include, but are
not limited to, glycosyltransferases, e.g., sialyltransferases. The
method includes contacting the peptide with: a) a modified sugar
donor; and b) an enzyme capable of transferring a modified sugar
moiety from the modified sugar donor onto an amino acid or glycosyl
residue of the peptide, under conditions appropriate to transfer a
modified sugar moiety from the donor to an amino acid or glycosyl
residue of the peptide, thereby synthesizing said peptide
conjugate.
[0033] In a preferred embodiment, prior to step a), the peptide is
contacted with a sialidase, thereby removing at least a portion of
the sialic acid on the peptide.
[0034] In another preferred embodiment, the peptide is contacted
with a sialidase, a glycosyltransferase and a modified sugar donor.
In this embodiment, the peptide is in contact with the sialidase,
glycosyltransferase and modified sugar donor essentially
simultaneously, no matter the order of addition of the various
components. The reaction is carried out under conditions
appropriate for the sialidase to remove a sialic acid residue from
the peptide; and the glycosyltransferase to transfer a modified
sugar moiety from the modified sugar donor to an amino acid or
glycosyl residue of the peptide.
[0035] In another preferred embodiment, the desialylation and
conjugation are performed in the same vessel, and the desialylated
peptide is preferably not purified prior to the conjugation step.
In another exemplary embodiment, the method further comprises a
`capping` step involving sialylation of the peptide conjugate. This
step is performed in the same reaction vessel that contains the
sialidase, sialyltransferase and modified sugar donor without prior
purification.
[0036] In another preferred embodiment, the desialylation of the
peptide is performed, and the asialo peptide is purified. The
purified asialo peptide is then subjected to conjugation reaction
conditions. In another exemplary embodiment, the method further
comprises a `capping` step involving sialylation of the peptide
conjugate. This step is performed in the same reaction vessel that
contains the sialidase, sialyltransferase and modified sugar donor
without prior purification.
[0037] In another exemplary embodiment, the capping step,
sialylation of the peptide conjugate, is performed in the same
reaction vessel that contains the sialidase, sialyltransferase and
modified sugar donor without prior purification.
[0038] In an exemplary embodiment, the contacting is for a time
less than 20 hours, preferably less than 16 hours, more preferably
less than 12 hours, even more preferably less than 8 hours, and
still more preferably less than 4 hours.
[0039] In a further aspect, the present invention provides a
peptide conjugate reaction mixture. The reaction mixture comprises:
a) a sialidase; b) an enzyme which is a member selected from
glycosyltransferase, exoglycosidase and endoglycosidase; c) a
modified sugar; and d) a peptide.
[0040] In another exemplary embodiment, the ratio of the sialidase
to the peptide is selected from 0.1 U/L:2 mg/mL to 10 U/L: 1 mg/mL,
preferably 0.5 U/L:2 mg/mL, more preferably 1.0 U/L:2 mg/mL, even
more preferably 10 U/L:2 mg/mL, still more preferably 0.1 U/L: 1
mg/mL, more preferably 0.5 U/L: 1 mg/mL, even more preferably 1.0
U/L: 1 mg/mL, and still more preferably 10 U/L: 1 mg/mL.
[0041] In an exemplary embodiment, at least 10%, 20%, 30%, 40%,
50%, 60%, 70% or 80% of said peptide conjugate includes at most two
PEG moieties. The PEG moieties can be added in a one-pot process,
or they can be added after the asialo peptide is purified.
[0042] In another exemplary embodiment, at least 10%, 20%, 30%,
40%, 50%, 60%, 70% or 80% of the peptide conjugate include at most
one PEG moiety. The PEG moiety can be added in a one-pot process,
or it can be added after the asialo peptide is purified.
[0043] In a further exemplary embodiment, the method further
comprises "capping", or adding sialic acid to the peptide
conjugate. In another exemplary embodiment, sialidase is added,
followed by a delay of 30 min, 1 hour, 1.5 hours, or 2 hours,
followed by the addition of the glycosyltransferase,
exoglycosidase, or endoglycosidase.
[0044] In another exemplary embodiment, sialidase is added,
followed by a delay of 30 min, 1 hour, 1.5 hours, or 2 hours,
followed by the addition of the glycosyltransfase, exoglycosidase,
or endoglycosidase. Other objects and advantages of the invention
will be apparent to those of skill in the art from the detailed
description that follows.
[0045] In another exemplary embodiment, the method includes: (a)
contacting a peptide comprising a glycosyl group selected from:
##STR11## with a modified sugar having the formula: ##STR12## and
an appropriate transferase which transfers the glycosyl linking
group onto a member selected from the GalNAc, Gal and the Sia of
said glycosyl group, under conditions appropriate for said
transfer. An exemplary modified sugar is CMP-sialic acid modified,
through a linker moiety, with a polymer, e.g., a straight chain or
branched poly(ethylene glycol) moiety.
[0046] The peptide can be acquired from essentially any source,
however, in one embodiment, prior to being modified as discussed
above, the peptide is expressed in a suitable host. Mammalian
(e.g., BHK, CHO), bacteria (e.g., E. coli) and insect cells (e.g.,
Sf-9) are exemplary expression systems providing peptides of use in
the compositions and methods set forth herein.
[0047] In exemplary embodiments, a peptide conjugate may be
administered to patients for the treatment of a tissue injury such
as ischemia, trauma, inflammation, or contact with toxic
substances. In other exemplary embodiments, a peptide conjugate may
be administered to patients for the treatment of a patient having
Hemophilia A, a patient with Hemophilia B, a patient having
Hemophilia A, wherein the patient also has antibodies to Factor
VIII, a patient having Hemophilia B, wherein the patient also has
antibodies to Factor IX, and a patient having liver cirrhosis.
[0048] In another exemplary embodiment, a peptide conjugate may be
administered to patients for the treatment of bleeding in
emergencies, elective surgery, cardiac surgery, spinal surgery,
liver transplantation, partial hepatectomies, pelvic-acetabular
fracture reconstruction, and allogeneic stem cell transplantation.
In another exemplary embodiment, a peptide conjugate may be
administered to patients for the treatment of acute intracerebral
haemorrhage, traumatic brain injury, variceal bleedings and upper
gastrointestinal bleeding.
[0049] In another aspect, the invention provides a pharmaceutical
formulation comprising a peptide conjugate and a pharmaceutically
acceptable carrier.
[0050] In the peptide conjugate, essentially each of the amino acid
residues to which the glycosyl linking group or modifying group is
bound has the same structure. For example, if one peptide includes
a Thr 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 linking
group covalently bound to the same Thr residue.
[0051] 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
[0052] FIG. 1 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 Factor VIIa-SA-PEG-10
KDa.
[0053] FIG. 2 is a synthetic scheme for producing an exemplary
PEG-glycosyl linking group precursor (modified sugar) of use in
preparing the conjugates of the invention.
[0054] FIG. 3 is a table providing exemplary sialyltransferases of
use in forming the glycoconjugates of the invention, e.g., to
glycoPEGylate peptides with a modified sialic acid.
[0055] FIG. 4, comprising FIGS. 4A to 4E, sets forth exemplary
schemes for remodeling glycan structures on Factor VII and Factor
VIIa. FIG. 4A is a diagram depicting the Factor VII and Factor VIIa
peptides indicating the residues which bind to glycans contemplated
for remodeling. FIG. 4B is a diagram depicting the Factor VII and
Factor VIIa peptides A (solid line) and B (dotted line) indicating
the residues which bind to glycans contemplated for remodeling, and
the formulas for the glycans. FIGS. 4C to 4E are diagrams of
contemplated remodeling steps of the glycan of the peptide in FIG.
4B based on the type of cell the peptide is expressed in and the
desired remodeled glycan structure.
[0056] FIG. 5, comprising FIGS. 5A and 5B, is an exemplary
nucleotide and corresponding amino acid sequence of Factor VIIa
(SEQ ID NOS: 1 and 2, respectively).
[0057] FIG. 6 is an image of an isoelectric focusing gel (pH 3-7)
of asialo-Factor VIIa. Lane 1 is Factor VIIa; lanes 2-5 are
asialo-Factor VIIa.
[0058] FIG. 7 is a graph of a MALDI spectra of Factor VIIa.
[0059] FIG. 8 is a graph of a MALDI spectra of Factor VIIa-SA-PEG-1
KDa.
[0060] FIG. 9 is a graph depicting a MALDI spectra of Factor
VIIa-SA-PEG-10 KDa.
[0061] FIG. 10 is an image of an SDS-PAGE gel of PEGylated Factor
VIIa. Lane 1 is asialo-Factor VIIa. Lane 2 is the product of the
reaction of asialo-Factor VIIa and CMP-SA-PEG-1 KDa with ST3Gal3
after 48 hr. Lane 3 is the product of the reaction of asialo-Factor
VIIa and CMP-SA-PEG-1 KDa with ST3Gal3 after 48 hr. Lane 4 is the
product of the reaction of asialo-Factor VIIa and CMP-SA-PEG-10 KDa
with ST3Gal3 at 96 hr.
[0062] FIG. 11A-B shows simultaneous desialylation, with less
sialidase, and PEGylation. These figures highlight that capping in
the presence of sialidase is efficient. FIG. 11A shows the reaction
course when the sialidase is at a level of 0.5 U/L. Lane 1
corresponds to native Factor VIIa while Lane 2 is asialo Factor
VIIa. From Lane 3 to Lane 7, there is an increasing amount of
PEGylated product as time progresses. In Lane 3, the major product
is monoPEGylated (see spot at 64), while aliquots assayed at later
times show the formation and increasing amounts of di (see spot
just below 97), tri (see spot just above 97), and higher PEGylated
products. Lanes 8 and 9 show the results of `capping`, or adding
sialic acid, to the reaction. When the reaction is capped, the
extent of reaction is stopped, as can be seen from the similar
PEGylated product distribution found in Lanes 5, 8 and 9. FIG. 11B
shows the reaction course when the sialidase is at a level of 0.1
U/L.
[0063] FIG. 12 shows the situation when the sialidase and the
glycosyltransferase are added at the same time.
[0064] FIG. 13 is a table of the peptides to which one or more
glycosyl linking groups can be attached to order to provide the
peptide conjugates of the invention.
[0065] FIG. 14A and B displays chromatograms showing the results of
HPLC experiments. FIG. 14A displays labeled chromatograms of Factor
VIIa-SA-PEG-10 KDa (top) and native Factor VIIa control (bottom)
analyzed by the light chain method. The separation of LC (light
chain), 1.times.10 KDa-PEG-LC, 2.times.10 KDa-PEG-LC, and
3.times.10 KDa-PEG-LC from other products is shown. FIG. 14B
displays labeled chromatograms of Factor VIIa-SA-PEG-10 KDa (top)
and native Factor VIIa control (bottom) analyzed by heavy chain
method. The separation of HC (heavy chain), 1.times.10 KDa-PEG-HC,
2.times.10 KDa-PEG-HC, and 3.times.10 KDa-PEG-HC from other
products is shown.
[0066] FIGS. 15A and B displays chromatograms showing the results
of HPLC experiments. FIG. 15A displays labeled chromatograms of
reduced native Factor VIIa control (top) and reduced Factor
VIIa-SA-PEG-40 KDa (bottom) analyzed by the light chain method. The
separation of LC (light chain), 1.times.40 KDa-PEG-LC, 2.times.40
KDa-PEG-LC, and 3.times.40 KDa-PEG-LC from other products is shown.
FIG. 15B displays labeled chromatograms of reduced native Factor
VIIa control (top) and Factor VIIa-SA-PEG-40 KDa (bottom) analyzed
by the heavy chain method. The separation of HC (heavy chain),
1.times.40 KDa-PEG-HC, 2.times.40 KDa-PEG-HC, and 3.times.40
KDa-PEG-HC from other products is shown.
DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED
EMBODIMENTS
Abbreviations
[0067] PEG, poly(ethyleneglycol); PPG, poly(propyleneglycol); Ara,
arabinosyl; Fru, fructosyl; Fuc, fucosyl; Gal, galactosyl; GalNAc,
N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc,
N-acetylglucosaminyl; Man, mannosyl; ManAc, mannosaminyl acetate;
Xyl, xylosyl; NeuAc, sialyl or N-acetylneuraminyl; Sia, sialyl or
N-acetylneuraminyl; and derivatives and analogues thereof.
Definitions
[0068] 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.
[0069] 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).
[0070] 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.
[0071] The term "sialic acid" or "sialyl" 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.
[0072] "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). A listing of some of
the peptides of the invention is provided in FIG. 13.
[0073] The term "peptide conjugate," refers to species of the
invention in which a peptide is conjugated with a modified sugar as
set forth herein.
[0074] 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.
[0075] As used herein, the term "modified sugar," or "modified
sugar residue", 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.
[0076] 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).
[0077] 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.
[0078] 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.
[0079] Many other polymers are also suitable for the invention.
Polymer backbones that are non-peptidic and water-soluble, within
about 2 to about 300 loci for attachment, 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(hydroxypropylmethacrylarnide), 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.
[0080] 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.
[0081] 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 may 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, D F A Crommelin and R D
Sindelar, eds., Harwood Publishers, Amsterdam, pp 101-120).
[0082] 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 G-CSF
peptide of the present invention. A subgenus of "glycoconjugation"
is "glyco-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.
[0083] 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.
[0084] 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-Schiff
base formation-reduction), or the glycosyl linking group may be
intact. An "intact glycosyl linking group" refers to a linking
group that is derived from a glycosyl moiety in which the
saccharide monomer that links the modifying group and to the
remainder of the conjugate is not degraded, e.g., oxidized, e.g.,
by sodium metaperiodate. "Intact glycosyl linking groups" of the
invention may be derived from a naturally occurring oligosaccharide
by addition of glycosyl unit(s) or removal of one or more glycosyl
unit from a parent saccharide structure.
[0085] The term, "non-glycosidic modifying group", as used herein,
refers to modifying groups which do not include a naturally
occurring sugar linked directly to the glycosyl linking group.
[0086] 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.
[0087] 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)).
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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).
[0092] 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.
[0093] 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%.
[0094] 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.
[0095] 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).
[0096] "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.
[0097] "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%.
[0098] 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.
[0099] "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. In the
fucosylated structures set forth herein, the Fuc-GlcNAc linkage is
generally 60 1,6 or .alpha.1,3, with .alpha.1,6 generally
preferred. 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.
[0100] 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.
[0101] 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--.
[0102] 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".
[0103] 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.
[0104] 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.
[0105] 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--.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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).
[0110] 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.
[0111] 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''').dbd.NR'''',
--NR--C(R'R'').dbd.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).
[0112] 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.
[0113] 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.u--U--, wherein T and U are
independently --NR--, --O--, --CRR'-- or a single bond, and u 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.z--X--(CR''R''').sub.d--, where z 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.
[0114] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
[0115] The invention is meant to include salts of the compounds of
the invention which are prepared with relatively nontoxic acids or
bases, depending on the particular substituents found on the
compounds described herein. When compounds of the present invention
contain relatively acidic functionalities, base addition salts can
be obtained by contacting the neutral form of such compounds with a
sufficient amount of the desired base, either neat or in a suitable
inert solvent. Examples of base addition salts include sodium,
potassium, lithium, calcium, ammonium, organic amino, or magnesium
salt, or a similar salt. When compounds of the present invention
contain relatively basic functionalities, acid addition salts can
be obtained by contacting the neutral form of such compounds with a
sufficient amount of the desired acid, either neat or in a suitable
inert solvent. Examples of acid addition salts include those
derived from inorganic acids like hydrochloric, hydrobromic,
nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, for
example, Berge et al., "Pharmaceutical Salts", Journal of
Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds
of the present invention contain both basic and acidic
functionalities that allow the compounds to be converted into
either base or acid addition salts.
[0116] The neutral forms of the compounds are preferably
regenerated by contacting the salt with a base or acid and
isolating the parent compounds in the conventional manner. The
parent form of the compound differs from the various salt forms in
certain physical properties, such as solubility in polar
solvents.
[0117] "Salt counterion", as used herein, refers to positively
charged ions that associate with a compound of the invention when
one of its moieties is negatively charged (e.g. COO--). Examples of
salt counterions include H.sup.+, H.sub.3O.sup.+, ammonium,
potassium, calcium, lithium, magnesium and sodium.
[0118] As used herein, the term "CMP-SA-PEG" is a cytidine
monophosphate molecule which is conjugated to a sialic acid which
comprises a polyethylene glycol moiety. If a length of the
polyethylene glycol chain is not specified, then any PEG chain
length is possible (e.g. 1 KDa, 2 KDa, 5 KDa, 10 KDa, 20 KDa, 30
KDa, 40 KDa). An exemplary CMP-SA-PEG is compound 5 in Scheme
1.
I. Introduction
[0119] To improve the effectiveness of a recombinant peptide used
for therapeutic purposes, the present invention provides conjugates
of glycosylated and unglycosylated peptides with a modifying group.
The modifying groups can be selected from polymeric modifying
groups such as, e.g., PEG (m-PEG), PPG (m-PPG), etc., therapeutic
moieties, diagnostic moieties, targeting moieties and the like.
Modification of the peptides, e.g., with a water-soluble polymeric
modifying group can improve the stability and retention time of the
recombinant peptide in a patient's circulation, and/or reduce the
antigenicity of recombinant peptide.
[0120] The peptide conjugates of the invention can be formed by the
enzymatic attachment of a modified sugar to the glycosylated or
unglycosylated peptide. A glycosylation site and/or a modified
glycosyl group provides a locus for conjugating a modified sugar
bearing a modifying group to the peptide, e.g., by
glycoconjugation.
[0121] The methods of the invention also make it possible to
assemble peptide conjugates and glycopeptide conjugates 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, particular
glycosyl residues, or combination of glycosyl residues of the
peptide. The methods are also practical for large-scale production
of peptide conjugates. Thus, the methods of the invention provide a
practical means for large-scale preparation of peptide conjugates
having preselected uniform derivatization patterns. The methods are
particularly well suited for modification of therapeutic peptides,
including but not limited to, glycopeptides that are incompletely
glycosylated during production in cell culture cells (e.g.,
mammalian cells, insect cells, plant cells, fungal cells, yeast
cells, or prokaryotic cells) or transgenic plants or animals.
[0122] The peptide conjugates can be produced as pharmaceutical
formulations comprising a peptide conjugate as well as a
pharmaceutically acceptable carrier. The peptide conjugates may be
administered to a patient selected from the group consisting of a
hemophiliac patient having a bleeding episode, a patient having
Hemophilia A, a patient with Hemophilia B, a patient having
Hemophilia A, wherein the patient also has antibodies to Factor
VIII, a patient having Hemophilia B, wherein the patient also has
antibodies to Factor IX, a patient having liver cirrhosis, a
cirrhotic patient having an orthotopic liver transplant, a
cirrhotic patient having upper gastrointestinal bleeding, a patient
having a bone marrow transplant, a patient having a liver
resection, a patient having a partial hepatectomy, a patient
undergoing pelvic-acetabular fracture reconstruction, a patient
bleeding from an acute intercerebral hemmorage, a patient
undergoing allogeneic stem cell transplantation, a patient bleeding
from traumatic brain injury, a patient bleeding in an emergency, a
patient having bleeding from trauma, a patient undergoing variceal
bleeding, a patient bleeding from elective surgery, a patient
bleeding from cardiac surgery, a patient bleeding from spinal
surgery, a liver resection a liver resection a liver resection. In
an exemplary embodiment, the patient is a human patient.
[0123] 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.
[0124] Determining optimal conditions for the preparation of
peptide conjugates with water-soluble polymers, e.g., involves the
optimization of numerous parameters, which are dependent on the
identity of the peptide and of the water-soluble polymer. For
example, when the polymer is poly(ethylene glycol), e.g., a
branched poly(ethylene glycol), a balance is preferably established
between the amount of polymer utilized in the reaction and the
viscosity of the reaction mixture attributable to the presence of
the polymer: if the polymer is too highly concentrated, the
reaction mixture becomes viscous, slowing the rate of mass transfer
and reaction.
[0125] Furthermore, though it is intuitively apparent to add an
excess of enzyme, the present inventors have recognized that, when
the enzyme is present in too great of an excess, the excess enzyme
becomes a contaminant whose removal requires extra purification
steps and material and unnecessarily increases the cost of the
final product.
[0126] Moreover, it is generally desired to produce a peptide with
a controlled level of modification. In some instances, it is
desireable to add one modified sugar preferentially. In other
instances, it is desirable to add two modified sugars
preferentially. Thus, the reaction conditions are preferably
controlled to influence the degree of conjugation of the modifying
groups to the peptide.
[0127] The present invention provides conditions under which the
yield of a peptide, having the desired level of conjugation, is
maximized. The conditions in the exemplary embodiments of the
inventions also recognize the expense of the various reagents and
the materials and time necessary to purify the product: the
reaction conditions set forth herein are optimized to provide
excellent yields of the desired product, while minimizing waste of
costly reagents.
II. The Compositions of Matter/Peptide Conjugates
[0128] In a first aspect, the present invention provides a
conjugate between a modified sugar and a peptide. The present
invention also provides a conjugate between a modifying group and a
peptide. A peptide conjugate can have one of several forms. In an
exemplary embodiment, a peptide conjugate can comprise a peptide
and a modifying group linked to an amino acid of the peptide
through a glycosyl linking group. In another exemplary embodiment,
a peptide conjugate can comprise a peptide and a modifying group
linked to a glycosyl reside of the peptide through a glycosyl
linking group. In another exemplary embodiment, the peptide
conjugate can comprise a peptide and a glycosyl linking group which
is bound to both a glycopeptide carbohydrate and directly to an
amino acid residue of the peptide backbone. In yet another
exemplary embodiment, a peptide conjugate can comprise a peptide
and a modifying group linked directly to an amino acid residue of
the peptide. In this embodiment, the peptide conjugate may not
comprise a glycosyl group. In any of these embodiments, the peptide
may or not be glycosylated.
[0129] The conjugates of the invention will typically correspond to
the general structure: ##STR13## 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", or modifying group, can be a
therapeutic agent, a bioactive agent, a detectable label, a
polymeric modifying group such as a water-soluble polymer (e.g.,
PEG, m-PEG, PPG, and m-PPG) or the like. The "agent", or modifying
group, 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." II.A. Peptide
[0130] The present invention should in no way be construed as
limited to a particular nucleic acid and amino acid sequences set
forth herein. Use of peptides of other sequences that are mutated
to increase or decrease a property or modify a structural feature
of the peptide are within the scope of the invention. For example,
mutant peptides of use in the invention include those that are
provided with additional 0-glycosylation sites or such sites at
other positions. Moreover, mutant peptides that include one or more
N-glycosylation site are of use in the invention. Perferably, the
amino acid sequence is at least about 96%, 97%, 98% or 99%
homologous to the amino acid sequences set forth herein.
[0131] In an exemplary embodiment, the amino acid residue to which
the glycosyl linking group is attached is a member selected from
serine, threonine and asparagine. In another exemplary embodiment,
the peptide has a sequence of SEQ. ID. NO 1. In another exemplary
embodiment, the amino acid residue is a member selected from Asn
145, Asn 322 and combinations thereof. In another exemplary
embodiment, the peptide is a bioactive peptide.
[0132] In yet another exemplary embodiment, the modified sugar
and/or PEG moiety on the peptide conjugate is located on the light
chain. In yet another exemplary embodiment, the modified sugar
and/or PEG moiety on the peptide conjugate is predominantly on the
heavy chain. In yet another exemplary embodiment, in a population
of peptide conjugates, the light chains predominantly contain a
modified sugar and/or PEG moiety. In yet another exemplary
embodiment, in a population of peptide conjugates, the heavy chains
predominantly contain a modified sugar and/or PEG moiety.
[0133] In another exemplary embodiment, the ratio of light chain:
heavy chain flinctionalization in the population is about 33:66. In
another exemplary embodiment, the ratio of light chain: heavy chain
flinctionalization in the population is about 35:65. In another
exemplary embodiment, the ratio of light chain: heavy chain
flinctionalization in the population is about 40:60. In another
exemplary embodiment, the ratio of light chain: heavy chain
functionalization in the population is about 45:55. In another
exemplary embodiment, the ratio is about 50:50. In another
exemplary embodiment, the ratio is about 55:45. In another
exemplary embodiment, the ratio is about 60:40. In another
exemplary embodiment, the ratio is about 65:35. In another
exemplary embodiment, the ratio is about 66:33. In another
exemplary embodiment, the ratio is about 70:30. In another
exemplary embodiment, the ratio is about 75:25. In another
exemplary embodiment, the ratio is about 80:20. In another
exemplary embodiment, the ratio is about 85:15. In another
exemplary embodiment, the ratio is about 90:10. In another
exemplary embodiment, the ratio of light chain: heavy chain
functionalization in the population is greater than about
90:10.
[0134] Methods for the expression and to determine the activity of
peptides are well known in the art, and are described in, for
example, U.S. Pat. No. 4,784,950. Briefly, expression of a peptide,
or variants thereof, can be accomplished in a variety of both
prokaryotic and eukaryotic systems, including E. coli, CHO cells,
BHK cells, insect cells using a baculovirus expression system, all
of which are well known in the art.
[0135] Assays for the activity of a peptide conjugate prepared
according to the methods of the present invention can be
accomplished using methods well known in the art. As a non-limiting
example, Quick et al. (Hemorragic Disease and Thrombosis, 2nd ed.,
Leat Febiger, Philadelphia, 1966), describes a one-stage clotting
assay useful for determining the biological activity of a peptide
prepared according to the methods of the present invention.
[0136] The peptides used in the invention are not limited to
peptide when the modifying group is: ##STR14## In these cases, the
peptide in the peptide conjugate is a member selected from the
peptides in FIG. 13. In these cases, the peptide in the peptide
conjugate is a member selected from Factor VII, Factor VIIa, Factor
VIII, Factor IX, Factor X, Factor XI, a peptide which is a member
selected from erythropoietin, granulocyte colony stimulating factor
(G-CSF), Granulocyte-Macrophage Colony Stimulating Factor
(GM-CSF)interferon alpha, interferon beta, interferon gamma,
.alpha..sub.1-antitrypsin (ATT, or .alpha.-1 protease inhibitor,
glucocerebrosidase, Tissue-Type Plasminogen Activator (TPA),
Interleukin-2 (IL-2), urokinase, human DNase, insulin, Hepatitis B
surface protein (HbsAg), human growth hormone, TNF Receptor-IgG Fc
region fusion protein (Enbrel.TM.), anti-HER2 monoclonal antibody
(Herceptin.TM.), monoclonal antibody to Protein F of Respiratory
Syncytial Virus (Synagis.TM.), monoclonal antibody to TNF-.alpha.
(Remicade.TM.), monoclonal antibody to glycoprotein IIb/IIIa
(Reopro.TM.), monoclonal antibody to CD20 (Rituxan.TM.),
anti-thrombin III (AT III), human Chorionic Gonadotropin (hCG),
alpha-galactosidase (Fabrazyme.TM.), alpha-iduronidase
(Aldurazyme.TM.), follicle stimulating hormone, beta-glucosidase,
anti-TNF-alpha monoclonal antibody (MLB 5075), glucagon-like
peptide-1 (GLP-1), beta-glucosidase (MLB 5064), alpha-galactosidase
A (MLB 5082) and fibroblast growth factor. In an exemplary
embodiment, the peptide is other than EPO.
[0137] In an exemplary embodiment, the polymeric modifying group
has a structure according to the following formulae: ##STR15##
[0138] The peptides used in the invention are also not limited when
the modifying group is: ##STR16## In an exemplary embodiment,
A.sup.1 and A.sup.2 are each members selected from --OH and
--OCH.sub.3.
[0139] Exemplary polymeric modifying groups according to this
embodiment include: ##STR17##
[0140] In an exemplary embodiment, in which the modifying group is
a branched water-soluble polymer, such as those shown above, it is
generally preferred that the concentration of sialidase is about
1.5 to about 2.5 U/L of reaction mixture. More preferably the
amount of sialidase is about 2 U/L.
[0141] In another exemplary embodiment, about 5 to about 9 grams of
peptide substrate is contacted with the amounts of sialidase set
forth above.
[0142] The modified sugar is present in the reaction mixture in an
amount from about 1 gram to about 6 grams, preferably from about 3
grams to about 4 grams. It is generally preferred to maintain the
concentration of a modified sugar having a branched water-soluble
polymer modifying moiety, e.g., the moiety shown above, at less
than about 0.5 mM. In a preferred embodiment, the modifying group
is a branched poly(ethylene glycol) having a molecular weight from
about 20 KDa to about 60 KDa, more preferably, from about 30 KDa to
about 50 KDa, and even more preferably about 40 KDa. An exemplary
modifying group having a molecular weight of about 40 KDa is one
that is from about 35 KDa to about 45 KDa.
[0143] Regarding the glycosyltransferase concentration, in a
presently preferred embodiment, using the modifying group set forth
above, the ratio of glycosyltransferase to peptide is about 40
.mu.g/mL transferase to about 200 .mu.M peptide.
II.B. Modified Sugar
[0144] In an exemplary embodiment, the peptides of the invention
are reacted with a modified sugar, thus forming a peptide
conjugate. A modified sugar comprises a "sugar donor moiety" as
well as a "sugar transfer moiety". The sugar donor moiety is any
portion of the modified sugar that will be attached to the peptide,
either through a glycosyl moiety or amino acid moiety, as a
conjugate of the invention. The sugar donor moiety includes those
atoms that are chemically altered during their conversion from the
modified sugar to the glycosyl linking group of the peptide
conjugate. The sugar transfer moiety is any portion of the modified
sugar that will be not be attached to the peptide as a conjugate of
the invention. For example, a modified sugar of the invention is
the PEGylated sugar nucleotide, PEG-sialic acid CMP. For PEG-sialic
acid CMP, the sugar donor moiety, or PEG-sialyl donor moiety,
comprises PEG-sialic acid while the sugar transfer moiety, or
sialyl transfer moiety, comprises CMP.
[0145] In modified sugars of use in the invention, the saccharyl
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 saccharyl 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.
[0146] Any saccharyl moiety can be utilized as the sugar donor
moiety of the modified sugar. The saccharyl moiety can be a known
sugar, such as mannose, galactose or glucose, or a species having
the stereochemistry of a known sugar. The general formulae of these
modified sugars are: ##STR18## Other saccharyl moieties that are
useful in forming the compositions of the invention include, but
are not limited to fucose and sialic acid, as well as amino sugars
such as glucosamine, galactosamine, mannosamine, the 5-amine
analogue of sialic acid and the like. The saccharyl moiety 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 modified sugar 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.
[0147] Examples of modified sugars of use in the invention are
described in PCT Patent Application No. PCT/US05/002522, which is
herein incorporated by reference.
[0148] 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 glycosyl
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: ##STR19## 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 peptide (--NH-(peptide)).
R.sup.14 is OR.sup.1, NHR.sup.1 or NH-L-R.sup.1. R.sup.1 and
NH-L-RL are as described above. II.C Glycosyl Linking Groups
[0149] In an exemplary embodiment, the invention provides a peptide
conjugate formed between a modified sugar of the invention and a
peptide. In another exemplary embodiment, when the modifying group
on the modified sugar is ##STR20## the peptide in the peptide
conjugate is a member selected from the peptides in FIG. 13. In yet
another exemplary embodiment, the peptide in the peptide conjugate
is a member selected from Factor VII, Factor VIIa, Factor VIII,
Factor IX, Factor X, Factor XI, erythropoietin, granulocyte colony
stimulating factor (G-CSF), Granulocyte-Macrophage Colony
Stimulating Factor (GM-CSF), interferon alpha, interferon beta,
interferon gamma, .alpha..sub.1-antitrypsin (ATT, or .alpha.-1
protease inhibitor, glucocerebrosidase, Tissue-Type Plasminogen
Activator (TPA), Interleukin-2 (IL-2), urokinase, human DNase,
insulin, Hepatitis B surface protein (HbsAg), human growth hormone,
TNF Receptor-IgG Fc region fusion protein (Enbrel.TM.), anti-HER2
monoclonal antibody (Herceptin.TM.), monoclonal antibody to Protein
F of Respiratory Syncytial Virus (Synagis.TM.), monoclonal antibody
to TNF-.alpha. (Remicade.TM.), monoclonal antibody to glycoprotein
IIb/IIIa (Reopro.TM.), monoclonal antibody to CD20 (Rituxan.TM.)
anti-thrombin III (AT III), human Chorionic Gonadotropin (hCG),
alpha-galactosidase (Fabrazyme.TM.), alpha-iduronidase
(Aldurazyme.TM.), follicle stimulating hormone, beta-glucosidase,
anti-TNF-alpha monoclonal antibody (MLB 5075), glucagon-like
peptide-1 (GLP-1), beta-glucosidase (MLB 5064), alpha-galactosidase
A (MLB 5082) and fibroblast growth factor. In this embodiment, the
sugar donor moiety (such as the saccharyl moiety and the modifying
group) of the modified sugar becomes a "glycosyl linking group".
The "glycosyl linking group" can alternatively refer to the
glycosyl moiety which is interposed between the peptide and the
modifying group.
[0150] In an exemplary embodiment, the polymeric modifying group
has a structure according to the following formulae: ##STR21##
[0151] In an exemplary embodiment, modifying group on the modified
sugar is: ##STR22## In an exemplary embodiment, Al and A.sup.2 are
each members selected from --OH and --OCH.sub.3.
[0152] Exemplary polymeric modifying groups according to this
embodiment include: ##STR23##
[0153] Due to the versatility of the methods available for adding
and/or modifying glycosyl residues on a peptide, the glycosyl
linking groups can have substantially any structure. 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
glycosyl linking groups and modified sugars. The glycosyl linking
group can comprise virtually any mono- or oligo-saccharide. The
glycosyl linking groups can be attached to an amino acid either
through the side chain or through the peptide backbone.
Alternatively the glycosyl linking groups can be attached to the
peptide through a saccharyl moiety. This saccharyl moiety can be a
portion of an O-linked or N-linked glycan structure on the
peptide.
[0154] In an exemplary embodiment, the invention provides a peptide
conjugate comprising an intact glycosyl linking group having a
formula that is selected from: ##STR24## In Formulae I R.sup.2 is
H, CH.sub.2OR.sup.7, COOR.sup.7 or OR', 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 a modifying group. This modifying group can be
a 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, the pyruvyl side chain is functionalized with the
polymeric modifying group. 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 group is a component of R.sup.5.
[0155] In an exemplary embodiment, the invention utilizes a
glycosyl linking group that has the formula: ##STR25## in which J
is a glycosyl moiety, L is a bond or a linker and R.sup.1 is a
modifying group, e.g., a polymeric modifying group. 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.
[0156] 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'). 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.
[0157] An exemplary species of NH-L-R.sup.1 has the formula:
--NH{C(O)(CH.sub.2).sub.aNH}s{C(O)(CH.sub.2).sub.b(OCH.sub.2CH.sub.2).sub-
.cO(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.
[0158] 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.cO(CH-
.sub.2).sub.dNHR.sup.1,
NHC(O)(CH.sub.2).sub.b(OCH.sub.2CH.sub.2).sub.cO(CH.sub.2).sub.dNHR.sup.1-
,
NHC(O)O(CH.sub.2).sub.b(OCH.sub.2CH.sub.2).sub.cO(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.cO(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.
[0159] In an exemplary embodiment, c is selected such that the PEG
moiety is approximately 1 kD, 5 kD, 10, kD, 15 kD, 20 kD, 25 kD, 30
kD, 35 kD, 40 kD or 45 kD.
[0160] For the purposes of convenience, the glycosyl linking groups
in the remainder of this section will be based on a sialyl moiety.
However, one of skill in the art will recognize that another
glycosyl moiety, such as mannosyl, galactosyl, glucosyl, or
fucosyl, could be used in place of the sialyl moiety.
[0161] In an exemplary embodiment, the 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: ##STR26##
[0162] 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.
[0163] In still a further exemplary embodiment, the conjugate is
formed between a peptide and a modified sugar in which the
modifying group is attached through a linker at the 6-carbon
position of the modified sugar. Thus, illustrative glycosyl linking
groups according to this embodiment have the formula: ##STR27## in
which the radicals are as discussed above. Glycosyl linking groups
include, without limitation, glucose, glucosamine,
N-acetyl-glucosamine, galactose, galactosamine,
N-acetyl-galactosamine, mannose, mannosamine, N-acetyl-mannosamine,
and the like.
[0164] In one embodiment, the present invention provides a peptide
conjugate comprising the following glycosyl linking group:
##STR28## 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--.
[0165] In one embodiment, the present invention provides a peptide
conjugate comprising the following glycosyl linking group:
##STR29## D is a member selected from --OH and R.sup.1-L-HN--; G is
a member selected from R.sup.1-L- and
--C(O)(C.sub.1-C.sub.6)alkyl-R.sup.1; R.sup.1 is a moiety
comprising a member selected from a straight-chain poly(ethylene
glycol) residue and branched poly(ethylene glycol) residue; and M
is a member selected from H, a salt counterion and a single
negative charge; L is a linker which is a member selected from a
bond, substituted or unsubstituted alkyl and substituted or
unsubstituted heteroalkyl. In an exemplary embodiment, when D is
OH, G is R.sup.1-L-. In another exemplary embodiment, when G is
--C(O)(C.sub.1-C.sub.6)alkyl, D is R.sup.1-L-NH--.
[0166] In any the compounds of the invention, a COOH group can
alternatively be COOM, wherein M is a member selected from H, a
negative charge, and a salt counterion.
[0167] The invention provides a peptide conjugate that includes a
glycosyl linking group having the formula: ##STR30##
[0168] In other embodiments, the glycosyl linking group has the
formula: ##STR31## in which the index t is 0 or 1.
[0169] In a still further exemplary embodiment, the glycosyl
linking group has the formula: ##STR32## in which the index t is 0
or 1.
[0170] In yet another embodiment, the glycosyl linking group has
the formula: ##STR33## in which the index p represents and integer
from 1 to 10; and a is either 0 or 1.
[0171] In another exemplary embodiment, the peptide conjugate
comprises a glycosyl moiety selected from the formulae: ##STR34##
##STR35## ##STR36## ##STR37## ##STR38## ##STR39## ##STR40##
##STR41## ##STR42## ##STR43## ##STR44## ##STR45## 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 indices t and a are independently
selected from 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. AA is an amino acid residue of the
peptide.
[0172] In an exemplary embodiment, the PEG moiety has a molecular
weight of about 20 KDa. In another exemplary embodiment, the PEG
moiety has a molecular weight of about 5 KDa. In another exemplary
embodiment, the PEG moiety has a molecular weight of about 10 KDa.
In another exemplary embodiment, the PEG moiety has a molecular
weight of about 40 KDa.
[0173] In an exemplary embodiment, the glycosyl linking group is a
branched SA-PEG-10 KDa moiety based on a cysteine residue, and one
or two of these glycosyl linking groups are covalently attached to
the peptide. In another exemplary embodiment, the glycosyl linking
group is a branched SA-PEG-10 KDa moiety based on a lysine residue,
and one or two of these glycosyl linking groups are covalently
attached to the peptide. In an exemplary embodiment, the glycosyl
linking group is a branched SA-PEG-10 KDa moiety based on a
cysteine residue, and one or two of these glycosyl linking groups
are covalently attached to the peptide. In an exemplary embodiment,
the glycosyl linking group is a branched SA-PEG-10 KDa moiety based
on a lysine residue, and one or two of these glycosyl linking
groups are covalently attached to the peptide. In an exemplary
embodiment, the glycosyl linking group is a branched SA-PEG-5 KDa
moiety based on a cysteine residue, and one, two or three of these
glycosyl linking groups are covalently attached to the peptide. In
an exemplary embodiment, the glycosyl linking group is a branched
SA-PEG-5 KDa moiety based on a lysine residue, and one, two or
three of these glycosyl linking groups are covalently attached to
the peptide. In an exemplary embodiment, the glycosyl linking group
is a branched SA-PEG-40 KDa moiety based on a cysteine residue, and
one or two of these glycosyl linking groups are covalently attached
to the peptide. In an exemplary embodiment, the glycosyl linking
group is a branched SA-PEG-40 KDa moiety based on a lysine residue,
and one or two of these glycosyl linking groups are covalently
attached to the peptide.
[0174] In an exemplary embodiment, a glycoPEGylated peptide
conjugate of the invention selected from the formulae set forth
below: ##STR46##
[0175] In the formulae above, the index t is an integer from 0 to 1
and the index p is an integer from 1 to 10. The symbol R 15
represents H, OH (e.g., Gal-OH), a sialyl moiety, a sialyl linking
group (i.e., sialyl linking group-polymeric modifying group
(Sia-L-R'), 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 peptide conjugate 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.
[0176] In an exemplary embodiment, the sialyl linking group is a
sialyl moiety to which is bound a polymer modified sialyl moiety
(e.g., Sia-Sia-L-R.sup.1) ("Sia-Sia.sup.p"). Here, the glycosyl
linking group is linked to a galactosyl moiety through a sialyl
moiety: ##STR47## 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.
[0177] In another exemplary embodiment, the glycans on the peptide
conjugates have a formula that is selected from the group:
##STR48## and combinations thereof.
[0178] In each of the formulae above, R.sup.15' is as discussed
above. Moreover, an exemplary peptide conjugate of the invention
will include at least one glycan with an R.sup.15 moiety having a
structure according to Formulae I or II.
[0179] In another exemplary embodiment, the glycosyl linking group
comprises at least one glycosyl linking group having the formula:
##STR49## wherein R.sup.15 is said sialyl linking group; and the
index p is an integer selected from 1 to 10.
[0180] In an exemplary embodiment, the glycosyl linking moiety has
the formula: ##STR50## in which b is an integer from 0 to 1. The
index s represents an integer from 1 to 10; and the index f
represents an integer from 1 to 2500.
[0181] In an exemplary embodiment, the polymeric modifying group is
PEG. In another exemplary embodiment, the PEG moiety has a
molecular weight of about 20 KDa. In another exemplary embodiment,
the PEG moiety has a molecular weight of about 5 KDa. In another
exemplary embodiment, the PEG moiety has a molecular weight of
about 10 KDa. In another exemplary embodiment, the PEG moiety has a
molecular weight of about 40 kDa. In another exemplary embodiment
the glycosyl linking group is attached to Asn145, Asn322, Ser52,
Ser60 or combinations thereof.
[0182] In an exemplary embodiment, the glycosyl linking group is a
linear SA-PEG-10 KDa moiety, and one or two of these glycosyl
linking groups are covalently attached to the peptide. In another
exemplary embodiment, the glycosyl linking group is a linear
SA-PEG-20 KDa moiety, and one or two of these glycosyl linking
groups are covalently attached to the peptide. In an exemplary
embodiment, the glycosyl linking group is a linear SA-PEG-5 KDa
moiety, and one, two or three of these glycosyl linking groups are
covalently attached to the peptide. In an exemplary embodiment, the
glycosyl linking group is a linear SA-PEG-40 KDa moiety, and one or
two of these glycosyl linking groups are covalently attached to the
peptide.
[0183] In another exemplary embodiment, the glycosyl linking group
is a sialyl linking group having the formula: ##STR51## In another
exemplary embodiment, Q is a member selected from H and CH.sub.3.
In another exemplary embodiment, wherein said glycosyl linking
group has the formula: ##STR52## wherein R.sup.15 is said sialyl
linking group; and the index p is an integer selected from 1 to 10.
In an exemplary embodiment, the glycosyl linking group comprises
the formula: ##STR53## wherein the index b is an integer selected
from 0 and 1. In an exemplary embodiment, the index s is 1; and the
index f is an integer selceted from about 200 to about 300. In
another exemplary embodiment, the glycosyl linking group is a
member selected from SA-PEG-10 KDa and SA-PEG-20 KDa, and wherein
the number of said glycosyl linking groups which are covalently
attached to the peptide is an integer selected from 1 to 2. In
another exemplary embodiment, the glycosyl linking group is member
selected from SA-PEG-5 KDa and SA-PEG-40 KDa, and wherein the
number of said glycosyl linking groups which are covalently
attached to the peptide is an integer selected from 1 to 3. II.D.
Modifying Groups
[0184] The peptide conjugates of the invention comprise a modifying
group. This group can be covalently attached to a peptide through
an amino acid or a glycosyl linking group. In another exemplary
embodiment, when the modifying group is ##STR54## the peptide in
the peptide conjugate is a member selected from the peptides in
FIG. 13. In another exemplary embodiment, the peptide in the
peptide conjugate is a member selected from Factor VII, Factor
VIIa, Factor VIII, Factor IX, Factor X, Factor XI, erythropoietin,
granulocyte colony stimulating factor (G-CSF),
Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF)interferon
alpha, interferon beta, interferon gamma, .alpha..sub.1-antitrypsin
(ATT, or .alpha.-1 protease inhibitor, glucocerebrosidase,
Tissue-Type Plasminogen Activator (TPA), Interleukin-2 (IL-2),
urokinase, human DNase, insulin, Hepatitis B surface protein
(HbsAg), human growth hormone, TNF Receptor-IgG Fc region fusion
protein (Enbrel.TM.), anti-HER2 monoclonal antibody
(Herceptin.TM.), monoclonal antibody to Protein F of Respiratory
Syncytial Virus (Synagis.TM.), monoclonal antibody to TNF-.alpha.
(Remicade.TM.), monoclonal antibody to glycoprotein IIb/IIIa
(Reopro.TM.), monoclonal antibody to CD20 (Rituxan.TM.),
anti-thrombin III (AT III), human Chorionic Gonadotropin (hCG),
alpha-galactosidase (Fabrazyme.TM.), alpha-iduronidase
(Aldurazyme.TM.), follicle stimulating hormone, beta-glucosidase,
anti-TNF-alpha monoclonal antibody (MLB 5075), glucagon-like
peptide-1 (GLP-1), beta-glucosidase (MLB 5064), alpha-galactosidase
A (MLB 5082) and fibroblast growth factor. "Modifying groups" can
encompass a variety of structures including targeting moieties,
therapeutic moieties, biomolecules. Additionally, "modifying
groups" include polymeric modifying groups, which are polymers
which can alter a property of the peptide such as its
bioavailability or its half-life in the body.
[0185] In an exemplary embodiment, the polymeric modifying group
has a structure according to the following formulae: ##STR55##
[0186] In another exemplary embodiment according to the formula
above, the polymeric modifying group has a structure according to
the following formula: ##STR56## In an exemplary embodiment,
A.sup.1 and A.sup.2 are each members selected from --OH and
--OCH.sub.3.
[0187] Exemplary polymeric modifying groups according to this
embodiment include: ##STR57##
[0188] For the purposes of convenience, the modifying groups in the
remainder of this section will be largely based on polymeric
modifying groups such as water soluble and water insoluble
polymers. However, one of skill in the art will recognize that
other modifying groups, such as targeting moieties, therapeutic
moieties and biomolecules, could be used in place of the polymeric
modifying groups.
II.D.i. Linkers of the Modifying Groups
[0189] The linkers of the modifying group serve to attach the
modifying group (ie polymeric modifying groups, targeting moieties,
therapeutic moieties and biomolecules) to the peptide. In an
exemplary embodiment, the polymeric modifying group is bound to a
glycosyl linking group, generally through a heteroatom, e.g,
nitrogen, on the core through a linker, L, as shown below:
##STR58## 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.
[0190] An exemplary compound according to the invention has a
structure according to Formulae I or II above, 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: ##STR59##
[0191] 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: ##STR60## in which s is an integer from 0 to 20 and
R.sup.1 is a linear polymeric modifying moiety.
[0192] In an exemplary embodiment, the polymeric modifying
group-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: ##STR61## 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.
[0193] 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.
[0194] 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 group 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.
[0195] In an exemplary embodiment, R.sup.5 includes the polymeric
modifying group. In another exemplary embodiment, R.sup.5 includes
both the polymeric modifying group and a linker, L, joining the
modifying group to the remainder of the molecule. As discussed
above, L can be a linear or branched structure. Similarly, the
polymeric modifying group can be branched or linear.
II.D.ii. Water-Soluble Polymers
[0196] 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.
[0197] 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)).
[0198] Exemplary 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."
[0199] 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).
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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 40,000.
II.D.iii. Branched Water Soluble Polymers
[0205] 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.
[0206] 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: ##STR62## 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.
[0207] In yet another embodiment, the polymeric modifying moiety is
a branched PEG moiety that 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: ##STR63##
in which the indices e, f and f' are independently selected
integers from 1 to 2500; and the indices q, q' and q'' are
independently selected integers from 1 to 20.
[0208] As will be apparent to those of skill, the branched polymers
of use in the invention include variations on the themes set forth
above. For example the di-lysine-PEG conjugate shown above can
include three polymeric subunits, the third bonded to the a-amine
shown as unmodified in the structure above. Similarly, the use of a
tri-lysine functionalized with three or four polymeric subunits
labeled with the polymeric modifying moiety in a desired manner is
within the scope of the invention.
[0209] 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 PEG containing peptide conjugates according to
this embodiment of the invention has the formula: ##STR64## Another
exemplary precursor of use to form the branched PEG containing
peptide conjugates according to this embodiment of the invention
has the formula: ##STR65##
[0210] 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, R.sup.16 and R.sup.17 are
independently selected from non-reactive groups (e.g., H,
unsubstituted alkyl, unsubstituted heteroalkyl) and 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.
[0211] 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.
[0212] In an exemplary embodiment, the precursor (Formula 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.
[0213] In an exemplary embodiment, the polymeric modifying group
has a structure according to the following formulae: ##STR66##
[0214] In another exemplary embodiment according to the formula
above, the branched polymer has a structure according to the
following formula: ##STR67## In an exemplary embodiment, A.sup.1
and A.sup.2 are each selected from --OH and --OCH.sub.3.
[0215] Exemplary polymeric modifying groups according to this
embodiment include: ##STR68##
[0216] In an exemplary embodiment, the moiety: ##STR69## ps 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: ##STR70##
[0217] 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 a
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.
[0218] In another exemplary embodiment, X.sup.a is a linking moiety
formed with another linker: ##STR71## 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.
[0219] 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.
[0220] 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.
[0221] In a further exemplary embodiment, the peptide conjugates of
the invention include a moiety, e.g., an R.sup.15 moiety that has a
formula that is selected from: ##STR72## ##STR73## 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.
[0222] In yet another exemplary embodiment, the invention provides
peptide conjugates having a moiety, e.g., an R.sup.15 moiety with
formula: ##STR74## 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.
[0223] In yet another exemplary embodiment, the peptide conjugate
includes a RI.sup.5 moiety with a formula which is a member
selected from: ##STR75## 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 the
indices h and j are independently selected integers from 0 to 10. A
further exemplary species is --C(O)NH--. The indices m and n are
integers independently selected from 0 to 5000. A.sup.1, A.sup.2,
A.sup.3, A.sup.4, A.sup.5, A.sup.6, A.sup.7, A.sup.8, A.sup.9,
A.sup.10 and A.sup.11 are members independently selected from H,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, --NA.sup.12A.sup.13,
--OA.sup.12 and --SiA.sup.12A.sup.13. A.sup.12 and A.sup.13 are
members independently selected from substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and
substituted or unsubstituted heteroaryl.
[0224] 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.
[0225] PEG of any molecular weight, e.g., 1 KDa, 2 KDa, 5 KDa, l0
KDa, 15 KDa, 20 KDa, 25 KDa, 30 KDa, 35 KDa, 40 KDa and 45 KDa is
of use in the present invention.
[0226] In an exemplary embodiment, the R.sup.15 moiety has a
formula that is a member selected from the group: ##STR76## In each
of the structures above, the linker fragment --NH(CH.sub.2).sub.a--
can be present or absent.
[0227] In other exemplary embodiments, the peptide conjugate
includes an R.sup.15 moiety selected from the group: ##STR77##
[0228] 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 KDa, 2 KDa, 5 KDa, 10 KDa, 15 KDa, 20 KDa, 25 KDa,
30 KDa, 35 KDa, 40 KDa and 45 KDa. 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.
[0229] Other branched polymers have structures based on di-lysine
(Lys-Lys) peptides, e.g.: ##STR78## and tri-lysine peptides
(Lys-Lys-Lys), e.g.: ##STR79## In each of the figures above, the
indices 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.
[0230] In another exemplary embodiment, the modifying group:
##STR80## has a formula that is a member selected from: ##STR81##
wherein Q is a member selected from H and substituted or
unsubstituted C.sub.1-C.sub.6 alkyl. The indices e and f are
integers independently selected from 1 to 2500, and the index q is
an integer selected from 0 to 20.
[0231] In another exemplary embodiment, the modifying group:
##STR82## has a formula that is a member selected from: ##STR83##
wherein Q is a member selected from H and substituted or
unsubstituted C.sub.1-C.sub.6 alkyl. The indices e, f and f' are
integers independently selected from 1 to 2500, and q and q' are
integers independently selected from 1 to 20.
[0232] In another exemplary embodiment, the branched polymer has a
structure according to the following formula: ##STR84## in which
the indices m and n are integers independently selected from 0 to
5000. A.sup.1, A.sup.2, A.sup.3, A.sup.4, A.sup.5, A.sup.6,
A.sup.7, A.sup.8, A.sup.9, A.sup.10 and A.sup.11 are members
independently selected from H, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, --NA.sup.12A.sup.13, --OA.sup.12 and
--SiA.sup.12A.sup.13. A.sup.12 and A.sup.13 are members
independently selected from substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and
substituted or unsubstituted heteroaryl.
[0233] Formula IIIa is a subset of Formula III. The structures
described by Formula IIIa are also encompassed by Formula III.
[0234] In an exemplary embodiment, the polymeric modifying group
has a structure according to the following formulae: ##STR85##
[0235] In another exemplary embodiment according to the formula
above, the branched polymer has a structure according to the
following formula: ##STR86## In an exemplary embodiment, A.sup.1
and A.sup.2 are members independently selected from --OH and
--OCH.sub.3.
[0236] Exemplary polymeric modifying groups according to this
embodiment include: ##STR87##
[0237] In an illustrative embodiment, the modified sugar is sialic
acid and selected modified sugar compounds of use in the invention
have the formulae: ##STR88## 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.
[0238] 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: ##STR89## The structures set forth above can be components
of R.sup.15.
[0239] 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.
[0240] In selected embodiments, R.sup.1 or L-R.sup.1 is a branched
PEG, for example, one of the species set forth above. In an
exemplary embodiment, the branched PEG structure is based on a
cysteine peptide. Illustrative modified sugars according to this
embodiment include: ##STR90## in which X.sup.4 is a bond or O. In
each of the structures above, the alkylamine linker
--(CH.sub.2).sub.aNH-- can be present or absent. The structures set
forth above can be components of R.sup.15/R.sup.15'.
[0241] 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: ##STR91## in which the indices q
and e are as discussed above.
[0242] Exemplary modified sugars are modified with water-soluble or
water-insoluble polymers. Examples of useful polymer are further
exemplified below.
[0243] In another exemplary embodiment, the peptide 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 a peptide that comprises at least one moiety
having the formula: ##STR92## in which the index t is an integer
from 0 to 1; the index s represents an integer from 1 to 10; and
the index f represents an integer from 1 to 2500.
[0244] In one embodiment, the present invention provides a peptide
conjugate comprising the following glycosyl linking group:
##STR93## D is a member selected from --OH and R.sup.1-L-HN--; G is
a member selected from R.sup.1-L- and
--C(O)(C.sub.1-C.sub.6)alkyl-R.sup.1; R.sup.1 is a moiety
comprising a member selected from a straight-chain poly(ethylene
glycol) residue and branched poly(ethylene glycol) residue; and M
is a member selected from H, a salt counterion and a single
negative charge; L is a linker which is a member selected from a
bond, substituted or unsubstituted alkyl and substituted or
unsubstituted heteroalkyl. In an exemplary embodiment, when D is
OH, G is R.sup.1-L-. In another exemplary embodiment, when G is
--C(O)(C.sub.1-C.sub.6)alkyl, D is R.sup.1-L-NH--.
[0245] In an exemplary embodiment, L-R.sup.1 has the formula:
##STR94## wherein a is an integer selected from 0 to 20.
[0246] In an exemplary embodiment, R.sup.1 has a structure that is
a member selected from: ##STR95## wherein e, f, m and n are
integers independently selected from 1 to 2500; and q is an integer
selected from 0 to 20.
[0247] an exemplary embodiment, R.sup.1 has a structure that is a
member selected from: ##STR96## wherein e, f and f' are integers
independently selected from 1 to 2500; and q and q' are integers
independently selected from 1 to 20.
[0248] In another exemplary embodiment, R.sup.1 has a structure
that is a member selected from: ##STR97## wherein e, f and f' are
integers independently selected from 1 to 2500; and q and q' are
integers independently selected from 1 to 20.
[0249] In another exemplary embodiment, R.sup.1 has a structure
that is a member selected from: ##STR98## wherein e and f are
integers independently selected from 1 to 2500
[0250] In another exemplary embodiment, the glycosyl linker has the
formula: ##STR99##
[0251] In another exemplary embodiment, the peptide conjugate
comprises at least one of said glycosyl linker according to a
formula selected from: ##STR100## wherein AA is an amino acid
residue of said peptide conjugate and t is an integer selected from
0 and 1.
[0252] In another exemplary embodiment, the peptide conjugate
comprises at least one of said glycosyl linker wherein each of said
glycosyl linker has a structure which is a member independently
selected from the following formulae: ##STR101## ##STR102## wherein
AA is an amino acid residue of said peptide conjugate and t is an
integer selected from 0 and 1.
[0253] In another exemplary embodiment, the peptide conjugate
comprises at least one of said glycosyl linker according to a
formula selected from: ##STR103## ##STR104## ##STR105## wherein AA
is an amino acid residue of said peptide conjugate and t is an
integer selected from 0 and 1. In an exemplary embodiment, a member
selected from 0 and 2 of the sialyl moieties which do not comprise
G are absent. In an exemplary embodiment, a member selected from 1
and 2 of the sialyl moieties which do not comprise G are
absent.
[0254] In another exemplary embodiment, the peptide conjugate
comprises at least one of said glycosyl linker according to a
formula selected from: ##STR106## ##STR107## ##STR108## wherein AA
is an amino acid residue of said peptide conjugate and t is an
integer selected from 0 and 1. In an exemplary embodiment, a member
selected from 0 and 2 of the sialyl moieties which do not comprise
G are absent. In an exemplary embodiment, a member selected from 1
and 2 of the sialyl moieties which do not comprise G are
absent.
[0255] In another exemplary embodiment, the peptide conjugate
comprises at least one said glycosyl linker according to a formula
selected from: ##STR109## ##STR110## wherein AA is an amino acid
residue of said peptide conjugate and t is an integer selected from
0 and 1. In an exemplary embodiment, a member selected from 0 and 2
of the sialyl moieties which do not comprise G are absent. In an
exemplary embodiment, a member selected from 1 and 2 of the sialyl
moieties which do not comprise G are absent.
[0256] In another exemplary embodiment, the peptide has the amino
acid sequence of SEQ. ID. NO:1. In another exemplary embodiment,
the glycosyl linker is attached to the peptide through an amino
acid residue selected from serine and threonine.
[0257] In another exemplary embodiment, the asparagine residue is a
member selected from N152, N322 and combinations thereof.
[0258] In another exemplary embodiment, the peptide is a bioactive
peptide.
[0259] In another exemplary embodiment, the glycosyl linker is
attached to said peptide through an amino acid residue which is an
asparagine residue.
[0260] In another exemplary embodiment, the invention provides a
peptide which is produced in a suitable host. The invention also
provides methods of expressing this peptide. In another exemplary
embodiment, the host is a mammalian expression system.
[0261] In another exemplary embodiment, the invention provides a
method of treating a condition in a subject in need thereof, said
condition characterized by compromised clotting potency in said
subject, said method comprising the step of administering to the
subject an amount of the peptide conjugate of invention, effective
to ameliorate said condition in said subject. In another exemplary
embodiment, the method comprises administering to said mammal an
amount of the peptide conjugate produced according to the methods
described herein.
[0262] In another aspect, the invention provides a method of making
a peptide conjugate comprising a glycosyl linker comprising a
modified sialyl residue having the formula: ##STR111## wherein
R.sup.2 is H, CH.sub.2OR.sup.7, COOR.sup.7 or OR.sup.7. 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. R.sup.8 and R.sup.9 are independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl or sialic acid. R16 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. X.sup.5 is a non-reactive group and
L.sup.a is a linker group. The method comprises (a) contacting a
peptide comprising the glycosyl moiety: ##STR112## with a
PEG-sialic acid donor moiety having the formula: ##STR113## and an
enzyme that transfers PEG-sialic acid onto the Gal of said glycosyl
moiety, under conditions appropriate for said transfer.
[0263] In another exemplary embodiment, the moiety: ##STR114## has
a formula that is a member selected from: ##STR115## wherein e, f,
m and n are integers independently selected from 1 to 2500; and
[0264] q is an integer selected from 0 to 20.
[0265] In another exemplary embodiment, the moiety: ##STR116## has
a formula that is a member selected from: ##STR117## wherein e, f
and f' are integers independently selected from 1 to 2500; and q
and q' are integers independently selected from 1 to 20.
[0266] In another exemplary embodiment, the glycosyl linker
comprises the formula: ##STR118##
[0267] In another exemplary embodiment, the peptide conjugate
comprises at least one glycosyl linker having the formula:
##STR119## wherein AA is an amino acid residue of said peptide; t
is an integer selected from 0 and 1; and R.sup.15 is the modified
sialyl moiety.
[0268] In another exemplary embodiment, the peptide has the amino
acid sequence of SEQ. ID. NO:1.
[0269] In another exemplary embodiment, the glycosyl linker is
attached to said peptide through an amino acid residue which is an
asparagine residue.
[0270] In another exemplary embodiment, the asparagine residue is a
member selected from N152, N322 and combinations thereof.
[0271] In another exemplary embodiment, the peptide is a bioactive
peptide.
[0272] In another exemplary embodiment, the method comprises, prior
to step (a): (b) expressing the peptide in a suitable host.
[0273] In another aspect, the invention provides a method of
treating a condition in a subject in need thereof, said condition
characterized by compromised clotting potency in said subject, said
method comprising the step of administering to the subject an
amount of the peptide conjugate produced according to the methods
described herein, effective to ameliorate said condition in said
subject. In another exemplary embodiment, the method comprises
administering to said mammal an amount of the peptide conjugate
produced according to the methods described herein.
[0274] In another aspect, the invention provides a method of
synthesizing a peptide conjugate, said method comprising combining
a) sialidase; b) enzyme which is a member selected from
glycosyltransferase, exoglycosidase and endoglycosidase; c)
modified sugar/modified sialyl residue; d) peptide thus
synthesizing said peptide conjugate. In an exemplary embodiment,
the combining is for a time less than 10 hours. In another
exemplary embodiment, the invention further comprises a capping
step.
II.D.iv. Water-Insoluble Polymers
[0275] 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.
[0276] 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.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] 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.
[0281] 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.
[0282] 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.
[0283] 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.
[0284] 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.
[0285] 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).
[0286] 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.
[0287] 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.
[0288] 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.
[0289] 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.
[0290] 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.
[0291] 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.
[0292] 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.
[0293] 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. No. 5,410,016, which issued on Apr. 25,
1995 and U.S. Pat. No. 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).
[0294] 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.
[0295] 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.
[0296] 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.
[0297] 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.
II.D.v. Methods of Producing the Polymeric Modifying Groups
[0298] The polymeric modifying groups can be activated for reaction
with a glycosyl or saccharyl moiety or an amino acid moiety.
Exemplary structures of activated species (e.g., carbonates and
active esters) include: ##STR120##
[0299] In the figure above, q is a member selected from 1-40. 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: ##STR121## PEG
molecules that are activated with these and other species and
methods of making the activated PEGs are set forth in WO
04/083259.
[0300] 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 a-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.
[0301] The branched PEG species set forth herein are readily
prepared by methods such as that set forth in the scheme below:
##STR122## 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 5 KDa, 10 KDa, 15 KDa,
20 KDa, 25 KDa, 30 KDa, 35 KDa, or 40 KDa in molecular weight.
[0302] 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.
[0303] In other exemplary embodiments, the urea moiety is replaced
by a group such as a amide.
II. E. Homodisperse Peptide Conjugate Compositions of Matter
[0304] In addition to providing peptide conjugates that are formed
through a chemically or enzymatically added glycosyl linking group,
the present invention provides compositions of matter comprising
peptide conjugates that are highly homogenous in their substitution
patterns. Using the methods of the invention, it is possible to
form peptide conjugates in which substantial proportion of the
glycosyl linking groups and glycosyl moieties across a population
of peptide conjugates 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 an exemplary peptide conjugate of the
invention, essentially each member of the water soluble polymer
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.
[0305] The present invention also provides conjugates analogous to
those described above in which the peptide is conjugated to a
modifying group, e.g. therapeutic moiety, diagnostic moiety,
targeting moiety, toxin moiety or the like via a glycosyl linking
group. Each of the above-recited modifying groups can be a small
molecule, natural polymer (e.g., polypeptide) or synthetic polymer.
When the modifying group is attached to a sialic acid, it is
generally preferred that the modifying group is substantially
non-fluorescent.
[0306] In an exemplary embodiment, the peptides of the invention
include at least one O-linked or N-linked glycosylation site, which
is glycosylated with a modified sugar that includes a polymeric
modifying group, e.g., a PEG moiety. In an exemplary embodiment,
the PEG is covalently attached to the peptide via an intact
glycosyl linking group, or via a non-glycosyl linker, e.g.,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl. 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.
[0307] The glycans on the peptides of the invention generally
correspond to those found on a peptide that is produced by
mammalian (BHK, CHO) cells or insect (e.g., Sf-9) cells, following
remodeling according to the methods set forth herein. For example,
insect-derived peptide 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.
[0308] 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.
II. F. Nucleotide Sugars
[0309] In another aspect of the invention, the invention also
provides sugar nucleotides. Exemplary species according to this
embodiment include: ##STR123## in which the index y is an integer
selected from 0, 1 and 2. Base is a nucleic acid base, such as
adenine, thymine, guanine, cytidine and uridine. R.sup.2, R.sup.3
and R.sup.4 are as described above. In an exemplary embodiment,
L-(R.sup.1).sub.w is a member selected from ##STR124## in which the
variables are as described above.
[0310] In an exemplary embodiment, L-(R.sup.1).sub.w has a
structure according to the following formula: ##STR125## In an
exemplary embodiment, A.sup.1 and A.sup.2 are each selected from
--OH and --OCH.sub.3.
[0311] Exemplary polymeric modifying groups according to this
embodiment include: ##STR126##
[0312] In another exemplary embodiment, the nucleotide sugars have
a formula which is a member selected from: ##STR127##
[0313] An exemplary nucleotide sugar according to this embodiment
has the structure: ##STR128##
[0314] An exemplary nucleotide sugar according to this embodiment
has the structure: ##STR129##
[0315] In another exemplary embodiment, the nucleotide sugar is
based upon the following formula: ##STR130## 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.
[0316] In an exemplary embodiment, L-R.sup.1 is a member selected
from ##STR131## in which the variables are as described above.
[0317] In an exemplary embodiment, L-R.sup.1 has a structure
according to the following formula: ##STR132## In an exemplary
embodiment, A.sup.1 and A.sup.2 are each selected from --OH and
--OCH.sub.3. III. The Methods
[0318] 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.
[0319] 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. The method of synthesizing a
peptide conjugate, comprising combining a) sialidase; b) an enzyme
capable of catalyzing the transfer of a glycosyl linking group such
as a glycosyltransferase, exoglycosidase or endoglycosidase; c)
modified sugar; d) a peptide, thus synthesizing the peptide
conjugate. 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.
[0320] 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.
[0321] 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: ##STR133## 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.
[0322] 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.: ##STR134## 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 O also falls within this general motif.
[0323] 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: ##STR135##
[0324] In another exemplary embodiment, the nucleotide sugar is
based on the stereochemistry of glucose. Exemplary species
according to this embodiment have the formulae: ##STR136##
[0325] Thus, in an illustrative embodiment in which the glycosyl
moiety is sialic acid, the method of the invention utilizes
compounds having the formulae: ##STR137## 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.
[0326] 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:
##STR138## in which X.sup.4 is O or a bond.
[0327] 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.
[0328] Useful reactive functional groups pendent from a sugar
nucleus or modifying group include, but are not limited to: [0329]
(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; [0330] (b)
hydroxyl groups, which can be converted to, e.g., esters, ethers,
aldehydes, etc. [0331] (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; [0332] (d)
dienophile groups, which are capable of participating in
Diels-Alder reactions such as, for example, maleimido groups;
[0333] (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; [0334] (f) sulfonyl halide groups for subsequent reaction
with amines, for example, to form sulfonamides; [0335] (g) thiol
groups, which can be, for example, converted to disulfides or
reacted with acyl halides; [0336] (h) amine or sulfhydryl groups,
which can be, for example, acylated, alkylated or oxidized; [0337]
(i) alkenes, which can undergo, for example, cycloadditions,
acylation, Michael addition, etc; and [0338] (j) epoxides, which
can react with, for example, amines and hydroxyl compounds.
[0339] 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.
[0340] 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)).
[0341] In an exemplary embodiment, the modified sugar is based upon
a 6-amino-N-acetyl-glycosyl moiety.
[0342] 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 KDa, 15 KDa or 20 KDa 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.
[0343] The 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.
[0344] 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.
[0345] 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.
[0346] 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.
[0347] 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).
[0348] 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.
[0349] 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.
[0350] 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.
[0351] 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.
[0352] 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).
[0353] 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).
[0354] 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. No. 5,876,980, 6,030,815, 5,728,554, and
5,922,577.
[0355] 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).
[0356] 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).
[0357] 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.
[0358] 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.
[0359] 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.
[0360] 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 a-hydroxy carboxylate 2. Compound 2 is converted to the
corresponding CMP derivative by the action of CMP-SA synthetase,
followed by catalytic hydrogenation of the CMP derivative to
produce compound 3. The amine introduced via formation of the
glycine adduct is utilized as a locus of PEG 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. ##STR139## In an exemplary embodiment, a
modified sugar can be attached to an 0-glycan binding site on a
peptide. The glycosyltransferases which can be used to produce this
conjugate include: for Ser56 (-Glc-(Xyl)n-Gal-SA-PEG--a
galactosyltransferase and sialyltransferase; for Ser56
-Glc-(Xyl)n-Xyl-PEG--a xylosyltransferase; and for
Ser60-Fuc-GlcNAc-(Gal)n-(SA)m-PEG--a GIcNAc transferase. III. A.
Conjugation of Modified Sugars to Peptides
[0361] 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. A list of preferred
sialyltransferases for use in the invention is provided in FIG.
3.
[0362] A number of methods of using glycosyltransferases to
synthesize desired oligosaccharide structures are known and are
generally applicable to the instant invention.
[0363] 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.
[0364] 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.
[0365] 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.
[0366] 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.
[0367] 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.
[0368] 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.
[0369] 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.
[0370] 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.
[0371] 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.
[0372] 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.
[0373] 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.
[0374] 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.
[0375] 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.
[0376] 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.
[0377] 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.
[0378] 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.
[0379] 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.
[0380] 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.
[0381] In yet a further example, a PEG moiety is added onto a Gal
residue using a modified sialic acid such as those discussed
above.
[0382] 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.
[0383] A high mannose structure can also be trimmed back to the
elementary tri-mannosyl core.
[0384] 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.
[0385] 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 GIcNAc-(Fuc)a residue is conjugated wit 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.
[0386] 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.
[0387] 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.
[0388] 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.
[0389] 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.
[0390] 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 peptide. 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.
[0391] 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).
[0392] 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.
[0393] 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.
[0394] 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.
[0395] 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.
[0396] 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.
[0397] In a selected embodiment, a 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.
[0398] 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.
[0399] 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.
[0400] 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 a peptide with an O-linked
modified glycan. The desialylated, modified peptide is optionally
partially or fully re-sialylated by using a sialyltransferase such
as ST3GalIII.
[0401] In another aspect, the invention provides a method of making
a PEGylated peptide conjugate of the invention. The method
includes: (a) contacting a peptide comprising a glycosyl group
selected from: ##STR140## with a PEG-sialic acid donor having the
formula which is a member selected from ##STR141## and an enzyme
that transfers PEG-sialic acid from said donor onto a member
selected from the GalNAc, Gal and 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. In an exemplary embodiment, the method further
comprises expressing the peptide in a host. In an exemplary
embodiment, the host is a mammalian cell or an insect cell. In
another exemplary embodiment, the mammalian cell is a member
selected from a BHK cell and a CHO cell and the insect cell is a
Spodoptera frugiperda cell.
[0402] 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.
[0403] In an exemplary embodiment, the PEG-sialic acid donor has
the formula: ##STR142##
[0404] In another exemplary embodiment, the PEG-sialic acid donor
has the formula: ##STR143##
[0405] In a further exemplary embodiment, the 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.
[0406] In an exemplary embodiment, the invention provides a method
of making a peptide conjugate comprising a glycosyl linker
comprising a modified sialyl residue having the formula: ##STR144##
wherein D is a member selected from --OH and R.sup.1-L-HN--; G is a
member selected from R.sup.1-L- and
--C(O)(C.sub.1-C.sub.6)alkyl-R.sup.1; R.sup.1 is a moiety
comprising a member selected from a straight-chain poly(ethylene
glycol) residue and branched poly(ethylene glycol) residue; M is a
member selected from H, a metal and a single negative charge; L is
a linker which is a member selected from a bond, substituted or
unsubstituted alkyl and substituted or unsubstituted heteroalkyl,
such that 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-- said method
comprising: (a) contacting a peptide comprising the glycosyl
moiety: ##STR145## with a PEG-sialic acid donor moiety having the
formula: ##STR146## and an enzyme that transfers said PEG-sialic
acid onto the Gal of said glycosyl moiety, under conditions
appropriate for said transfer.
[0407] In an exemplary embodiment, L-R.sup.1 has the formula:
##STR147## wherein a is an integer selected from 0 to 20.
[0408] In another exemplary embodiment, R.sup.1 has a structure
that is a member selected from: ##STR148## wherein e, f, m and n
are integers independently selected from 1 to 2500; and q is an
integer selected from 0 to 20.
[0409] Large scale or small scale amounts of peptide conjugate can
be produced by the methods described herein. In an exemplary
embodiment, the amount of peptide is a member selected from about
0.5 mg to about 100 kg. In an exemplary embodiment, the amount of
peptide is a member selected from about 0.1 kg to about 1 kg. In an
exemplary embodiment, the amount of peptide is a member selected
from about 0.5 kg to about 10 kg. In an exemplary embodiment, the
amount of peptide is a member selected from about 0.5 kg to about 3
kg. In an exemplary embodiment, the amount of peptide is a member
selected from about 0.1 kg to about 5 kg. In an exemplary
embodiment, the amount of peptide is a member selected from about
0.08 kg to about 0.2 kg. In an exemplary embodiment, the amount of
peptide is a member selected from about 0.05 kg to about 0.4 kg. In
an exemplary embodiment, the amount of peptide is a member selected
from about 0.1 kg to about 0.7 kg. In an exemplary embodiment, the
amount of peptide is a member selected from about 0.3 kg to about
1.75 kg. In an exemplary embodiment, the amount of peptide is a
member selected from about 25 kg to about 65 kg.
[0410] The concentration of peptide utilized in the reactions
described herein is a member selected from about 0.5 to about 10 mg
peptide/mL reaction mixture. In an exemplary embodiment, the
peptide concentration is a member selected from about 0.5 to about
1 mg peptide/mL reaction mixture. In an exemplary embodiment, the
peptide concentration is a member selected from about 0.8 to about
3 mg peptide/mL reaction mixture. In an exemplary embodiment, the
peptide concentration is a member selected from about 2 to about 6
mg peptide/mL reaction mixture. In an exemplary embodiment, the
peptide concentration is a member selected from about 4 to about 9
mg peptide/mL reaction mixture. In an exemplary embodiment, the
peptide concentration is a member selected from about 1.2 to about
7.8 mg peptide/mL reaction mixture. In an exemplary embodiment, the
peptide concentration is a member selected from about 6 to about
9.5 mg peptide/mL reaction mixture.
[0411] The concentration of CMP-SA-PEG that can be utilized in the
reactions described herein is a member selected from about 0.1 to
about 1.0 mM. Factors which may increase or decrease the
concentration include the size of the PEG, time of incubation,
temperature, buffer components, as well as the type, and
concentration, of glycosyltransferase used. In an exemplary
embodiment, CMP-SA-PEG concentration is a member selected from
about 0.1 to about 1.0 mM. In an exemplary embodiment, CMP-SA-PEG
concentration is a member selected from about 0.1 to about 0.5 mM.
In an exemplary embodiment, CMP-SA-PEG concentration is a member
selected from about 0.1 to about 0.3 mM. In an exemplary
embodiment, CMP-SA-PEG concentration is a member selected from
about 0.2 to about 0.7 mM. In an exemplary embodiment, CMP-SA-PEG
concentration is a member selected from about 0.3 to about 0.5 mM.
In an exemplary embodiment, CMP-SA-PEG concentration is a member
selected from about 0.4 to about 1.0 mM. In an exemplary
embodiment, CMP-SA-PEG concentration is a member selected from
about 0.5 to about 0.7 mM. In an exemplary embodiment, CMP-SA-PEG
concentration is a member selected from about 0.8 to about 0.95 mM.
In an exemplary embodiment, CMP-SA-PEG concentration is a member
selected from about 0.55 to about 1.0 mM.
[0412] The molar equivalents of CMP-SA-PEG that can be utilized in
the reactions described herein are based on the theoretical number
of SA-PEGs that can be added to the protein. The theoretical number
of SA-PEGs is based on the theoretical number of sialation sites on
the protein as well as the MW of the protein when compared to the
MW and therefore moles of CMP-SA-PEG. For a protein, that is about
four or five PEGs based on N-glycans that are primarily bi- and
tri-antennary with only two glycan sites. In an exemplary
embodiment, the molar equivalents of CMP-SA-PEG is an integer
selected from 1 to 20. In an exemplary embodiment, the molar
equivalents of CMP-SA-PEG is an integer selected from 1 to 20. In
an exemplary embodiment, the molar equivalents of CMP-SA-PEG is an
integer selected from 2 to 6. In an exemplary embodiment, the molar
equivalents of CMP-SA-PEG is an integer selected from 3 to 17. In
an exemplary embodiment, the molar equivalents of CMP-SA-PEG is an
integer selected from 4 to 11. In an exemplary embodiment, the
molar equivalents of CMP-SA-PEG is an integer selected from 5 to
20. In an exemplary embodiment, the molar equivalents of CMP-SA-PEG
is an integer selected from 1 to 10. In an exemplary embodiment,
the molar equivalents of CMP-SA-PEG is an integer selected from 12
to 20. In an exemplary embodiment, the molar equivalents of
CMP-SA-PEG is an integer selected from 14 to 17. In an exemplary
embodiment, the molar equivalents of CMP-SA-PEG is an integer
selected from 7 to 15. In an exemplary embodiment, the molar
equivalents of CMP-SA-PEG is an integer selected from 8 to 16.
III. B. Simultaneous Desialylation and GlycoPEGylation of a
Peptide
[0413] The present invention provides a "one-pot" method of
glycopegylating a peptide. The one-pot method is distinct from
other exemplary processes to make a peptide conjugate, which employ
a sequential de-sialylation with sialidase, subsequent purification
of the asialo peptide on an anion exchange column, then
glycoPEGylation using CMP-sialic acid-PEG and a glycosyltransferase
(such as ST3Gal3), exoglycosidase or an endoglycosidase. The
peptide conjugate is then purified via anion exchange followed by
size exclusion chromatography to produce the purified peptide
conjugate.
[0414] The one-pot method is an improved method to manufacture a
peptide conjugate. In this method, the de-sialylation and
glycoPEGylation reactions are combined in a one-pot reaction which
obviates the first anion exchange chromatography step used in the
previously described process to purify the asialo peptide. This
reduction in process steps produces several advantages. First, the
number of process steps required to produce the peptide conjugate
is reduced, which also reduces the operating complexity of the
process. Second, the process time for the production of the peptide
conjugates is reduced e.g., from 4 to 2 days. This reduces the raw
material requirements and quality control costs associated with
in-process controls. Third, the invention utilizes less sialidase,
e.g., up to 20-fold less sialidase, e.g., 500 mU/L is required to
produce the peptide conjugate relative to the process. This
reduction in the use of sialidase significantly reduces the amount
of contaminants, such as sialidase, in the reaction mixture.
[0415] In an exemplary embodiment, a peptide conjugate is prepared
by the following method. In a first step, a peptide is combined
with a sialidase, a modified sugar of the invention, and an enzyme
capable of catalyzing the transfer of the glycosyl linking group
from the modified sugar to the peptide, thus preparing the peptide
conjugate. Any sialidase may be used in this method. Exemplary
sialidases of use in the invention can be found in the CAZY
database (see http://afmb.cnrs-mrs.fr/CAZY/index.html and
www.cazv.org/CAZY). Exemplary sialidases can be purchased from any
number of sources (QA-Bio, Calbiochem, Marukin, Prozyme, etc.). In
an exemplary embodiment, the sialidase is a member selected from
cytoplasmic sialidases, lysosomal sialidases, exo-a sialidases, and
endosialidases. In another exemplary embodiment, the sialidase used
is produced from bacteria such as Clostridium perfringens or
Streptococcus pneumoniae, or from a virus such as an adenovirus. In
an exemplary embodiment, the enzyme capable of catalyzing the
transfer of the glycosyl linking group from the modified sugar to
the peptide is a member selected from a glycosyltransferase, such
as sialyltransferases and fucosyltransferases, as well as
exoglycosidases and endoglycosidases. In an exemplary embodiment,
the enzyme is a glycosyltransferase, which is ST3Gal3. In another
exemplary embodiment, the enzyme used is produced from bacteria
such as Escherichia Coli or a fungus such as Aspergillus niger. In
another exemplary embodiment, the sialidase is added to the peptide
before the glycosyltransferase for a specified time, allowing the
sialidase reaction to proceed before initiating the GlycoPEGylation
reaction with addition of the PEG-sialic acid reagent and the
glycosyltransferase. Many of these examples are discussed herein.
Finally, any modified sugar described herein can be utilized in
this reaction.
[0416] In another exemplary embodiment, the method further
comprises a `capping` step. In this step, additional non-PEGylated
sialic acid is added to the reaction mixture. In an exemplary
embodiment, this sialic acid is added to the peptide or peptide
conjugate thus preventing further addition of PEG-sialic acid. In
another exemplary embodiment, this sialic acid impedes the function
of the glycosyltransferase in the reaction mixture, effectively
stopping the addition of glycosyl linking groups to the peptides or
peptide conjugates. Most importantly, the sialic acid that is added
to the reaction mixture caps the unglycoPEGylated glycans thereby
providing a peptide conjugate that has improved pharmaceokinetics.
In addition, this sialidase can be added directly the the
glycoPEGylation reaction mixture when the extent of PEGylation to
certain amounts is desired without prior purification.
[0417] In an exemplary embodiment, after the capping step, less
than about 50% of the sialylation sites on the peptide or peptide
conjugate does not comprise a sialyl moiety. In an exemplary
embodiment, after the capping step, less than about 40% of the
sialylation sites on the peptide or peptide conjugate does not
comprise a sialyl moiety. In an exemplary embodiment, after the
capping step, less than about 30% of the sialylation sites on the
peptide or peptide conjugate do not comprise a sialyl moiety. In an
exemplary embodiment, after the capping step, less than about 20%
of the sialylation sites on the peptide or peptide conjugate do not
comprise a sialyl moiety. In an exemplary embodiment, after the
capping step, less than about 10% of the sialylation sites on the
peptide or peptide conjugate do not comprise a sialyl moiety. In an
exemplary embodiment, between about 20% and about 5% of the
sialylation sites on the peptide or peptide conjugate does not
comprise a sialyl moiety. In an exemplary embodiment, between about
25% and about 10% of the sialylation sites on the peptide or
peptide conjugate does not comprise a sialyl moiety. In an
exemplary embodiment, after the capping step, essentially all of
the sialylation sites on the peptide or peptide conjugate comprise
a sialyl moiety.
III.C. Desialylation and Selective Modification of Peptides
[0418] In another exemplary embodiment, the present invention
provides a method for desialylating a peptide. The method
preferably provides a peptide that is at least about 40%,
preferably 45%, preferably about 50%, preferably about 55%,
preferably about 60%, preferably about 65%, preferably about 70%,
preferably about 75%, preferably about 80%, preferably at least
85%, more preferably at least 90%, still more preferably, at least
92%, preferably at least 94%, even more preferably at least 96%,
still more preferably at least 98%, and still more preferably 100%
disialylated.
[0419] The method includes contacting the peptide with a sialidase,
preferably for a time period. The preselected time period is
sufficient to desialylate the peptide to the degree desired. In a
preferred embodiment, the desialylated peptide is separated from
the sialidase when the desired degree of desialylation is achieved.
An exemplary desialylation reaction and purification cycle is set
forth herein.
[0420] Those of skill are able to determine an appropriate
preselected time period over which to conduct the desialylation
reaction. In an exemplary embodiment, the period is less than 24
hours, preferably less than 8 hours, more preferably less than 6
hours, more preferably less than 4 hours, still more preferably
less than 2 hours and even more preferably less than 1 hour.
[0421] In another exemplary embodiment, in the preparation at the
end of the desialylation reaction, at least 10% of the members of
the population of peptides has only a single sialic acid attached
thereto, preferably at least 20%, more preferably at least 30%,
still more preferably at least 40%, even still more preferably at
least 50% and more preferably at least 60%, and still more
preferably completely desialylated.
[0422] In yet a further exemplary embodiment, in the preparation at
the end of the desialylation reaction, at least 10% of the members
of the population of peptides is fully desialylated, preferably at
least 20%, more preferably at least 30%, even more preferably at
least 40%, still more preferably at least 50% and even still more
preferably at least 60%.
[0423] In still another exemplary embodiment, in the preparation at
the end of the desialylation reaction, at least 10%, 20%, 30%, 40%,
50% or 60% of the members of the peptide population has only a
single sialic acid, and at least 10%, 20%, 30%, 40%, 50% or 60% of
the peptide is fully disialylated.
[0424] In a preferred embodiment, in the preparation at the end of
the desialylation reaction, at least 50% of the population of
peptides is fully disialylated and at least 40% of the members of
the peptide population bears only a single sialic acid moiety.
[0425] Following desialylation, the peptide is optionally
conjugated with a sugar or modified sugar. An exemplary modified
sugar includes a saccharyl moiety bound to a branched or linear
poly(ethylene glycol) moiety. The conjugation is catalyzed by an
enzyme that transfers the modified sugar from a modified sugar
donor onto an amino acid or glycosyl residue of the peptide. An
exemplary modified sugar donor is a CMP-sialic acid that bears a
branched or linear poly(ethylene glycol) moiety. An exemplary
poly(ethylene glycol) moiety has a molecular weight of at least
about 2 KDa, more preferably at least about 5 KDa, more preferably
at least about 10 KDa, preferably at least about 20 KDa, more
preferably at least about 30 KDa, and more preferably at least
about 40 KDa.
[0426] In an exemplary embodiment, the enzyme utilized to transfer
the modified sugar moiety from the modified sugar donor is a
glycosyltransferase, e.g., sialyltransferase. An exemplary
sialyltransferase of use in the methods of the invention is
ST3Gal3.
[0427] An exemplary method of the invention results in a modified
peptide bearing at least one, preferably at least two, preferably
at least three modifying groups. In one embodiment, the peptide
produced bears a single modifying group on the light chain of the
peptide. In another embodiment, the method provides a modified
peptide that bears a single modifying group on the heavy chain. In
still another embodiment, the method provides a modified peptide
with a single modifying group on the light chain and a single
modifying group on the heavy chain.
[0428] In another aspect, the invention provides a method of
preparing a modified peptide. The method includes contacting the
peptide with a modified sugar donor bearing a modifying group and
an enzyme capable of transferring a modified sugar moiety from the
modified sugar donor onto an amino acid or glycosyl residue of the
peptide.
[0429] In an exemplary embodiment, the method provides a population
of modified peptides in which at least 40%, preferably at least
50%, preferably at least 60%, more preferably at least 70% and even
more preferably at least 80% of the population members are
mono-conjugated on the light chain of the peptide.
[0430] In an exemplary embodiment, the method provides a population
of modified peptides in which at least 40%, preferably at least
50%, preferably at least 60%, more preferably at least 70% and even
more preferably at least 80% of the population members are
di-conjugated on the light chain of the peptide.
[0431] In an exemplary embodiment of this aspect, the method
provides a population of modified peptides in which no more than
50%, preferably no more than 30%, preferably no more than 20%, more
preferably no more than 10% of the population members are
mono-conjugated on the heavy chain of the peptide.
[0432] In an exemplary embodiment of this aspect, the method
provides a population of modified peptides in which no more than
50%, preferably no more than 30%, preferably no more than 20%, more
preferably no more than 10% of the population members are
di-conjugated on the heavy chain of the peptide.
[0433] The peptide can be subjected to the action of a sialidase
prior to the contacting step, or the peptide can be used without
prior desialylation. When the peptide is contacted with a sialidase
it can be either essentially completely desialylated or only
partially desialylated. In a preferred embodiment, the peptide is
at least partially desialylated prior to the contacting step. The
peptide may be essentially completely desialylated (essentially
asialo) or only partially desialylated. In a preferred embodiment,
the desialylated peptide is one of the desialylated embodiments
described hereinabove.
[0434] In yet another aspect, the invention provides a method
altering glycosylation of a peptide. The method includes, (a)
contacting the peptide with a sialidase, thereby removing at least
one sialic acid moiety from the peptide. The peptide can be
partially or wholly desialylated as discussed above. In step (b),
the product from step (a) with at least one glycosyltransferase and
at least one sugar donor. The at least one sugar donor is a
substrate for the at least one glycosyltransferase. Preferably,
step (b) results in transfer of a sugar residue from the donor onto
an amino acid or glycosyl residue of the peptide. The sugar residue
can be a modified or unmodifed sugar residue. The conditions of
step (b) are maintained until a desired level of glycosylation of
the peptide is achieved, as discussed above. Following step (b),
the sialidase content of the reaction mixture is reduced and
preferably, essentially all of the sialidase is removed. In an
exemplary embodiment, the removal of sialidase is effected by a
chromatography (e.g., ion exchange, e.g., anion exchange) or
filtration step (e.g., nanofiltration). When the sialidase is
removed, the resulting glycosylated peptide (i.e., produced in step
(a)) is optionally contacted with a further at least one sugar
donor and at least one glycosyltransferase, transferring at least
one sugar residue onto an amino acid or glycosyl moiety of the
peptide. In a preferred embodiment, the sugar residue is sialic
acid. In a still further preferred embodiment, the sugar donor is
CMP-sialic acid. The glycosyltransferase is typically a
sialyltransferase.
[0435] An exemplary sialidase is that of Arthrobacter ureafaciens.
Other sialidases of use in the present invention are known in the
art. (See, e.g.,
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db-Protein&cmd=search-
&term=sialidase).
[0436] Exemplary peptides with which the above method can be
practiced are those set forth in the figures appended hereto, e.g,
EPO, Blood Factors (FVII, FVIII, FIX, FXIII, etc.), interferon
1-.beta., G-CSF, hGH, GLP-1, and BMP-7.
III. D. Additional Aliquots of Reagents Added in the Synthesis of
Peptide Conjugates
[0437] In an exemplary embodiment of the synthesis of the peptide
conjugates described herein, one or more additional aliquots of a
reaction component/reagent is added to the reaction mixture after a
selected period of time. In another exemplary embodiment, the
reaction component/reagent added is a modified sugar nucleotide.
Introduction of a modified sugar nucleotide into the reaction will
increase the likelihood of driving the GlycoPEGylation reaction to
completion. In an exemplary embodiment, the nucleotide sugar is a
CMP-SA-PEG described herein. In an exemplary embodiment, the
reaction component/reagent added is a sialidase. In an exemplary
embodiment, the reaction component/reagent added is a
glycosyltransferase. In an exemplary embodiment, the reaction
component/reagent added is magnesium. In an exemplary embodiment,
the additional aliquot added represents about 10%, or 20%, or 30%,
or 40%, or 50%, or 60%, or 70%, or 80% or 90% of the original
amount in added at the start of the reaction. In an exemplary
embodiment, the reaction component/reagent is added to the reaction
about 3 hours, or 6 hours, or 8 hours, or 10 hours, or 12 hours, or
18 hours, or 24 hours, or 30 hours, or 36 hours after its
start.
III.E. Selective Production of light chain PEGylated Peptide
Coniukates
[0438] In an exemplary embodiment, the invention provides a method
of increasing the production of peptide conjugates which are
modified on the light chain over the heavy chain. This method
involves the inactivation or sequestering of the heavy chain, thus
allowing GlycoPEGylation to preferentially occur on the light
chain. The serine protease activity of the heavy chain of the
peptide can be exploited as the basis for this sequestration.
Adding a benzamidine matrix and/or pseudoaffinity resin for serine
proteases to a GlycoPEGylation reaction mixture results in
sequestration of the heavy chain, while GlycoPEGylation proceeds on
the light chain. The light chain can then be purified away from the
heavy chain by standard techniques known in the art. The heavy
chain can be removed from the matrix by the addition of benzamidine
or removed from the resin by lowering the pH of the solution.
Benzamidine impurities introduced in this step can be removed by
diafiltration.
III.E. Purification of Peptide Conjugates
[0439] 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 peptides 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. In certain instances, the
molecular weight cutoff differences between the impurity and the
product will be utilized in order to ensure product purification.
For example, in order to purify product Peptide-SA-PEG-40 KDa from
unreacted CMP-SA-PEG-40 KDa, a filter must be chosen that will
allow, for example, Peptide-SA-PEG-40 KDa to remain in the
retentate while allowing CMP-SA-PEG-40 KDa to flow into the
filtrate. Nanofiltration or reverse osmosis can then be used to
remove salts and/or purify the product saccharides (see, e.g., WO
98/15581). Nanofilter membranes are a class of reverse osmosis
membranes that pass monovalent salts but retain polyvalent salts
and uncharged solutes larger than about 100 to about 2,000 Daltons,
depending upon the membrane used. Thus, in a typical application,
saccharides prepared by the methods of the present invention will
be retained in the membrane and contaminating salts will pass
through.
[0440] 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. Purification can be
used to separate one chain of the peptide conjugate from the other,
as further described later in this section.
[0441] 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.
[0442] A protease inhibitor 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. The
protease inhibitors used in the foregoing steps may be low
molecular weight inhibitors, including antipain,
alpha-1-antitrypsin, anti-thrombin, leupeptin, amastatin,
chymostatin, banzamidin, as well as other serine protease
inhibitors (i.e. serpins). Generally, serine protease inhibitors
should be used in concentrations ranging from 0.5-100 .mu.M,
although chymostatin in cell culture may be used in concentrations
upward of 200 .mu.M. Other serine protease inhibitors will include
inhibitors specific to the chymotyypsin-like, the subtilisin-like,
the alphalbeta hydrolase, or the signal peptidase clans of serine
proteases. Besides serine proteases, other types of protease
inhibitors may also be used, including cysteine protease inhibitors
(1-10 .mu.M) and aspartic protease inhibitors (1-5 .mu.M), as well
as non-specific protease inhibitors such as pepstatin (0.1-5
.mu.M). Protease inhibitors used in this invention may also include
natural protease inhibitors, such as the hirustasin inhibitor
isolated from leech. In some embodiments, protease inhibitors will
comprise synthetic peptides or antibodies that are able to bind
with specificity to the protease catalytic site to stabilize the
peptide without interfering with a glycoPEGylation reaction.
[0443] 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.
[0444] 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-P1, filed
May 6, 2005).
[0445] 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.
[0446] 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.
[0447] In an exemplary embodiment, the purification is accomplished
by the methods set forth in commonly owned, co-assigned U.S.
Provisional Patent No. 60/665,588, filed Mar. 24, 2005.
[0448] According to the present invention, pegylated peptides are
produced either via sequential de-sialylation or simultaneous
sialylation can be purified or resolved by using magnesium chloride
gradient.
[0449] In an exemplary embodiment, the peptide conjugates can be
separated into a light chain and a heavy chain, and one chain can
be purified away from the other. In another exemplary embodiment, a
product is obtained in which at least 80% of the peptide conjugate
in the product is the light chain portion of the peptide conjugate.
In another exemplary embodiment, a product is obtained in which at
least 90% of the peptide conjugate in the product is the light
chain portion of the peptide conjugate. In another exemplary
embodiment, a product is obtained in which at least 95% of the
peptide conjugate in the product is the light chain portion of the
peptide conjugate. In another exemplary embodiment, a product is
obtained in which essentially all of the peptide conjugate in the
product is the light chain portion of the peptide conjugate. This
product is possible for any compound of the invention.
[0450] In another exemplary embodiment, a product is obtained in
which at least 80% of the peptide conjugate in the product is the
heavy chain portion of the peptide conjugate. In another exemplary
embodiment, a product is obtained in which at least 90% of the
peptide conjugate in the product is the heavy chain portion of the
peptide conjugate. In another exemplary embodiment, a product is
obtained in which at least 95% of the peptide conjugate in the
product is the heavy chain portion of the peptide conjugate. In
another exemplary embodiment, a product is obtained in which
essentially all of the peptide conjugate in the product is the
heavy chain portion of the peptide conjugate. This product is
possible for any compound of the invention.
III.F. Properties of Peptide Conjugates
[0451] In an exemplary embodiment, the peptide conjugates of the
invention possess essentially the same biochemical properties as a
native peptide. In an exemplary embodiment, the peptide conjugates
of the invention possess reduced, or enhanced biochemical
properties over a native peptide depending on the site of
PEGylation, the size of the PEG added and the number of PEGs
added.
IV. Pharmaceutical Compositions
[0452] 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.
[0453] 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).
[0454] In an exemplary embodiment, the pharmaceutical formulation
comprises a peptide conjugate and a pharmaceutically acceptable
diluent which is a member selected from sodium chloride, calcium
chloride dihydrate, glycylglycine, polysorbate 80, and mannitol. In
another exemplary embodiment, the pharmaceutically acceptable
diluent is sodium chloride and glycylglycine. In another exemplary
embodiment, the pharmaceutically acceptable diluent is calcium
chloride dihydrate and polysorbate 80. In another exemplary
embodiment, the pharmaceutically acceptable diluent is
mannitol.
[0455] 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.
[0456] 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.
[0457] 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.
[0458] 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).
[0459] 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.
[0460] 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.
[0461] 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.
[0462] The active ingredient used in the pharmaceutical
compositions of the present invention is peptide conjugates having
the biological properties of stimulating blood clot production.
Preferably, the peptide conjugate are administered parenterally.
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 (.about.7 U) to 100
(.about.7000 U) .mu.g/kg body weight of the active material.
Preferable doses for treatment of anemic conditions are about 50 to
about 300 Units/kg three times a week. Because the present
invention provides a composition of matter comprising a peptide
with an enhanced in vivo residence time, the stated dosages are
optionally lowered when a composition of the invention is
administered.
[0463] 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.
EXAMPLES
Example 1
Purification of PEGylated Factor VIIa.
[0464] GlycoPEGylated samples of Factor VIIa were purified with a
modified anion-exchange method. Samples were handled at 5.degree.
C. Immediately before loading the column, 1 g Chelex 100 (BioRad)
per 10 mL Factor VIIa solution was added to the remodeled sample.
After stirring for 10 min, the suspension was filtered on a
cellulose acetate membrane (0.2 .mu.m) with a vacuum system. The
retained chelator resin on the filter was washed once with 1-2 mL
water per 10 mL bulk. The conductivity of the filtrate was adjusted
to 10 mS/cm at 5.degree. C., and adjusted to pH 8.6, if
necessary.
[0465] Anion exchange was performed at 8-10.degree. C. A column
containing Q Sepharose FF was prepared before loading by washing
with 1 M NaOH (10 column volumes), water (5 column volumes), 2 M
NaCl, 50 mM HOAc, pH 3 (10 column volumes), and equilibrating with
175 mM NaCl, 10 mM glycylglycine, pH 8.6 (10 column volumes). For
each PEGylation reaction, 15-20 mg Factor VIIa was loaded on to an
XK16 column (Amersham Biosciences) with 10 mL Q Sepharose FF (no
more than 2 mg protein per mL resin) at a flow rate of 100 cm/h.
For the 2 KDa linear PEG, 20 mg Factor VIIa was loaded on to an
XK26 column (Amersham Biosciences) with 40 mL Q Sepharose FF (0.5
mg protein per mg resin) at a flow rate of 100 cm/h.
[0466] After loading, the column was washed with 175 mM NaCl, 10 mM
glycylglycine, pH 8.6 10 column volumes) and 50 mM NaCl, 10 mM
glycylglycine, pH 8.6 (2 column volumes). Elution was performed
with a step gradient of 15 mM CaCl.sub.2 by using 50 mM NaCl, 10 mM
glycylglycine, 15 mM CaCI.sub.2, pH 8.6 (5 column volumes). The
column was then washed with 1 M NaCl, 10 mM glycylglycine, pH 8.6
(5 column volumes). The effluent was monitored by absorbance at 280
run. Fractions (5 mL) were collected during the flow-through and
the two washes; 2.5 mL fractions were collected during the
CaCl.sub.2 and 1M salt elutions. Fractions containing Factor VIIa
were analyzed by non-reducing SDS-PAGE (Tris-glycine gels and/or
NUPAGE gels) and a Colloidal Blue Staining Kit. The appropriate
fractions with Factor VIIa were pooled, and the pH was adjusted to
7.2 with 4 M HCl.
[0467] Factor VIIa-SA-PEG-10 KDa was purified as described above,
except for the following changes. EDTA (10 mM) was added to to the
PEGylated Factor VIIa solution, the pH was adjusted to pH 6, and
the conductivity was adjusted to 5 mS/cm, at 5.degree. C. About 20
mg of Factor VIIa-SA-PEG-10 KDa was loaded on to an XK16 column
(Amersham Biosciences) with 10 mL Poros 50 Micron HQ resin (no more
than 2 mg protein per mL, resin) at a flow rate of 100 cm/h. After
loading, the column was washed with 175 mM NaCl, 10 mM histidine pH
6 (10 column volumes) and 50 mM NaCl, 10 mM histidine, pH 6 (2
column volumes). Elution was performed with a step gradient of 20
mM CaCl.sub.2 in 50 mM NaCl, 10 mM histidine, pH 6 (5 column
volumes). The column was then washed with 1 M NaCl, 10 mM
histidine, pH 6 (5 column volumes).
[0468] The anion-exchange eluate containing Factor VIIa-SA-PEG-10
KDa (25mL) was concentrated to 5-7 mL by using an Amicon Ultra-15
10K centrifugal filter device, according to the manufacturer's
directions (Millipore). Following concentration, size exclusion
chromatography was performed. The sample (5-7 mL) was loaded onto a
column containing Superdex 200 (HiLoad 16/60, prep grade; Amersham
Biosciences) equilibrated in 50 mM NaCl, 10 mM glycylglycine, 15 mM
CaCl.sub.2, pH 7.2 for most of the PEGylated variants. Factor
VIIa-SA-PEG-10 KDa was separated from the unmodified, asialo-Factor
VIIa at a flow rate of 1 mL/min, and the absorbance was monitored
at 280 nm. Fractions (1 mL) containing Factor VIIa were collected
and analyzed by non-reducing SDS-PAGE (Tris-glycine gels and/or
NuPAGE gels) and a Colloidal Blue Staining Kit. Fractions
containing the targeted PEGylated isoform and devoid of the
unmodified, asialo-Factor VIIa were pooled and concentrated to 1
mg/mL using an Amicon Ultra-15 10K centrifugal filter device.
Protein concentration was determined from absorbance readings at
280 nm using an extinction coefficient of 1.37
(mg/mL).sup.-1cm.sup.-1.
Example 2
Determination of PEGylated Isoforms by Reversed Phase HPLC
Analysis.
[0469] PEGylated Factor VIIa was analyzed by HPLC on a
reversed-phase column (Zorbax 300SB-C3, 5 .mu.m particle size,
2.1.times.150 mm). The eluants were A) 0.1 TFA in water and B)
0.09% TFA in acetonitrile. Detection was at 214 nm. The gradient,
flow rate, and column temperature depended on the PEG length (40
KDa, 20 KDa, and 10 KDa PEG: 35-65% B in 30 min, 0.5 mL/min,
45.degree. C.; 10 KDa PEG: 35-60% B in 30 min, 0.5 mL/min,
45.degree. C.; 5 KDa: 40-50%B in 40 min, 0.5 mL/min, 45.degree. C.;
2 KDa: 38-43% B in 67 min, 0.6 mL/min, 55.degree. C.). The identity
of each peak was assigned based on two or more of four different
pieces of evidence: the known retention time of native Factor VIIa,
the SDS-PAGE migration of the isolated peak, the MALDI-TOF mass
spectrum of the isolated peak, and the orderly progression of the
retention time of each peak with increasing number of attached
PEG.
Example 3
Determination of Site of PEG Attachment by Reversed-phase HPLC.
[0470] Factor VIIa and PEGylated Factor VIIa variants were reduced
by mixing sample (10 .mu.L at a concentration of 1 mg/mL) with
reducing buffer (40 .mu.L, 50 mM NaCl, 10 mM glycylglycine, 15 mM
EDTA, 8 M urea, 20 mM DTT, pH 8.6) for 15 min at room temperature.
Water (50 .mu.L) was added and the sample cooled to 4.degree. C.
until injected on the HPLC (<12 hrs). The HPLC column, eluants,
and detection were as described above for non-reduced samples. The
flow rate was 0.5 mL/min and the gradient was 30-55% B in 90 min,
followed by a brief wash cycle up to 90% B. The identity of each
peak was assigned as described in Example 2.
Example 4
Factor VIIa Clotting Assay.
[0471] PEGylated samples and standards were tested in duplicate,
and were diluted in 100 mM NaCl, 5 mM CaCl.sub.2. 0.1% BSA
(wt/vol), 50 mM Tris, pH 7.4. The standard and samples were assayed
over a range from 0.1 to 10 ng/mL. Equal volumes of diluted
standards and samples were mixed with Factor VIIa deficient plasma
(Diagnostica Stago), and stored on ice for no greater than 4 hours
before they were assayed.
[0472] Clotting times were measured with a STart4 coagulometer
(Diagnostica Stago). The coagulometer measured the time elapsed
until an in vitro clot was formed, as indicated by the stopping of
the gentle back-and-forth movement of a magnetic ball in a sample
cuvette.
[0473] Into each cuvette, one magnetic ball was deposited, plus 100
.mu.L Factor VIIa sample/deficient plasma and 100 .mu.L of a
diluted rat brain cephalin solution (stored on ice for no greater
than 4 hours). Each reagent was added with 5 seconds between each
well, and the final mixture was incubated for 300 seconds at
37.degree. C. Diluted rat brain cephalin (RBC) solution was made
from 2 mL RBC stock solution (1 vial RBC stock, from Haemachem,
plus 10 mL 150 mM NaCl) and 4 mL 100 mM NaCl, 5 mM CaCl.sub.2, 0.1%
BSA (wt/vol), 50 mM Tris, pH 7.4.
[0474] At 300 seconds, the assay was started by the addition of 100
.mu.L of a pre-heated (37.degree. C.) solution of soluble tissue
factor (2 .mu.g/mL; amino acids 1-209) in 100 mM NaCl. 12.5 mM
CaCl.sub.2, 0.1% BSA (wt/vol), 50 mM Tris, pH 7.4. Again, this next
solution was added with a 5 second interval between samples.
[0475] The clotting times from the diluted standards were used to
generate a standard curve (log clot time versus log Factor VIIa
concentration). The resulting linear regression from the curve was
used to determine the relative clotting activities of PEGylated
variants. PEGylated Factor VIIa variants were compared against an
aliquotted stock of Factor VIIa.
Example 5
Factor VIIa-SA-PEG-10 KDa: One Pot Method
[0476] Factor VIIa (5 mg diluted in the product formulation buffer
to a final concentration of 1 mg/mL), CMP-SA-PEG-10 KDa (10 mM, 60
.mu.L) and A. niger enzyme ST3Gal3 (33 U/L) and 10 mM histidine, 50
mM NaCl, 20 mM CaCl.sub.2 were combined in a reaction vessel along
with either 10 U/L, 1 U/L, 0.5 U/L or 0.1 U/L of sialidase
(CalBiochem). The ingredients were mixed and incubated at
32.degree. C. Reaction progress was measured by analyzing aliquots
at 30 minute intervals for the first four hours. An aliquot was
then removed at the 20 hour timepoint and subjected to SDS-PAGE.
Extent of PEGylation was determined by removing 1 mL at 1.5, 2.5
and 3.5 hour timepoint and purifying the sample on a Poros 50HQ
column.
[0477] For the reaction conditions containing 10 U/L of sialidase,
no appreciable amount of Factor VIIa-SA-PEG product was formed. For
the reaction conditions containing 1 U/L of sialidase, about 17.6%
of the Factor VIIa in the reaction mixture was either mono or
diPEGylated after 1.5 hours. This increased to 29% after 2.5 hours,
and 40.3% after 3.5 hours. For the reaction conditions containing
0.5 U/L of sialidase, about 44.5% of the Factor VIIa in the
reaction mixture was either mono or diPEGylated after 3 hours, and
0.8% was triPEGylated or greater. After 20 hours, 69.4% was either
mono or diPEGylated, and 18.3% was triPEGylated or greater.
[0478] For the reaction conditions containing 0.1 U/L of sialidase,
about 29.6% of the Factor VIIa in the reaction mixture was either
mono or diPEGylated after 3 hours. After 20 hours, 71.3% was either
mono or diPEGylated, and 15.1% was triPEGylated or greater.
[0479] Results are shown in FIG. 11 and FIG. 12.
Example 6
[0480] Preparation of Cysteine-PEG.sub.2 (2) ##STR149## a.
Synthesis of Compound 1
[0481] Potassium hydroxide (84.2 mg, 1.5 mmol, as a powder) was
added to a solution of L-cysteine (93.7mg, 0.75 mmol) in anhydrous
methanol (20 L) 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).
[0482] 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.
b. Synthesis of Cysteine-PEG.sub.2 (2)
[0483] Triethylamine (.about.0.5 mL) was added dropwise to a
solution of compound 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 hour
at room temperature. The reaction mixture was stirred at room
temperature for 24 hours. 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 hours 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).
[0484] 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 7
Factor VIIa-SA-PEG-40 KDa
[0485] GlycoPEGylation of Factor VIIa (One Pot with Capping).
GlycoPEGylation of Factor VIIa was accomplished in a one-pot
reaction where desialation and PEGylation occur simultaneously,
followed by capping with sialic acid. The reaction was performed in
a jacketed glass vessel controlled at 32.degree. C. by a
recirculating waterbath. First, the concentrated 0.2 .mu.m-filtered
Factor VIIa was introduced into the vessel and heated to 32.degree.
C. by mixing with a stir bar for 20 minutes. A solution of
sialidase was made from dry powder in 10 mM histidine/50 mM NaCl/20
mM CaCl.sub.2, pH 6.0 at a concentration of 4,000 U/L. Once the
Factor VIIa reached 32.degree. C., the sialidase was added to the
Factor VIIa, and the reaction was mixed for approximately 5 minutes
to ensure a uniform solution after time which the mixing was
stopped. The desialation was allowed to proceed for 1.0 h at
32.degree. C. During the desialation reaction, the CMP-SA-PEG-40
KDa was dissolved into 1 0 mM histidine/50 mM NaCl/20 mM
CaCl.sub.2, pH 6.0 buffer, and the concentration of was determined
by UV absorbance at 271 nm. After the CMP-SA-PEG-40 KDa was
dissolved, the CMP-SA-PEG-40 KDa was added to the reaction, as well
as the ST3Gal3, and the reaction was mixed for approximately 15
minutes with a stir bar to ensure a uniform solution. An additional
volume of 85 mL of buffer was added to make the reaction 1.0 L. The
reaction was allowed to proceed without stirring for 24 hours
before CMP-SA was added to a concentration of 4.3 mM to quench the
reaction and cap the remaining terminal galactose residues with
sialic acid. The quenching was allowed to proceed with mixing for
30 minutes at 32.degree. C. The total volume of the reaction was
1.0 L before quenching. Timepoint samples (1 mL) were taken at 0,
4.5, 7.5, and 24 h, quenched with CMP-SA, and analyzed by RP-HPLC
and SDS-PAGE.
[0486] Purification of Factor VIIa-SA-PEG-40 KDa. After capping,
the solution was diluted with 2.0 L of 10 mM histidine, pH 6.0 that
had been stored overnight at 4.degree. C. and the sample was
filtered through a 0.2 .mu.m Millipak 60 filter. The resulting load
volume was 3.1 L. The AEX2 chromatography was performed at
20-25.degree. C. (ambient room temperature) on an Akta Pilot
system. After loading, a 10 column volumes wash with equilibration
buffer was performed, and the product was eluted from the column
using a 10 column volume gradient of MgCl.sub.2 which resulted in
resolution of PEGylated-Factor VIIa species from unPEGylated Factor
VIIa. The loading for this column was intentionally kept low,
targeting <2 mg Factor VIIa/mL resin. SDS-PAGE gels were run in
addition to RP-HPLC analysis of selected fractions and pools of
fractions in order to make the pool of bulk product. Pooled
fractions were pH adjusted to 6.0 with 1M NaOH and stored in the
cold room at 2-8.degree. C. overnight.
[0487] Final Concentration/Diafiltration, aseptic filtration and
aliquoting. The pooled fractions were filtered through a Millipak
20 0.2 .mu.m filter and stored overnight at 2-8.degree. C. To
perform the concentration/diafiltration, a Millipore 0.1 m.sup.2 30
KDa regenerated cellulose membrane was used in a system fitted with
a peristaltic pump and silicone tubing. The system was assembled
and flushed with water, then sanitized with 0.1M NaOH for at least
1 hour, and then stored in 0.1M NaOH until equilibration with 10 mM
histidine/5 mM CaCl.sub.2/100 mM NaCl pH 6.0 diafiltration buffer
immediately before use. The product was concentrated to
approximately 400 mL and then diafiltered at constant volume with
approximately 5 diavolumes of buffer. The product was then
concentrated to approximately 300 mL and recovered after a low
pressure recirculation for 5 minutes, and the membranes were rinsed
with 200 mL of diafiltration buffer by a recirculation for 5
minutes. The wash was recovered with product, and another 50 mL of
buffer was recirculated for another 5 minutes for a final wash. The
resulting bulk was approximately 510 mL, and that was filtered
through a 1 L vacuum filter fitted with a 0.2.mu.m PES membrane
(Millipore). The aseptically-filtered bulk was then aliquoted into
25 mL aliquots in 50 mL sterile falcon tubes and frozen at
-80.degree. C.
[0488] Analysis of the PEGylation Reaction by HPLC (Example 7)
TABLE-US-00001 Purification Conjugation Reaction Time After 0 hrs
4.5 hrs 7.5 hrs 24 hrs Chromatography % Unpegylated 94.7 76.1 66.6
51.0 0.6 % Monopegylated 0.9 17.9 26.1 39.1 85.6 % Dipegylated 0.1
0.9 1.9 5.1 5.1 % Tripegylated 0.0 0.0 0.0 0.2 0.2
After 24 hours, the bulk product PEG-state distribution was: 0.7%
unpegylated, 85.3% mono-pegylated, 11.5% di-pegylated, and 0.3%
tri-pegylated. Column chromatography is the main step in the
process that generates the product distribution, largely through
removing unpegylated material from mono- and di-pegylated
species.
Example 8
Factor VIIa-SA-PEG-10 KDa
[0489] The following example describes a procedure for determining
the number of modified sugar attachments to light and heavy chains
of Factor VIIa-SA-PEG-10 KDa by reverse phase HPLC.
[0490] Factor VIIa-SA-PEG-10 KDa was subjected to reducing
conditions in order to separate the heavy chain from the light
chain. After separation, the heavy and light chains were subjected
to separate reverse phase HPLC experiments. Peaks were assigned
based on their position relative to the non-modified Factor VIIa
peaks in the chromatograms of the native Factor VIIa control.
[0491] The following table describes the HPLC solvent gradient
parameters for the light chain. The column temperature was
39.degree. C. TABLE-US-00002 Time, min Solvent B, % Flow rate,
mL/min Comment 0 30 0.5 Initial condition 60 47 0.5 Gradient
elution 60.2 90 0.5 Start wash 70 90 0.5 Wash
[0492] The chromatograms of light chain Factor VIIa-SA-PEG-10 KDa
(top) and native light chain Factor VIIa (bottom) are provided in
FIG. 14A.
[0493] The following table describes the HPLC solvent gradient
parameters for the heavy chain. The column temperature was
52.degree. C. TABLE-US-00003 Time, min Solvent B, % Flow rate,
ml/min Comment 0 42.5 0.5 Initial condition 36 52.5 0.5 Gradient
elution 36.1 90 0.5 Start wash 41 90 0.5 wash
[0494] The chromatograms of heavy chain Factor VIIa-SA-PEG-10 KDa
(top) and native heavy chain Factor VIIa (bottom) are provided in
FIG. 14B.
Example 9
Factor VIIa-SA-PEG-40 KDa
[0495] The following example describes a procedure for determining
the number of modified sugar attachments to light and heavy chains
of Factor VIIa-SA-PEG-40 KDa by reverse phase HPLC.
[0496] Factor VIIa-SA-PEG-40 KDa was subjected to reducing
conditions in order to separate the heavy chain from the light
chain. After separation, the heavy and light chains were subjected
to separate reverse phase HPLC experiments. Peaks were assigned
based on their position relative to the non-modified sugar peaks in
the chromatograms of the native Factor VIIa control.
[0497] The following table describes the HPLC solvent gradient
parameters for the light chain. The column temperature was
25.degree. C. TABLE-US-00004 Time (min) Eluent B (%) Comment 0 30
Initial conditions 60 47 Gradient elution 60.5 90 Begin wash 65.5
90 End wash 66 42.5 Begin heavy chain method equilibration 70 42.5
End of Run
[0498] The chromatograms of light chain Factor VIIa-SA-PEG-40 KDa
(bottom) and native light chain Factor VIIa (top) are provided in
FIG. 15A.
[0499] The following table describes the HPLC solvent gradient
parameters for the heavy chain. The column temperature was
40.degree. C. TABLE-US-00005 Time (min) Eluent B (%) Comment 0 42.5
Initial conditions 36 52.5 Gradient elution 36.5 90 Begin wash 41.5
90 End wash 42 30 Begin light chain method equilibration 47 30 End
Run
[0500] The chromatograms of heavy chain Factor VIIa-SA-PEG-40 KDa
(bottom) and native heavy chain Factor VIIa (top) are provided in
FIG. 15B.
[0501] 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.
* * * * *
References