U.S. patent application number 10/579621 was filed with the patent office on 2007-11-01 for glycopegylated erythropoietin.
Invention is credited to Robert J. Bayer, Shawn DeFrees, David A. Zopf.
Application Number | 20070254834 10/579621 |
Document ID | / |
Family ID | 34637500 |
Filed Date | 2007-11-01 |
United States Patent
Application |
20070254834 |
Kind Code |
A1 |
DeFrees; Shawn ; et
al. |
November 1, 2007 |
Glycopegylated Erythropoietin
Abstract
The present invention provides conjugates between erythropoietin
and PEG moieties. The conjugates are linked via an intact glycosyl
linking group interposed between and covalently attached to the
peptide and the modifying group. The conjugates are formed from
glycosylated peptides by the action of a glycosyltransferase. The
glycosyltransferase ligates a modified sugar moiety onto a glycosyl
residue on the peptide. Also provided are methods for preparing the
conjugates, methods for treating various disease conditions with
the conjugates, and pharmaceutical formulations including the
conjugates.
Inventors: |
DeFrees; Shawn; (North
Wales, PA) ; Bayer; Robert J.; (San Diego, CA)
; Zopf; David A.; (Wayne, PA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP (SF)
2 PALO ALTO SQUARE
3000 El Camino Real, Suite 700
PALO ALTO
CA
94306
US
|
Family ID: |
34637500 |
Appl. No.: |
10/579621 |
Filed: |
November 24, 2004 |
PCT Filed: |
November 24, 2004 |
PCT NO: |
PCT/US04/39712 |
371 Date: |
February 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60524989 |
Nov 24, 2003 |
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60539387 |
Jan 26, 2004 |
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60555504 |
Mar 22, 2004 |
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60590573 |
Jul 23, 2004 |
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60592744 |
Jul 29, 2004 |
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60614518 |
Sep 29, 2004 |
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60623387 |
Oct 29, 2004 |
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Current U.S.
Class: |
514/54 ;
514/12.2; 514/15.1; 514/7.7; 530/395 |
Current CPC
Class: |
A61P 17/02 20180101;
C07H 19/10 20130101; C07K 14/505 20130101; A61P 7/06 20180101; A61K
38/00 20130101; A61P 7/00 20180101; A61K 47/60 20170801 |
Class at
Publication: |
514/008 ;
530/395 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 7/00 20060101 A61P007/00; C07K 1/00 20060101
C07K001/00 |
Claims
1. An erythropoietin peptide comprising the moiety: ##STR68##
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 is a moiety comprising a member selected a moiety
comprising a straight-chain or branched poly(ethylene glycol)
residue; and 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--.
2. The peptide according to claim 1, wherein L-R.sup.1 has the
formula: ##STR69## wherein a is an integer from 0 to 20.
3. The peptide according to claim 1, wherein R.sup.1 has a
structure that is a member selected from: ##STR70## wherein e and f
are integers independently selected from 1 to 2500; and q is an
integer from 0 to 20.
4. The peptide according to claim 1, wherein R.sup.1 has a
structure that is a member selected from: ##STR71## 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.
5. The peptide according to claim 1, wherein R.sup.1 has a
structure that is a member selected from: ##STR72## wherein e, f
and f' are integers independently selected from 1 to 2500; and q,
q' and q'' are integers independently selected from 1 to 20.
6. The peptide according to claim 1 wherein R.sup.1 has a structure
that is a member selected from: ##STR73## wherein e and f are
integers independently selected from 1 to 2500.
7. The peptide according to claim 1, wherein said moiety has the
formula: ##STR74##
8. The peptide according to claim 1, wherein said moiety has the
formula: ##STR75##
9. The peptide according to claim 1, wherein said moiety has the
formula: ##STR76## wherein AA is an amino acid residue of said
peptide.
10. The peptide according to claim 9, wherein said amino acid
residue is a member selected from serine or threonine.
11. The peptide according to claim 10, wherein said peptide has the
amino acid sequence of SEQ. ID. NO:1.
12. The peptide according to claim 11, wherein said amino acid
residue is a serine at position 126 of SEQ. ID. NO:1.
13. The peptide according to claim 1, wherein said peptide
comprises at least one of said moiety according to a formula
selected from: ##STR77## wherein AA is an amino acid residue of
said peptide and t is an integer equal to 0 or 1.
14. The peptide according to claim 13, wherein said amino acid
residue is an asparagine residue.
15. The peptide according to claim 14, wherein said peptide has the
amino acid sequence of SEQ ID NO:1, and wherein said amino acid
residue is an asparagine residue which is a member selected from
N24, N38, N83, and combinations thereof.
16. The peptide according to claim 1 wherein said peptide comprises
at least one of said moiety according to the formula: ##STR78##
wherein AA is an amino acid residue of said peptide, and t is an
integer equal to 0 or 1.
17. The peptide according to claim 16, wherein said amino acid
residue is an arginine residue.
18. The peptide according to claim 17, wherein said peptide has the
amino acid sequence of SEQ ID NO:1, and wherein said amino acid
residue is an asparagine residue which is a member selected from
N24, N38, N83, and combinations thereof.
19. The peptide of claim 1, wherein said peptide comprises at least
one of said moiety according to a formula selected from: ##STR79##
##STR80## ##STR81## wherein AA is an amino acid residue of said
peptide, and t is an integer equal to 0 or 1.
20. The peptide according to claim 1 wherein said peptide comprises
at least one said moiety according to a formula selected from:
##STR82## ##STR83## wherein AA is an amino acid residue of said
peptide, and t is an integer equal to 0 or 1.
21. The peptide according to claim 20, wherein said amino acid
residue is an asparagine residue.
22. The peptide according to claim 21, wherein said peptide has the
amino acid sequence of SEQ ID NO:1, and wherein said amino acid
residue is an asparagine residue which is a member selected from
N24, N38, N83, and combinations thereof.
23. The peptide according to claim 1, wherein said peptide is a
bioactive erythropoietin peptide.
24. The peptide according to claim 23, wherein said peptide is
erythropoietically active.
25. The peptide according to claim 24, wherein said peptide is
essentially non-erythropoietically active.
26. The peptide according to claim 25, wherein said peptide is
tissue protective.
27. A method of making a PEG-ylated erythropoietin comprising the
moiety: ##STR84## wherein R.sup.1 is a moiety comprising
straight-chain or branched poly(ethylene glycol) residue; and L is
a linker which is a member selected from substituted or
unsubstituted alkyl and substituted or unsubstituted heteroalkyl,
said method comprising: (a) contacting a substrate erythropoietin
peptide comprising the glycosyl moiety: ##STR85## with a PEG-sialic
acid donor moiety having the formula: ##STR86## and an enzyme that
transfers said PEG-sialic acid onto the Gal of said glycosyl
moiety, under conditions appropriate to for said transfer.
28. The method of claim 27, further comprising, prior to step (a):
(b) expressing said substrate erythropoietin peptide in a suitable
host.
29. The method of claim 28, wherein said host is selected from an
insect cell and a mammalian cell.
30. The method of claim 29, wherein said insect cell is a
Spodoptera frugiperda cell line.
31. A method of treating a condition in a subject in need thereof,
said condition characterized by compromised red blood cell
production in said subject, said method comprising the step of
administering to the subject an amount of a peptide according to
claim 1, effective to ameliorate said condition in said
subject.
32. A method of enhancing red blood cell production in a mammal,
said method comprising administering to said mammal an peptide
according to claim 1.
33. A method of treating a tissue injury in a subject in need
thereof, said injury characterized by damage resulting from
ischemia, trauma, inflammation or contact with toxic substances,
said method comprising the step of administering to the subject an
amount of an erythropoietin peptide according to claim 1, effective
to ameliorate the damage associated with the tissue injury in said
subject.
34. A pharmaceutical formulation comprising the erythropoietin
peptide according to claim 1, and a pharmaceutically acceptable
carrier.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Patent Application No. 60/524,989, filed Nov. 24, 2003; U.S.
Provisional Patent Application No. 60/555,504, filed Mar. 22, 2004;
U.S. Provisional Patent Application No. 60/590,573, filed Jul. 23,
2004; U.S. Provisional Patent Application No. 60/592,744, filed
Jul. 29, 2004; U.S. Provisional Patent Application No. 60/614,518,
filed Sep. 29, 2004; and U.S. Provisional Patent Application No.
60/623,387 each of which is incorporated herein by reference in
their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] Erythropoietin (EPO) is a cytokine produced by the kidney
and liver which acts on hematopoietic stem cells to stimulate the
production of red blood cells. The protein exists in two forms: one
being a 165 amino acid peptide, and the other is a 166 amino acid
peptide. The 166 amino acid peptide has the same sequence as the
165 amino acid peptide except that the 166 amino acid peptide has
an additional arginine in the most C-terminal position. The mature
165 amino acid peptide is a 34 kD glycoprotein comprising three
N-glycosylation sites (Asn-24, Asn-38, and Asn-83), and 1
O-glycosylation site (Ser-126), and some variants are
"hyperglycosylated" comprising 5 N-linked glycosylation sites.
[0003] Erythropoietin synthesis is induced by conditions that
effectively create tissue hypoxia, such as lowering of the arterial
O.sub.2 tension or increasing the oxygen affinity of the blood.
Under usual conditions of homeostasis, hematocrit and the
concentration of hemoglobin in blood are maintained constant with
erythropoiesis counterbalancing the permanent destruction of aged
red blood cells by macrophages in bone marrow, spleen and liver.
Quantitatively, about 1% of the red cell mass, which is about
2-3.times.10.sup.11 red blood cells, is renewed each day. However,
in situations that effectively generate tissue hypoxia, such as
blood loss or location to high altitudes, the induction of EPO may
stimulate erythropoiesis 10-fold or more over normal levels.
[0004] Because EPO stimulates red blood cell production, it is an
effective therapy for many diseases and conditions associated with
reduced hematocrit. Initial trials of replacement therapy with
recombinant human EPO to restore the hematocrit in patients with
end-stage renal failure were first reported about 20 years ago (see
e.g., Winearls, C. G.; et al. (1986) Lancet, 2, 1175-1178, and
Eschbach, J. W.; et al. (1987) N. Engl. J. Med., 316, 73-78). This
work provided an impetus for further studies into the
pathophysiology and pharmacology of EPO (see e.g., Jelkmann, W. and
Gross, A. (1989) Erythropoietin; Springer, Berlin Heidelberg New
York).
[0005] Since those early studies, recombinant human EPO has been
used successfully to treat numerous pathological conditions. For
example, the pharmacological application of recombinant human EPO
to surgical patients can lower the severity and duration of
postoperative anemia. The administration of recombinant human EPO
has also proven to be effective therapy for patients suffering from
several non-renal diseases, such as chronic inflammation,
malignancy and AIDS, wherein a relative lack of endogenous EPO
contributes to the development of anemia (see e.g., Means, R. T.
and Krantz, S. B. (1992) Blood, 80, 1639-1647, and Jelkmann, W.
(1998) J. Interf. Cytokine Res., 18, 555-559). Furthermore, it has
been reported that EPO is tissue protective in ischemic, traumatic,
toxic and inflammatory injuries (see e.g., Brines M., et al. (2004)
PNAS USA 101:14907-14912 and Brines, M. L., et al. (2000). Proc.
Natl. Acad. Sci. USA 97, 10526-10531).
[0006] The usefulness and effectiveness of EPO for the treatment of
anemias and other conditions arising from such a wide variety of
causes makes recombinant human EPO perhaps the best selling drug in
the world. Indeed, estimated sales amount to more than 5 billion US
dollars per year.
[0007] Only one recombinant human EPO, produced in Chinese Hamster
Ovary (CHO) cell line, is used extensively as a therapeutic. Since
mammals all produce glycans of similar structure, Chinese Hamster
Ovary (CHO), Baby Hamster Kidney (BHK), and Human Embryonic
Kidney-293 (HEK-293) are the preferred host cells for production of
glycoprotein therapeutics. As is known in the art, proper
glycosylation is a critically important factor influencing the in
vivo the half life and immunogenicity of therapeutic peptides.
Indeed, poorly glycosylated proteins are recognized by the liver as
being "old" and thus, are more quickly eliminated from the body
than are properly glycosylated proteins.
[0008] Unfortunately, one frustrating, and well known aspect of
protein glycosylation is the phenomenon of microheterogeneity.
Thus, even the preferred host cells for production of human
therapeutic glycoproteins such as EPO, typically produce peptides
comprising a range of variations in the precise structure of the
glycan. The extent of this heterogeneity can vary considerably from
glycosylation site to glycosylation site, from protein to protein,
and from cell type to cell type. Therefore, numerous glycoforms,
each of which each is effectively a distinct molecular species,
typically exist in any given glycoprotein preparation.
[0009] The problem of microheterogeneity thus poses numerous
problems for the large industrial scale production of therapeutic
glycoproteins. In particular, since each glycoform can represent a
distinct molecular species, preparations of therapeutic
glycoproteins must be fractionated to purify the desired single
glycoform. Further complications arise from the fact that different
production batches may vary with respect to the percentage of the
desired glycoform comprising the batch of glycoprotein therapeutic.
Thus, large, not always predictable portions of each preparation
may be have to be discarded, so that ultimately the final yield of
a desired glycoform can be low. Overall, the problem of
microheterogeneity means that therapeutic glycopeptides produced by
mammalian cell culture require higher production costs, which
ultimately translate to higher health care costs than might be
necessary if a more efficient method for making longer lasting,
more effective glycoprotein therapeutics was available.
[0010] One solution to the problem of providing cost effective
glycopeptide therapeutics has been to provide peptides with longer
in vivo half lives. For example, glycopeptide therapeutics with
improved pharmacokinetic properties have been produced by attaching
synthetic polymers to the peptide backbone. An exemplary polymer
that has been conjugated to peptides is poly(ethylene glycol)
("PEG"). The use of PEG to derivatize peptide therapeutics has been
demonstrated to reduce the immunogenicity of the peptides. For
example, U.S. Pat. No. 4,179,337 (Davis et al.) discloses
non-immunogenic polypeptides such as enzymes and peptide hormones
coupled to polyethylene glycol (PEG) or polypropylene glycol. In
addition to reduced immunogenicity, the clearance time in
circulation is prolonged due to the increased size of the
PEG-conjugate of the polypeptides in question.
[0011] 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).
[0012] In these non-specific methods, poly(ethyleneglycol) is added
in a random, non-specific manner to reactive residues on a peptide
backbone. Of course, random addition of PEG molecules has its
drawbacks, including a lack of homogeneity of the final product,
and the possibility for reduction in the biological or enzymatic
activity of the peptide. Therefore, for the production of
therapeutic peptides, a derivitization strategy that results in the
formation of a specifically labeled, readily characterizable,
essentially homogeneous product is superior. Such methods have been
developed.
[0013] 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).
[0014] Erythropoietin (EPO) is an extremely valuable therapeutic
peptide. Although commercially available forms of EPO are in use
today, these peptides are less than maximally effective due factors
including microheterogeneity of the glycoprotein product which
increases production costs, poor pharmacokinetics of the resulting
isolated glycoprotein product, or a combination of the two. Thus,
there remains a need in the art for long lasting EPO peptides with
improved effectiveness and better pharmacokinetics. Furthermore, to
be effective for the largest number of individuals, it must be
possible to produce, on an industrial scale, an EPO peptide with
improved therapeutic pharmacokinetics that has a predictable,
essentially homogeneous, structure which can be readily reproduced
over, and over again.
[0015] Fortunately, EPO peptides with improved the therapeutic
effectiveness and methods for making them have now been discovered.
Indeed, the invention provides EPO peptides with improved
pharmacokinetics. The invention also provides industrially
practical and cost effective methods for the production of modified
EPO peptides. The EPO peptides of the invention comprise modifying
groups such as PEG moieties, therapeutic moieties, biomolecules and
the like. The present invention therefore fulfills the need for EPO
peptides with improved the therapeutic effectiveness and improved
pharmacokinetics for the treatment of conditions and diseases
wherein EPO provides effective therapy.
SUMMARY OF THE INVENTION
[0016] It has now been discovered that the controlled modification
of erythropoietin (EPO) with one or more poly(ethylene glycol)
moieties affords novel EPO derivatives with improved
pharmacokinetic properties. Furthermore, cost effective methods for
reliable production of the modified EPO peptides of the invention
have been discovered and developed.
[0017] In one aspect, the present invention provides an
erythropoietin peptide comprising the moiety: ##STR1## 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
is a moiety comprising a member selected a moiety comprising a
straight-chain or branched poly(ethylene glycol) residue; and 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--. In one
embodiment, a R.sup.1-L has the formula: ##STR2## wherein a is an
integer from 0 to 20. In another embodiment, R.sup.1 has a
structure that is a member selected from: ##STR3## wherein e and f
are integers independently selected from 1 to 2500; and q is an
integer from 1 to 20. In other embodiments R.sup.1 has a structure
that is a member selected from: ##STR4## 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.
[0018] In still another embodiment, the invention provides a
peptide wherein R.sup.1 has a structure that is a member selected
from: ##STR5## wherein e, f and f' are integers independently
selected from 1 to 2500; and q, q' and q'' are integers
independently selected from 1 to 20. In other embodiments, R.sup.1
has a structure that is a member selected from: ##STR6## wherein e
and f are integers independently selected from 1 to 2500.
[0019] In another aspect, the invention provides a peptide
comprising a moiety having the formula: ##STR7##
[0020] In other embodiments, the moiety has the formula:
##STR8##
[0021] In another exemplary embodiment the peptide comprises a
moiety according to the formula ##STR9## wherein AA is an amino
acid residue of said peptide. In some embodiments the amino acid
residue is a member selected from serine, threonine and tyrosine.
In a preferred embodiment the amino acid residue is a serine at
position 126 of SEQ. ID. NO:1.
[0022] In another exemplary embodiment, the invention provides an
erythropoietin peptide wherein the peptide comprises at least one
moiety that has the formula: ##STR10## wherein t is an integer from
equal to 0 or 1. Thus, in this embodiment, the modified sialic acid
moiety may occur on either branch of the biantennary structure.
[0023] In another related embodiment, the invention provides an
erythropoietin peptide wherein the peptide comprises at least one
moiety that has the formula: ##STR11##
[0024] In another embodiment, the invention provides an
erythropoietin peptide wherein the peptide comprises at least one
moiety that has a formula according to: ##STR12## In this
embodiment, the modified sialic acid moiety may occur on any one or
more of the branches of the either form of the triantennary
structure.
[0025] In still another embodiment, the invention provides an
erythropoietin peptide wherein the peptide comprises at least one
moiety that has the formula: ##STR13## In this embodiment, the
modified sialic acid moiety may occur on any one or more of the
branches of the tetra antennary structure.
[0026] In another aspect the invention provides an erythropoietin
peptide that is a bioactive erythropoietin peptide. In one
embodiment, the erythropoietin peptide is erythropoietically
active. In another embodiment, the erythropoietin peptide is
essentially non-erythropoietically active. In another embodiment,
the erythropoietin peptide is tissue protective.
[0027] In another aspect, the invention provides a method of making
a PEG-ylated erythropoietin comprising the moiety: ##STR14##
wherein R.sup.1 is a moiety comprising straight-chain or branched
poly(ethylene glycol) residue; and L is a linker which is a member
selected from substituted or unsubstituted alkyl and substituted or
unsubstituted heteroalkyl. The method comprises contacting a
substrate erythropoietin peptide comprising the glycosyl moiety:
##STR15## with a PEG-sialic acid donor moiety having the formula:
##STR16## and an enzyme that transfers said PEG-sialic acid onto
the Gal of said glycosyl moiety, under conditions appropriate to
for the transfer. In one embodiment, the erythropoietin peptide is
expressed in a suitable host. In one embodiment the host is
mammalian cell, and in another embodiment the host cell is an
insect cell.
[0028] In another aspect, the invention provides a method of
treating a condition in a subject in need thereof, wherein the
condition is characterized by compromised red blood cell production
in the subject. The method comprises the step of administering to
the subject an amount of the erythropoietin peptide of the
invention effective to ameliorate the condition in the subject.
[0029] In another aspect, the invention provides a method of
enhancing red blood cell production in a mammal. The method
comprises administering to the mammal an amount of the
erythropoietin peptide of the invention effective to enhance red
blood cell production in the mammal.
[0030] In another aspect, the invention provides a method of
treating a tissue injury in a subject in need thereof, said injury
characterized by damage resulting from ischemia, trauma,
inflammation or contact with toxic substances, said method
comprising the step of administering to the subject an amount of an
erythropoietin peptide of the invention effective to ameliorate
said tissue injury in the subject.
[0031] In another aspect, the invention provides a pharmaceutical
formulation comprising the erythropoietin peptide of the invention
and a pharmaceutically acceptable carrier.
[0032] In the o-linked erythropoietin conjugates of the invention,
essentially each of the amino acid residues to which the polymer is
bound has the same structure. For example, if one peptide includes
a Ser linked glycosyl residue, at least about 70%, 80%, 90%, 95%,
97%, 99%, 99.2%, 99.4%, 99.6%, or more preferably 99.8% of the
peptides in the population will have the same glycosyl residue
covalently bound to the same Ser residue.
[0033] 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
[0034] FIG. 1. FIG. 1 illustrates some exemplary modified sugar
nucleotides useful in the practice of the invention.
[0035] FIG. 2. FIG. 2 illustrates further exemplary modified sugar
nucleotides useful in the practice of the invention.
[0036] FIG. 3. FIG. 3 illustrates exemplary modified sialic acid
nucleotides useful in the practice of the invention. A: Structure
of 40 kilodalton CMP-Sialic acid-PEG. B: Structure of 30 kilodalton
CMP-Sialic acid-PEG.
[0037] FIG. 4. FIG. 4 presents a schematic representation of
exemplary glycopegylated EPO isoforms isolated from Chinese Hamster
Ovary cells. A. An exemplary 40 kilodalton O-linked pegylated
glycoform. B: One of several 30 kilodalton N-linked pegylated
glycoforms. The modified sialic acid moiety comprising the PEG
molecule, may occur on any one or more of any of the branches of
the N-linked glycosyl residue. Furthermore the illustration is
exemplary in that any glycosylated EPO molecule may comprise any
mixture of mono-, bi- tri-, or tetra-antennary N-linked glycosyl
residues and any one or more of the branches may further comprise a
modified sialic acid moiety of the invention.
[0038] FIG. 5. FIG. 5 illustrates an exemplary CHO-derived EPO
peptide in its non-glycopegylated form. As discussed in the legend
to FIG. 4 (above) the illustration is exemplary in that any
glycosylated EPO molecule may comprise any mixture of mono-, bi-
tri-, or tetra-antennary N-linked glycosyl residues.
[0039] FIG. 6. FIG. 6 shows the results of experiments comparing
the pharmacokinetics of two CHO-derived non-glycopegylated EPO
forms, and two different CHO-derived glycopegylated EPO forms.
[0040] FIG. 7. FIG. 7 illustrates an insect-derived glycopegylated
EPO peptide according to the invention.
[0041] FIG. 8. FIG. 8 shows the results of experiments comparing
the pharmacokinetics of a CHO-derived non-glycopegylated EPO form,
an insect-derived non-glycopegylated EPO form, with their
corresponding glycopegylated forms.
[0042] FIG. 9. FIG. 9 shows the relative activities of two forms of
non-glycopegylated EPO (A and B) versus two glycoPEGylated variants
(the 30 kilodalton and 40 kilodalton variants of FIGS. 4 A and B)
and a hyperglycosylated EPO variant in stimulating proliferation of
EPO receptor-bearing TF1 cells in culture.
[0043] FIG. 10. FIG. 10 shows inhibition of binding of
isotope-labeled EPO to a recombinant chimeric EPO receptor by
various concentrations of two non-pegylated EPO variants (A and B)
and two glycoPEGylated variants (the 30 kilodalton and 40
kilodalton variants of FIGS. 4 A and B).
DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED
EMBODIMENTS
Abbreviations
[0044] PEG, poly(ethyleneglycol); PPG, poly(propyleneglycol); Ara,
arabinosyl; Fru, fructosyl; Fuc, fucosyl; Gal, galactosyl; GalNAc,
N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc,
N-acetylglucosarninyl; Man, mannosyl; ManAc, mannosaminyl acetate;
Xyl, xylosyl; and NeuAc, sialyl(N-acetylneuraminyl); M6P,
mannose-6-phosphate.
Definitions
[0045] 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.
[0046] 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).
[0047] 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.
[0048] The term "sialic acid" refers to any member of a family of
nine-carbon carboxylated sugars. The most common member of the
sialic acid family is N-acetyl-neuraminic acid
(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic
acid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member
of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in
which the N-acetyl group of NeuAc is hydroxylated. A third sialic
acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano
et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J.
Biol. Chem. 265: 21811-21819 (1990)). Also included are
9-substituted sialic acids such as a 9-O--C.sub.1-C.sub.6
acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,
9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of
the sialic acid family, see, e.g., Varki, Glycobiology 2: 2540
(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 sialyation procedure is disclosed
in international application WO 92/16640, published Oct. 1,
1992.
[0049] "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).
[0050] The term "peptide conjugate," refers to species of the
invention in which a peptide is conjugated with a modified sugar as
set forth herein.
[0051] 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 .alpha. 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.
[0052] As used herein, the term "modified sugar," refers to a
naturally- or non-naturally-occurring carbohydrate that is
enzymatically added onto an amino acid or a glycosyl residue of a
peptide in a process of the invention. The modified sugar is
selected from a number of 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.
[0053] 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).
[0054] 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.
[0055] 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.
[0056] Many other polymers are also suitable for the invention.
Polymer backbones that are non-peptidic and water-soluble, with
from 2 to about 300 termini, are particularly useful in the
invention. Examples of suitable polymers include, but are not
limited to, other poly(alkylene glycols), such as poly(propylene
glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol
and the like, poly(oxyethylated polyol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide),
poly(.alpha.-hydroxy acid), poly(vinyl alcohol), polyphosphazene,
polyoxazoline, poly(N-acryloylmorpholine), such as described in
U.S. Pat. No. 5,629,384, which is incorporated by reference herein
in its entirety, and copolymers, terpolymers, and mixtures thereof.
Although the molecular weight of each chain of the polymer backbone
can vary, it is typically in the range of from about 100 Da to
about 100,000 Da, often from about 6,000 Da to about 80,000 Da.
[0057] 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.
[0058] The term "half-life" or "t1/2", as used herein in the
context of administering a peptide drug to a patient, is defined as
the time required for plasma concentration of a drug in a patient
to be reduced by one half. There may be more than one half-life
associated with the peptide drug depending on multiple clearance
mechanisms, redistribution, and other mechanisms well known in the
art. Usually, alpha and beta half-lives are defined such that the
alpha phase is associated with redistribution, and the beta phase
is associated with clearance. However, with protein drugs that are,
for the most part, confined to the bloodstream, there can be at
least two clearance half-lives. For some glycosylated peptides,
rapid beta phase clearance may be mediated via receptors on
macrophages, or endothelial cells that recognize terminal
galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, or
fucose. Slower beta phase clearance may occur via renal glomerular
filtration for molecules with an effective radius <2 nm
(approximately 68 kD) and/or specific or non-specific uptake and
metabolism in tissues. GlycoPEGylation may cap terminal sugars
(e.g., galactose or N-acetylgalactosamine) and thereby block rapid
alpha phase clearance via receptors that recognize these sugars. It
may also confer a larger effective radius and thereby decrease the
volume of distribution and tissue uptake, thereby prolonging the
late beta phase. Thus, the precise impact of glycoPEGylation on
alpha phase and beta phase half-lives will vary depending upon the
size, state of glycosylation, and other parameters, as is well
known in the art. Further explanation of "half-life" is found in
Pharmaceutical Biotechnology (1997, D F A Crommelin and R D
Sindelar, eds., Harwood Publishers, Amsterdam, pp 101-120).
[0059] The term "glycoconjugation," as used herein, refers to the
enzymatically mediated conjugation of a modified sugar species to
an amino acid or glycosyl residue of a polypeptide, e.g., an
Erythropoietin peptide of the present invention. A subgenus of
"glycoconjugation" is "glycol-PEGylation," in which the modifying
group of the modified sugar is poly(ethylene glycol), and alkyl
derivative (e.g., m-PEG) or reactive derivative (e.g., H2N-PEG,
HOOC-PEG) thereof.
[0060] 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.
[0061] The term, "glycosyl linking group," as used herein refers to
a glycosyl residue to which a modifying group (e.g., PEG moiety,
therapeutic moiety, biomolecule) is covalently attached; the
glycosyl linking group joins the modifying group to the remainder
of the conjugate. In the methods of the invention, the "glycosyl
linking group" becomes covalently attached to a glycosylated or
unglycosylated peptide, thereby linking the agent to an amino acid
and/or glycosyl residue on the peptide. A "glycosyl linking group"
is generally derived from a "modified sugar" by the enzymatic
attachment of the "modified sugar" to an amino acid and/or glycosyl
residue of the peptide. The glycosyl linking group can be a
saccharide-derived structure that is degraded during formation of
modifying group-modified sugar cassette (e.g.,
oxidation.fwdarw.Schiff base formation.fwdarw.reduction), or the
glycosyl linking group may be intact. An "intact glycosyl linking
group" refers to a linking group that is derived from a glycosyl
moiety in which the saccharide monomer that links the modifying
group and to the remainder of the conjugate is not degraded, e.g.,
oxidized, e.g., by sodium metaperiodate. "Intact glycosyl linking
groups" of the invention may be derived from a naturally occurring
oligosaccharide by addition of glycosyl unit(s) or removal of one
or more glycosyl unit from a parent saccharide structure.
[0062] 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.
[0063] 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)).
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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).
[0068] 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.
[0069] The term "tissue protective" refers to the defense of a
tissue against the effects of cellular damage that are typically
associated with the experience by a tissue or organ of
ischemia/hypoxia, trauma, toxicity and/or inflammation. Cellular
damage may lead to apoptosis and/or necrosis (i.e., toxic cell
death). Thus, a "tissue protective" effect guards a tissue from
experiencing the degree of apoptosis and/or toxic cell death
normally associated with a given traumatic, inflammatory, toxic or
ischemic injury. For example, EPO reduces the area of infarct after
middle cerebral artery occlusion in a rodent model (Siren, A. L. et
al. (2001). Proc. Natl. Acad. Sci. U.S.A. 98, 4044-4049). Thus,
under such conditions EPO provides a "tissue protective" effect by
effectively reducing the necrosis and/or apotosis normally
associated with the ischemic injury (e.g., ischemic stroke).
"Tissue protective" also refers to the defense of a tissue against
the effects of cellular damage and the ensuing cell death
associated with degenerative diseases such as retinopathy, or
neurodegenerative disease.
[0070] 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%.
[0071] 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.
[0072] 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).
[0073] "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.
[0074] "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%.
[0075] 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.
[0076] "Substantially uniform glycoform" or a "substantially
uniform glycosylation pattern," when referring to a glycopeptide
species, refers to the percentage of acceptor moieties that are
glycosylated by the glycosyltransferase of interest (e.g.,
fucosyltransferase). For example, in the case of a .alpha.1,2
fucosyltransferase, a substantially uniform fucosylation pattern
exists if substantially all (as defined below) of the
Gal.beta.1,4-GlcNAc-R and sialylated analogues thereof are
fucosylated in a peptide conjugate of the invention. It will be
understood by one of skill in the art, that the starting material
may contain glycosylated acceptor moieties (e.g., fucosylated
Gal.beta.1,4-GlcNAc-R moieties). Thus, the calculated percent
glycosylation will include acceptor moieties that are glycosylated
by the methods of the invention, as well as those acceptor moieties
already glycosylated in the starting material.
[0077] 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.
[0078] 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--.
[0079] 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".
[0080] 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.
[0081] 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.
[0082] 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
--H.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--.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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).
[0087] 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.
[0088] 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(NR'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).
[0089] 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.
[0090] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CRR').sub.q-U-, wherein T and U are
independently --NR--, --O--, --CRR'-- or a single bond, and q is an
integer of from 0 to 3. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula -A-(CH.sub.2).sub.r-B-,
wherein A and B are independently --CRR'--, --O--, --NR--, --S--,
--S(O)--, --S(O).sub.2--, --S(O).sub.2NR'-- or a single bond, and r
is an integer of from 1 to 4. One of the single bonds of the new
ring so formed may optionally be replaced with a double bond.
Alternatively, two of the substituents on adjacent atoms of the
aryl or heteroaryl ring may optionally be replaced with a
substituent of the formula --(CRR').sub.s--X--(CR''R''').sub.d--,
where s and d are independently integers of from 0 to 3, and X is
--O--, --NR'--, --S--, --S(O)--, --S(O).sub.2--, or
--S(O).sub.2NR'--. The substituents R, R', R'' and R''' are
preferably independently selected from hydrogen or substituted or
unsubstituted (C.sub.1-C.sub.6)alkyl.
[0091] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
Introduction
[0092] Erythropoietin (EPO) is a glycoprotein which serves as the
principal regulator of red blood cell synthesis. Erythropoietin is
produced in the kidney and acts by stimulating precursor cells in
the bone marrow causing them to divide and differentiate into
mature red blood cells. EPO may exist as a either a 165 or 166
amino acid glycoprotein. The 166 amino acid variant is
distinguished from the 165 amino acid variant by the presence of an
additional arginine residue at the C-terminal end of the
protein.
[0093] Recombinant EPO has been available for some time as an
effective therapeutic agent in the treatment of various forms of
anemia, including anemias associated with chronic renal failure,
zidovidine treated HIV infected patients, and cancer patients on
chemotherapy. The glycoprotein is administered parenterally, either
as an intravenous (IV) or subcutaneous (SC) injection.
[0094] To improve the effectiveness of recombinant erythropoietin
used for therapeutic purposes, the present invention provides
conjugates of glycosylated and unglycosylated erythropoietin
peptides. The conjugates may be additionally modified by further
conjugation with diverse species such as therapeutic moieties,
diagnostic moieties, targeting moieties and the like.
[0095] The conjugates of the invention are formed by the enzymatic
attachment of a modified sugar to the glycosylated or
unglycosylated peptide. Glycosylation sites provide loci for
conjugating modifying groups to the peptide, e.g., by
glycoconjugation. An exemplary modifying group is a water-soluble
polymer, such as poly(ethylene glycol), e.g., methoxy-poly(ethylene
glycol). Modification of the EPO peptides can improve the stability
and retention time of the recombinant EPO in a patient's
circulation, or reduce the antigenicity of recombinant EPO.
[0096] The methods of the invention make it possible to assemble
peptides and glycopeptides that have a substantially homogeneous
derivatization pattern. The enzymes used in the invention are
generally selective for a particular amino acid residue,
combination of amino acid residues, or particular glycosyl residues
of the peptide. The methods are also practical for large-scale
production of modified peptides and glycopeptides. Thus, the
methods of the invention provide a practical means for large-scale
preparation of glycopeptides having preselected uniform
derivatization patterns.
[0097] The present invention also provides conjugates of
glycosylated and unglycosylated peptides with increased therapeutic
half-life due to, for example, reduced clearance rate, or reduced
rate of uptake by the immune or reticuloendothelial system (RES).
Moreover, the methods of the invention provide a means for masking
antigenic determinants on peptides, thus reducing or eliminating a
host immune response against the peptide. Selective attachment of
targeting agents can also be used to target a peptide to a
particular tissue or cell surface receptor that is specific for the
particular targeting agent.
The Conjugates
[0098] In a first aspect, the present invention provides a
conjugate between a selected modifying group and an EPO
peptide.
[0099] The link between the peptide and the modifying group
includes a glycosyl linking group interposed between the peptide
and the selected moiety. As discussed herein, the selected moiety
is essentially any species that can be attached to a saccharide
unit, resulting in a "modified sugar" that is recognized by an
appropriate transferase enzyme, which appends the modified sugar
onto the peptide. The saccharide component of the modified sugar,
when interposed between the peptide and a selected moiety, becomes
a "glycosyl linking group," e.g., an "intact glycosyl linking
group." The glycosyl linking group is formed from any mono- or
oligo-saccharide that, after modification with the modifying group,
is a substrate for an enzyme that adds the modified sugar to an
amino acid or glycosyl residue of a peptide.
[0100] The glycosyl linking group can be, or can include, a
saccharide moiety that is degradatively modified before or during
the addition of the modifying group. For example, the glycosyl
linking group can be derived from a saccharide residue that is
produced by oxidative degradation of an intact saccharide to the
corresponding aldehyde, e.g., via the action of metaperiodate, and
subsequently converted to a Schiff base with an appropriate amine,
which is then reduced to the corresponding amine.
[0101] The conjugates of the invention will typically correspond to
the general structure: ##STR17## in which the symbols a, b, c, d
and s represent a positive, non-zero integer; and t is either 0 or
a positive integer. The "agent" is a therapeutic agent, a bioactive
agent, a detectable label, water-soluble moiety (e.g., PEG, m-PEG,
PPG, and m-PPG) or the like. The "agent" can be a peptide, e.g.,
enzyme, antibody, antigen, etc. The linker can be any of a wide
array of linking groups, infra. Alternatively, the linker may be a
single bond or a "zero order linker."
[0102] In an exemplary embodiment, the selected modifying group is
a water-soluble polymer, e.g., m-PEG. The water-soluble polymer is
covalently attached to the peptide via a glycosyl linking group.
The glycosyl linking group is covalently attached to an amino acid
residue or a glycosyl residue of the peptide. The invention also
provides conjugates in which an amino acid residue and a glycosyl
residue are modified with a glycosyl linking group.
[0103] An exemplary water-soluble polymer is poly(ethylene glycol),
e.g., methoxy-poly(ethylene glycol). The poly(ethylene glycol) used
in the present invention is not restricted to any particular form
or molecular weight range. For unbranched poly(ethylene glycol)
molecules the molecular weight is preferably between 500 and
100,000. A molecular weight of 2000-60,000 is preferably used and
preferably of from about 5,000 to about 30,000.
[0104] 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 equal to or greater than about 40,000
daltons.
[0105] In addition to providing conjugates that are formed through
an enzymatically added glycosyl linking group, the present
invention provides conjugates that are highly homogenous in their
substitution patterns. Using the methods of the invention, it is
possible to form peptide conjugates in which essentially all of the
modified sugar moieties across a population of conjugates of the
invention are attached to multiple copies of a structurally
identical amino acid or glycosyl residue. Thus, in a second aspect,
the invention provides a peptide conjugate having a population of
water-soluble polymer moieties, which are covalently bound to the
peptide through an intact glycosyl linking group. In a preferred
conjugate of the invention, essentially each member of the
population is bound via the glycosyl linking group to a glycosyl
residue of the peptide, and each glycosyl residue of the peptide to
which the glycosyl linking group is attached has the same
structure.
[0106] Also provided is a peptide conjugate having a population of
water-soluble polymer moieties covalently bound thereto through a
glycosyl linking group. In a preferred embodiment, essentially
every member of the population of water soluble polymer moieties is
bound to an amino acid residue of the peptide via a glycosyl
linking group, and each amino acid residue having a glycosyl
linking group attached thereto has the same structure.
[0107] The present invention also provides conjugates analogous to
those described above in which the peptide is conjugated to a
therapeutic moiety, diagnostic moiety, targeting moiety, toxin
moiety or the like via an intact glycosyl linking group. Each of
the above-recited moieties can be a small molecule, natural polymer
(e.g., polypeptide) or synthetic polymer.
[0108] Essentially any erythropoietin peptide having any sequence
is of use as a component of the conjugates of the present
invention. In an exemplary embodiment, the peptide has the
sequence: TABLE-US-00001 (SEQ ID NO:1) H.sub.2N-APPRLIDSR
VLERYLLEAK EAETTTGA EHSLNEIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL
SEAVLRGQAL LVSSQPWEP LQLHVDKAVS GLRSLTTLLR ALGAQKEAIS PPDAAAAPL
RTITADTFRK LFRVYSNFLR GKLKLYTGEA RTGD-COOH.
[0109] In another exemplary embodiment the peptide has the
sequence: TABLE-US-00002 (SEQ ID NO:2) H.sub.2N-APPRLIDSR
VLERYLLEAK EAETTTGA EHSLNEIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL
SEAVLRGQAL LVSSQPWEP LQLHVDKAVS GLRSLTTLLR ALGAQKEAIS PPDAAAAPL
RTITADTFRK LFRVYSNFLR GKLKLYTGEA RTGDR-COOH.
[0110] In the sequences set forth above, there are two disulfide
bonds, one at C7-C161 and another at C29-C33. The cysteine residues
are shown above in bold italics.
[0111] Preferably, neither terminus is derivatized.
[0112] The peptides of the invention include at least one N-linked
or O-linked glycosylation site, which is glycosylated with a
glycosyl residue that includes a PEG moiety. The PEG is covalently
attached to the peptide via an intact glycosyl linking group. The
glycosyl linking group is covalently attached to either an amino
acid residue or a glycosyl residue of the peptide. Alternatively,
the glycosyl linking group is attached to one or more glycosyl
units of a glycopeptide. The invention also provides conjugates in
which the glycosyl linking group is attached to both an amino acid
residue and a glycosyl residue.
[0113] The PEG moiety is attached to an intact glycosyl linker
directly, or via a non-glycosyl linker, e.g., substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl.
[0114] In a preferred embodiment, the erythropoietin peptide
comprises the moiety shown in Formula I ##STR18##
[0115] In Formula I, 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 is a moiety comprising a
member selected a moiety comprising a straight-chain or branched
poly(ethylene glycol) residue; and 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--.
The Compositions
[0116] As discussed above, the invention provides saccharides
bearing a modifying group, activated analogues of these species and
conjugates formed between species such as peptides and lipids and a
modified saccharide of the invention.
Modified Sugars
[0117] The present invention provides modified sugars, modified
sugar nucleotides and conjugates of the modified sugars. In
modified sugar compounds of the invention, the sugar moiety is
preferably a saccharide, a deoxy-saccharide, an amino-saccharide,
or an N-acyl saccharide. The term "saccharide" and its equivalents,
"saccharyl," "sugar," and "glycosyl" refer to monomers, dimers,
oligomers and polymers. The sugar moiety is also functionalized
with a modifying group. The modifying group is conjugated to the
sugar moiety, typically, through conjugation with an amine,
sulfhydryl or hydroxyl, e.g., primary hydroxyl, moiety on the
sugar. In an exemplary embodiment, the modifying group is attached
through an amine 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.
[0118] Any sugar can be utilized as the sugar core of the
conjugates of the invention. Exemplary sugar cores that are useful
in forming the compositions of the invention include, but are not
limited to, glucose, galactose, mannose, fucose, and sialic acid.
Other useful sugars include amino sugars such as glucosamine,
galactosamine, mannosamine, the 5-amine analogue of sialic acid and
the like. The sugar core can be a structure found in nature or it
can be modified to provide a site for conjugating the modifying
group. For example, in one embodiment, the invention provides a
sialic acid derivative in which the 9-hydroxy moiety is replaced
with an amine. The amine is readily derivatized with an activated
analogue of a selected modifying group.
[0119] In the discussion that follows the invention is illustrated
by reference to the use of selected derivatives of sialic acid.
Those of skill in the art will recognize that the focus of the
discussion is for clarity of illustration and that the structures
and compositions set forth are generally applicable across the
genus of saccharide groups, modified saccharide groups, activated
modified saccharide groups and conjugates of modified saccharide
groups.
[0120] In an exemplary embodiment, the invention provides a
modified sugar amine that has the formula: ##STR19## in which G is
a glycosyl moiety, L is a bond or a linker and R.sup.1 is the
modifying group. Exemplary bonds are those that are formed between
a reactive group on the glycosyl moiety, e.g., NH.sub.2, SH, or OH,
and a group of complementary reactivity on the modifying group.
Thus, exemplary bonds include, but are not limited to NHR.sup.1,
OR.sup.1, SR.sup.1 and the like. For example, when R.sup.1 includes
a carboxylic acid moiety, this moiety may be activated and coupled
with an NH.sub.2 moiety on the glycosyl residue affording a bond
having the structure NHC(O)R.sup.1. Similarly, the OH and SH groups
can be converted to the corresponding ether or thioether
derivatives, respectively.
[0121] Exemplary linkers include alkyl and heteroalkyl moieties.
The linkers include linking groups, for example acyl-based linking
groups, e.g., --C(O)NH--, --OC(O)NH--, and the like. The linking
groups are bonds formed between components of the species of the
invention, e.g., between the glycosyl moiety and the linker (L), or
between the linker and the modifying group (R.sup.1). Other 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.
[0122] Another exemplary linker is a PEG moiety or a PEG moiety
that is functionalized with an amino acid residue. The PEG is to
the glycosyl group through the amino acid residue at one PEG
terminus and bound to R.sup.1 through the other PEG terminus.
Alternatively, the amino acid residue is bound to R.sup.1 and the
PEG terminus not bound to the amino acid is bound to the glycosyl
group.
[0123] An exemplary species for L-R.sup.1 has the formula:
--NH{C(O)(CH.sub.2).sub.aNH}.sub.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 the --NH
moiety is replaced by, for example, --S, --O and --CH.sub.2.
[0124] More particularly, the invention provides compounds in which
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 2500.
[0125] In an illustrative embodiment, G is sialic acid and selected
compounds of the invention have the formulae: ##STR20## As those of
skill in the art will appreciate, the sialic acid moiety in the
exemplary compounds above can be replaced with any other
amino-saccharide including, but not limited to, glucosamine,
galactosamine, mannosamine, their N-acetyl derivatives, and the
like.
[0126] 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: ##STR21##
[0127] In a further exemplary embodiment, the invention provides
modified sugars in which the 6-hydroxyl position is converted to
the corresponding amine moiety, which bears a linker-modifying
group cassette such as those set forth above. Exemplary saccharyl
groups that can be used as the core of these modified sugars
include Gal, GalNAc, Glc, GlcNAc, Fuc, Xyl, Man, and the like. A
representative modified sugar according to this embodiment has the
formula: ##STR22## in which R.sup.3-R.sup.5 and R.sup.7 are members
independently selected from H, OH, C(O)CH.sub.3, NH, and
NHC(O)CH.sub.3. R.sup.6 is OR.sup.1, NHR.sup.1 or L-R.sup.1, which
is as described above.
[0128] Selected conjugates of the invention are based on mannose,
galactose or glucose, or on species having the stereochemistry of
mannose, galactose or glucose. The general formulae of these
conjugates are: ##STR23##
[0129] In another exemplary embodiment, the invention provides
compounds as set forth above that are activated as the
corresponding nucleotide sugars. Exemplary sugar nucleotides that
are used in the present invention in their modified form include
nucleotide mono-, di- or triphosphates or analogs thereof. In a
preferred embodiment, the modified sugar nucleotide is selected
from a UDP-glycoside, CMP-glycoside, or a GDP-glycoside. Even more
preferably, the sugar nucleotide portion of the modified sugar
nucleotide is selected from UDP-galactose, UDP-galactosamine,
UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic
acid, or CMP-NeuAc. In an exemplary embodiment, the nucleotide
phosphate is attached to C-1.
[0130] Thus, in an illustrative embodiment in which the glycosyl
moiety is sialic acid, the invention provides compounds having the
formulae: ##STR24## 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. Exemplary modified sugar
nucleotide compounds according to these embodiments are set forth
in FIG. 1 and FIG. 2.
[0131] In another exemplary embodiment, the invention provides a
conjugate formed between a modified sugar of the invention and a
substrate, e.g., a peptide, lipid, aglycone, etc., more
particularly between a modified sugar and a glycosyl residue of a
glycopeptide or a glycolipid. In this embodiment, the sugar moiety
of the modified sugar becomes a glycosyl linking group interposed
between the substrate and the modifying group. An exemplary
glycosyl linking group is an intact glycosyl linking group, in
which the glycosyl moiety or moieties forming the linking group are
not degraded by chemical (e.g., sodium metaperiodate) or enzymatic
processes (e.g., oxidase). Selected conjugates of the invention
include a modifying group that is attached to the amine moiety of
an amino-saccharide, e.g., mannosamine, glucosamine, galactosamine,
sialic acid etc. Exemplary modifying group-intact glycosyl linking
group cassette according to this motif is based on a sialic acid
structure, such as that having the formulae: ##STR25## In the
formulae above, R.sup.1, L.sup.1 and L.sup.2 are as described
above.
[0132] In still a further exemplary embodiment, the conjugate is
formed between a substrate and the 1-position of a saccharyl moiety
that in which the modifying group is attached through a linker at
the 6-carbon position of the saccharyl moiety. Thus, illustrative
conjugates according to this embodiment have the formulae:
##STR26## in which the radicals are as discussed above. Those of
skill will appreciate that the modified saccharyl moieties set
forth above can also be conjugated to a substrate at the 2, 3, 4,
or 5 carbon atoms.
[0133] Illustrative compounds according to this embodiment include
compounds having the formulae: ##STR27## in which the R groups and
the indices are as described above.
[0134] The invention also provides sugar nucleotides modified with
L-R.sup.1 at the 6-carbon position. Exemplary species according to
this embodiment include: ##STR28## in which the R groups, and L,
represent moieties as discussed above. The index "y" is 0, 1 or
2.
[0135] A further exemplary nucleotide sugar of the invention, based
on a species having the stereochemistry of GDP mannose. An
exemplary species according to this embodiment has the structure:
##STR29##
[0136] In a still further exemplary embodiment, the invention
provides a conjugate, based on the stereochemistry of UDP
galactose. An exemplary compound according to this embodiment has
the structure: ##STR30##
[0137] In another exemplary embodiment, the nucleotide sugar is
based on the stereochemistry of glucose. Exemplary species
according to this embodiment have the formulae: ##STR31##
[0138] The modifying group, R.sup.1, is any of a number of species
including, but not limited to, water-soluble polymers,
water-insoluble polymers, therapeutic agents, diagnostic agents and
the like. The nature of exemplary modifying groups is discussed in
greater detail hereinbelow.
Modifying Groups
Water-Soluble Polymers
[0139] 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.
[0140] 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)).
[0141] Preferred water-soluble polymers are those in which a
substantial proportion of the polymer molecules in a sample of the
polymer are of approximately the same molecular weight; such
polymers are "homodisperse."
[0142] 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).
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] Exemplary poly(ethylene glycol) molecules of use in the
invention include, but are not limited to, those having the
formula: ##STR32## in which R.sup.8 is H, OH, NH.sub.2, substituted
or unsubstituted alkyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted
heteroalkyl, e.g., acetal, OHC--, H.sub.2N--(CH.sub.2).sub.q--,
HS--(CH.sub.2).sub.q, or --(CH.sub.2).sub.qC(Y)Z.sup.1. The index
"e" represents an integer from 1 to 2500. The indices b, d, and q
independently represent integers from 0 to 20. The symbols Z and Z'
independently represent OH, NH.sub.2, leaving groups, e.g.,
imidazole, p-nitrophenyl, HOBT, tetrazole, halide, S--R.sup.9, the
alcohol portion of activated esters; --(CH.sub.2).sub.pC(Y.sup.1)V,
or --(CH.sub.2).sub.pU(CH.sub.2).sub.sC(Y.sup.1).sub.v. The symbol
Y represents H(2), .dbd.O, .dbd.S, .dbd.N--R.sup.10. The symbols X,
Y, Y.sup.1, A.sup.1, and U independently represent the moieties O,
S, N--R.sup.11. The symbol V represents OH, NH.sub.2, halogen,
S--R.sup.12, the alcohol component of activated esters, the amine
component of activated amides, sugar-nucleotides, and proteins. The
indices p, q, s and v are members independently selected from the
integers from 0 to 20. The symbols R.sup.9, R.sup.10, R.sup.11 and
R.sup.12 independently represent H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heterocycloalkyl
and substituted or unsubstituted heteroaryl.
[0148] In other exemplary embodiments, the poly(ethylene glycol)
molecule is selected from the following: ##STR33##
[0149] The poly(ethylene glycol) useful in forming the conjugate of
the invention is either linear or branched. Branched poly(ethylene
glycol) molecules suitable for use in the invention include, but
are not limited to, those described by the following formula:
##STR34## in which R.sup.8 and R.sup.8' are members independently
selected from the groups defined for R.sup.8, above. A.sup.1 and
A.sup.2 are members independently selected from the groups defined
for A.sup.1, above. The indices e, f, o, and q are as described
above. Z and Y are as described above. X.sup.1 and X.sup.1' are
members independently selected from S, SC(O)NH, HNC(O)S, SC(O)O, O,
NH, NHC(O), (O)CNH and NHC(O)O, OC(O)NH.
[0150] In other exemplary embodiments, the branched PEG is based
upon a cysteine, serine or di-lysine core. Thus, further exemplary
branched PEGs include: ##STR35##
[0151] In yet another embodiment, the branched PEG moiety is based
upon a tri-lysine peptide. The tri-lysine can be mono-, di-, tri-,
or tetra-PEG-ylated. Exemplary species according to this embodiment
have the formulae: ##STR36## in which e, f and f' are independently
selected integers from 1 to 2500; and q, q' and q'' are
independently selected integers from 1 to 20.
[0152] In exemplary embodiments of the invention, the PEG is m-PEG
(5 kD, 10 kD, or 20 kD). An exemplary branched PEG species is a
serine- or cysteine-(m-PEG).sub.2 in which the m-PEG is a 20 kD
m-PEG.
[0153] As will be apparent to those of skill, the branched polymers
of use in the invention include variations on the themes set forth
above. For example the di-lysine-PEG conjugate shown above can
include three polymeric subunits, the third bonded to the
.alpha.-amine shown as unmodified in the structure above.
Similarly, the use of a tri-lysine functionalized with three or
four polymeric subunits is within the scope of the invention.
[0154] Specific embodiments according to the invention include:
##STR37## and carbonates and active esters of these species, such
as: ##STR38##
[0155] 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: ##STR39## PEG molecules that are activated with these and
other species and methods of making the activated PEGs are set
forth in WO 04/083259.
[0156] Those of skill in the art will appreciate that one or more
of the m-PEG arms of the branched polymer can be replaced by a PEG
moiety with a different terminus, e.g., OH, COOH, NH.sub.2,
C.sub.2-C.sub.10-alkyl, etc. Moreover, the structures above are
readily modified by inserting alkyl linkers (or removing carbon
atoms) between the .alpha.-carbon atom and the functional group of
the 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.
[0157] The branched PEG species set forth herein are readily
prepared by methods such as that set forth in the scheme below:
##STR40## in which X.sup.a 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.
[0158] 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.a. 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.
[0159] In an exemplary embodiment, the modifying group is a PEG
moiety, however, any modifying group, e.g., water-soluble polymer,
water-insoluble polymer, therapeutic moiety, etc., can be
incorporated in a glycosyl moiety through an appropriate linkage.
The modified sugar is formed by enzymatic means, chemical means or
a combination thereof, thereby producing a modified sugar. In an
exemplary embodiment, the sugars are substituted with an active
amine at any position that allows for the attachment of the
modifying moiety, yet still allows the sugar to function as a
substrate for an enzyme capable of coupling the modified sugar to
the peptide. In an exemplary embodiment, when galactosamine is the
modified sugar, the amine moiety is attached to the carbon atom at
the 6-position.
[0160] The present invention also provides nucleotide sugars in
which the sugar moiety is modified. An exemplary modified sugar
nucleotide bears a sugar group that is modified through an amine
moiety on the sugar. Modified sugar nucleotides, e.g.,
saccharyl-amine derivatives of a sugar nucleotide, are also of use
in the methods of the invention. For example, a saccharyl amine
(without the modifying group) can be enzymatically conjugated to a
peptide (or other species) and the free saccharyl amine moiety
subsequently conjugated to a desired modifying group.
Alternatively, the modified sugar nucleotide can function as a
substrate for an enzyme that transfers the modified sugar to a
saccharyl acceptor on a substrate, e.g., a peptide, glycopeptide,
lipid, aglycone, glycolipid, etc.
[0161] In one embodiment in which the saccharide core is galactose
or glucose, R.sup.5 is NHC(O)Y.
[0162] In an exemplary embodiment, the modified sugar is based upon
a 6-amino-N-acetyl-glycosyl moiety. As shown below for
N-acetylgalactosamine, the 6-amino-sugar moiety is readily prepared
by standard methods. ##STR41##
[0163] In the scheme above, the index n represents an integer from
1 to 2500, preferably from 10 to 1500, and more preferably from 10
to 1200. The symbol "A" represents an activating group, e.g., a
halo, a component of an activated ester (e.g., a
N-hydroxysuccinimide ester), a component of a carbonate (e.g.,
p-nitrophenyl carbonate) and the like. Those of skill in the art
will appreciate that other PEG-amide nucleotide sugars are readily
prepared by this and analogous methods.
[0164] In other exemplary embodiments, the amide moiety is replaced
by a group such as a urethane or a urea.
[0165] In still further embodiments, R.sup.1 is a branched PEG, for
example, one of those species set forth above. Illustrative
compounds according to this embodiment include: ##STR42## in which
X.sup.4 is a bond or O.
[0166] Moreover, as discussed above, the present invention provides
nucleotide sugars that are modified with a water-soluble polymer,
which is either straight-chain or branched. For example, compounds
having the formula shown below are within the scope of the present
invention: ##STR43## in which X.sup.4 is O or a bond.
[0167] Similarly, the invention provides nucleotide sugars of those
modified sugar species in which the carbon at the 6-position is
modified: ##STR44## in which X.sup.4 is a bond or O.
[0168] Also provided are conjugates of peptides and glycopeptides,
lipids and glycolipids that include the compositions of the
invention. For example, the invention provides conjugates having
the following formulae: ##STR45##
Water-Insoluble Polymers
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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).
[0179] 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(phosphazenes), poly(phosphoesters), poly(thioesters),
polysaccharides and mixtures thereof. More preferably still, the
bioresorbable polymer includes a poly(hydroxy) acid component. Of
the poly(hydroxy) acids, polylactic acid, polyglycolic acid,
polycaproic acid, polybutyric acid, polyvaleric acid and copolymers
and mixtures thereof are preferred.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] Bio-compatible hydrogel compositions whose integrity can be
controlled through crosslinking are known and are presently
preferred for use in the methods of the invention. For example,
Hubbell et al., U.S. Pat. Nos. 5,410,016, which issued on Apr. 25,
1995 and 5,529,914, which issued on Jun. 25, 1996, disclose
water-soluble systems, which are crosslinked block copolymers
having a water-soluble central block segment sandwiched between two
hydrolytically labile extensions. Such copolymers are further
end-capped with photopolymerizable acrylate functionalities. When
crosslinked, these systems become hydrogels. The water soluble
central block of such copolymers can include poly(ethylene glycol);
whereas, the hydrolytically labile extensions can be a
poly(.alpha.-hydroxy acid), such as polyglycolic acid or polylactic
acid. See, Sawhney et al., Macromolecules 26: 581-587 (1993).
[0187] In another preferred embodiment, the gel is a
thermoreversible gel. Thermoreversible gels including components,
such as pluronics, collagen, gelatin, hyaluronic acid,
polysaccharides, polyurethane hydrogel, polyurethane-urea hydrogel
and combinations thereof are presently preferred.
[0188] 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, which issued on Jun. 11, 1985. For example, liposome
formulations may be prepared by dissolving appropriate lipid(s)
(such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl
choline, arachidoyl 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.
[0189] 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,
are of use in the present invention.
[0190] The structural formats discussed above in the context of the
water-soluble polymers, both straight-chain and branched are
generally applicable with respect to the water-insoluble polymers
as well. Thus, for example, the cysteine, serine, dilysine, and
trilysine branching cores can be functionalized with two
water-insoluble polymer moieties. The methods used to produce these
species are generally closely analogous to those used to produce
the water-soluble polymers.
The Methods
[0191] 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.
[0192] Thus, the invention provides a method of forming a covalent
conjugate between a selected moiety and a peptide, a glycolipid or
an aglycone (e.g., ceramide or sphingosine). For clarity of
illustration, the invention is illustrated with reference to a
conjugate formed between a peptide and the modified glycosyl moiety
of an activated modified sugar of the invention. Those of skill
will appreciate that the invention equally encompasses methods of
forming conjugates of glycolipids, and aglycones with an activated
modified sugar of the invention.
[0193] In exemplary embodiments, the conjugate is formed between a
water-soluble polymer, a therapeutic moiety, targeting moiety or a
biomolecule, and a glycosylated or non-glycosylated peptide. The
polymer, therapeutic moiety or biomolecule is conjugated to the
peptide via a glycosyl linking group, which is interposed between,
and covalently linked to both the peptide and the modifying group
(e.g., water-soluble polymer). The method includes contacting the
peptide with a mixture containing a modified sugar and an enzyme,
e.g., a glycosyltransferase, that conjugates the modified sugar to
the substrate (e.g., peptide, aglycone, glycolipid). The reaction
is conducted under conditions appropriate to form a covalent bond
between the modified sugar and the peptide (or other substrate).
The sugar moiety of the modified sugar is preferably selected from
nucleotide sugars.
[0194] The acceptor peptide is typically synthesized de novo, or
recombinantly expressed in a prokaryotic cell (e.g., bacterial
cell, such as E. coli) or in a eukaryotic cell such as a mammalian,
yeast, insect, fungal or plant cell. The peptide can be either a
full-length protein or a fragment. Moreover, the peptide can be a
wild type or mutated peptide. In an exemplary embodiment, the
peptide includes a mutation that adds one or more N- or O-linked
glycosylation sites to the peptide sequence.
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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).
[0199] 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.
[0200] 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.
[0201] 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.
[0202] The present invention also provides means of adding (or
removing) one or more selected glycosyl residues to a peptide,
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.
[0203] Addition or removal of any carbohydrate moieties present on
the glycopeptide is accomplished either chemically or
enzymatically. Chemical deglycosylation is preferably 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).
[0204] Chemical addition of glycosyl moieties is carried out by any
art-recognized method. Enzymatic addition of sugar moieties is
preferably achieved using a modification of the methods set forth
herein, substituting native glycosyl units for the modified sugars
used in the invention. Other methods of adding sugar moieties are
disclosed in U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and
5,922,577.
[0205] 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).
[0206] 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).
[0207] 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.
[0208] 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
glycosylunits, 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.
Preparation of Modified Sugars
[0209] 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.
[0210] Useful reactive functional groups pendent from a sugar
nucleus or modifying group include, but are not limited to: [0211]
(a) carboxyl groups and various derivatives thereof including, but
not limited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazole
esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl
esters, alkyl, alkenyl, alkynyl and aromatic esters; [0212] (b)
hydroxyl groups, which can be converted to, e.g., esters, ethers,
aldehydes, etc. [0213] (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; [0214] (d)
dienophile groups, which are capable of participating in
Diels-Alder reactions such as, for example, maleimido groups;
[0215] (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; [0216] (f) sulfonyl halide groups for subsequent reaction
with amines, for example, to form sulfonamides; [0217] (g) thiol
groups, which can be, for example, converted to disulfides or
reacted with acyl halides; [0218] (h) amine or sulfhydryl groups,
which can be, for example, acylated, alkylated or oxidized; [0219]
(i) alkenes, which can undergo, for example, cycloadditions,
acylation, Michael addition, etc; and [0220] (j) epoxides, which
can react with, for example, amines and hydroxyl compounds.
[0221] 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.
[0222] 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)).
[0223] 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.
[0224] In Scheme 1, the amino glycoside 1, is treated with the
active ester of a protected amino acid (e.g., glycine) derivative,
converting the sugar amine residue into the corresponding protected
amino acid amide adduct. The adduct is treated with an aldolase to
form .alpha.-hydroxy carboxylate 2. Compound 2 is converted to the
corresponding CMP derivative by the action of CMP-SA synthetase,
followed by catalytic hydrogenation of the CMP derivative to
produce compound 3. The amine introduced via formation of the
glycine adduct is utilized as a locus of PEG attachment by reacting
compound 3 with an activated PEG or PPG derivative (e.g.,
PEG-C(O)NHS, PEG-OC(O)O-p-nitrophenyl), producing species such as 4
or 5, respectively. ##STR46##
[0225] Table 1 sets forth representative examples of sugar
monophosphates that are derivatized with a PEG moiety. Certain of
the compounds of Table 1 are prepared by the method of Scheme 1.
Other derivatives are prepared by art-recognized methods. See, for
example, Keppler et al., Glycobiology 11: 11R (2001); and Charter
et al., Glycobiology 10: 1049 (2000)). Other amine reactive PEG and
PPG analogues are commercially available, or they can be prepared
by methods readily accessible to those of skill in the art.
TABLE-US-00003 TABLE 1 ##STR47## ##STR48## ##STR49## ##STR50##
##STR51## ##STR52## ##STR53## ##STR54## ##STR55## ##STR56##
[0226] The modified sugar phosphates of use in practicing the
present invention can be substituted in other positions as well as
those set forth above. Presently preferred substitutions of sialic
acid are set forth in Formula II: ##STR57## in which X is a linking
group, which is preferably selected from --O--, --N(H)--, --S,
CH.sub.2--, and --N(R).sub.2, in which each R is a member
independently selected from R.sup.1-R.sup.5. The symbols Y, Z, A
and B each represent a group that is selected from the group set
forth above for the identity of X. X, Y, Z, A and B are each
independently selected and, therefore, they can be the same or
different. The symbols R.sup.1, R.sup.2, R.sup.3, R.sup.4 and
R.sup.5 represent H, a PEG moiety, therapeutic moiety, biomolecule
or other moiety. Alternatively, these symbols represent a linker
that is bound to a PEG moiety, therapeutic moiety, biomolecule or
other moiety.
[0227] Exemplary moieties attached to the conjugates disclosed
herein include, but are not limited to, PEG derivatives (e.g.,
acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl-PEG, aryl-PEG),
PPG derivatives (e.g., acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPG
carbamoyl-PPG, aryl-PPG), therapeutic moieties, diagnostic
moieties, mannose-6-phosphate, heparin, heparan, SLe.sub.x,
mannose, mannose-6-phosphate, Sialyl Lewis X, FGF, VFGF, proteins,
chondroitin, keratan, dermatan, albumin, integrins, antennary
oligosaccharides, peptides and the like. Methods of conjugating the
various modifying groups to a saccharide moiety are readily
accessible to those of skill in the art (POLY (ETHYLENE GLYCOL
CHEMISTRY: BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, J. Milton
Harris, Ed., Plenum Pub. Corp., 1992; POLY (ETHYLENE GLYCOL)
CHEMICAL AND BIOLOGICAL APPLICATIONS, J. Milton Harris, Ed., ACS
Symposium Series No. 680, American Chemical Society, 1997;
Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,
1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY
SYSTEMS, ACS Symposium Series Vol. 469, American Chemical Society,
Washington, D.C. 1991).
Linker Groups (Cross-Linking Groups)
[0228] Preparation of the modified sugar for use in the methods of
the present invention includes attachment of a PEG moiety to a
sugar residue and preferably, forming a stable adduct, which is a
substrate for a glycosyltransferase. Thus, it is often preferred to
use a linker, e.g., one formed by reaction of the PEG and sugar
moiety with a cross-linking agent to conjugate the PEG and the
sugar. Exemplary bifunctional compounds which can be used for
attaching modifying groups to carbohydrate moieties include, but
are not limited to, bifunctional poly(ethyleneglycols), polyamides,
polyethers, polyesters and the like. General approaches for linking
carbohydrates to other molecules are known in the literature. See,
for example, Lee et al., Biochemistry 28: 1856 (1989); Bhatia et
al., Anal. Biochem. 178: 408 (1989); Janda et al., J. Am. Chem.
Soc. 112: 8886 (1990) and Bednarski et al., WO 92/18135. In the
discussion that follows, the reactive groups are treated as benign
on the sugar moiety of the nascent modified sugar. The focus of the
discussion is for clarity of illustration. Those of skill in the
art will appreciate that the discussion is relevant to reactive
groups on the modifying group as well.
[0229] An exemplary strategy involves incorporation of a protected
sulfhydryl onto the sugar using the heterobifunctional crosslinker
SPDP (n-succinimidyl-3-(2-pyridyldithio)propionate and then
deprotecting the sulfhydryl for formation of a disulfide bond with
another sulfhydryl on the modifying group.
[0230] If SPDP detrimentally affects the ability of the modified
sugar to act as a glycosyltransferase substrate, one of an array of
other crosslinkers such as 2-iminothiolane or N-succinimidyl
S-acetylthioacetate (SATA) is used to form a disulfide bond.
2-iminothiolane reacts with primary amines, instantly incorporating
an unprotected sulfhydryl onto the amine-containing molecule. SATA
also reacts with primary amines, but incorporates a protected
sulfhydryl, which is later deacetylated using hydroxylamine to
produce a free sulfhydryl. In each case, the incorporated
sulfhydryl is free to react with other sulfhydryls or protected
sulfhydryl, like SPDP, forming the required disulfide bond.
[0231] The above-described strategy is exemplary, and not limiting,
of linkers of use in the invention. Other crosslinkers are
available that can be used in different strategies for crosslinking
the modifying group to the peptide. For example,
TPCH(S-(2-thiopyridyl)-L-cysteine hydrazide and TPMPH
((S-(2-thiopyridyl)mercapto-propionohydrazide) react with
carbohydrate moieties that have been previously oxidized by mild
periodate treatment, thus forming a hydrazone bond between the
hydrazide portion of the crosslinker and the periodate generated
aldehydes. TPCH and TPMPH introduce a 2-pyridylthione protected
sulfhydryl group onto the sugar, which can be deprotected with DTT
and then subsequently used for conjugation, such as forming
disulfide bonds between components.
[0232] If disulfide bonding is found unsuitable for producing
stable modified sugars, other crosslinkers may be used that
incorporate more stable bonds between components. The
heterobifunctional crosslinkers GMBS
(N-gama-malimidobutyryloxy)succinimide) and SMCC (succinimidyl
4-(N-maleimido-methyl)cyclohexane) react with primary amines, thus
introducing a maleimide group onto the component. The maleimide
group can subsequently react with sulfhydryls on the other
component, which can be introduced by previously mentioned
crosslinkers, thus forming a stable thioether bond between the
components. If steric hindrance between components interferes with
either component's activity or the ability of the modified sugar to
act as a glycosyltransferase substrate, crosslinkers can be used
which introduce long spacer arms between components and include
derivatives of some of the previously mentioned crosslinkers (i.e.,
SPDP). Thus, there is an abundance of suitable crosslinkers, which
are useful; each of which is selected depending on the effects it
has on optimal peptide conjugate and modified sugar production.
[0233] A variety of reagents are used to modify the components of
the modified sugar with intramolecular chemical crosslinks (for
reviews of crosslinking reagents and crosslinking procedures see:
Wold, F., Meth. Enzymol. 25: 623-651, 1972; Weetall, H. H., and
Cooney, D. A., In: ENZYMES AS DRUGS. (Holcenberg, and Roberts,
eds.) pp. 395-442, Wiley, New York, 1981; Ji, T. H., Meth. Enzymol.
91: 580-609, 1983; Mattson et al., Mol. Biol. Rep. 17: 167-183,
1993, all of which are incorporated herein by reference). Preferred
crosslinking reagents are derived from various zero-length,
homo-bifunctional, and hetero-bifunctional crosslinking reagents.
Zero-length crosslinking reagents include direct conjugation of two
intrinsic chemical groups with no introduction of extrinsic
material. Agents that catalyze formation of a disulfide bond belong
to this category. Another example is reagents that induce
condensation of a carboxyl and a primary amino group to form an
amide bond such as carbodiimides, ethylchloroformate, Woodward's
reagent K (2-ethyl-5-phenylisoxazolium-3'-sulfonate), and
carbonyldiimidazole. In addition to these chemical reagents, the
enzyme transglutaminase (glutamyl-peptide
.gamma.-glutamyltransferase; EC 2.3.2.13) may be used as
zero-length crosslinking reagent. This enzyme catalyzes acyl
transfer reactions at carboxamide groups of protein-bound
glutaminyl residues, usually with a primary amino group as
substrate. Preferred homo- and hetero-bifunctional reagents contain
two identical or two dissimilar sites, respectively, which may be
reactive for amino, sulfhydryl, guanidino, indole, or nonspecific
groups.
[0234] i. Preferred Specific Sites in Crosslinking Reagents
[0235] 1. Amino-Reactive Groups
[0236] In one preferred embodiment, the sites on the cross-linker
are amino-reactive groups. Useful non-limiting examples of
amino-reactive groups include N-hydroxysuccinimide (NHS) esters,
imidoesters, isocyanates, acylhalides, arylazides, p-nitrophenyl
esters, aldehydes, and sulfonyl chlorides.
[0237] NHS esters react preferentially with the primary (including
aromatic) amino groups of a modified sugar component. The imidazole
groups of histidines are known to compete with primary amines for
reaction, but the reaction products are unstable and readily
hydrolyzed. The reaction involves the nucleophilic attack of an
amine on the acid carboxyl of an NHS ester to form an amide,
releasing the N-hydroxysuccinimide. Thus, the positive charge of
the original amino group is lost.
[0238] Imidoesters are the most specific acylating reagents for
reaction with the amine groups of the modified sugar components. At
a pH between 7 and 10, imidoesters react only with primary amines.
Primary amines attack imidates nucleophilically to produce an
intermediate that breaks down to amidine at high pH or to a new
imidate at low pH. The new imidate can react with another primary
amine, thus crosslinking two amino groups, a case of a putatively
monofunctional imidate reacting bifunctionally. The principal
product of reaction with primary amines is an amidine that is a
stronger base than the original amine. The positive charge of the
original amino group is therefore retained.
[0239] Isocyanates (and isothiocyanates) react with the primary
amines of the modified sugar components to form stable bonds. Their
reactions with sulfhydryl, imidazole, and tyrosyl groups give
relatively unstable products.
[0240] Acylazides are also used as amino-specific reagents in which
nucleophilic amines of the affinity component attack acidic
carboxyl groups under slightly alkaline conditions, e.g. pH
8.5.
[0241] Arylhalides such as 1,5-difluoro-2,4-dinitrobenzene react
preferentially with the amino groups and tyrosine phenolic groups
of modified sugar components, but also with sulfhydryl and
imidazole groups.
[0242] p-Nitrophenyl esters of mono- and dicarboxylic acids are
also useful amino-reactive groups. Although the reagent specificity
is not very high, .alpha.- and .epsilon.-amino groups appear to
react most rapidly.
[0243] Aldehydes such as glutaraldehyde react with primary amines
of modified sugar. Although unstable Schiff bases are formed upon
reaction of the amino groups with the aldehydes of the aldehydes,
glutaraldehyde is capable of modifying the modified sugar with
stable crosslinks. At pH 6-8, the pH of typical crosslinking
conditions, the cyclic polymers undergo a dehydration to form
.alpha.-.beta. unsaturated aldehyde polymers. Schiff bases,
however, are stable, when conjugated to another double bond. The
resonant interaction of both double bonds prevents hydrolysis of
the Schiff linkage. Furthermore, amines at high local
concentrations can attack the ethylenic double bond to form a
stable Michael addition product.
[0244] Aromatic sulfonyl chlorides react with a variety of sites of
the modified sugar components, but reaction with the amino groups
is the most important, resulting in a stable sulfonamide
linkage.
[0245] 2. Sulfhydryl-Reactive Groups
[0246] In another preferred embodiment, the sites are
sulfhydryl-reactive groups. Useful, non-limiting examples of
sulfhydryl-reactive groups include maleimides, alkyl halides,
pyridyl disulfides, and thiophthalimides.
[0247] Maleimides react preferentially with the sulfhydryl group of
the modified sugar components to form stable thioether bonds. They
also react at a much slower rate with primary amino groups and the
imidazole groups of histidines. However, at pH 7 the maleimide
group can be considered a sulfhydryl-specific group, since at this
pH the reaction rate of simple thiols is 1000-fold greater than
that of the corresponding amine.
[0248] Alkyl halides react with sulfhydryl groups, sulfides,
imidazoles, and amino groups. At neutral to slightly alkaline pH,
however, alkyl halides react primarily with sulfhydryl groups to
form stable thioether bonds. At higher pH, reaction with amino
groups is favored.
[0249] Pyridyl disulfides react with free sulfhydryls via disulfide
exchange to give mixed disulfides. As a result, pyridyl disulfides
are the most specific sulfhydryl-reactive groups.
[0250] Thiophthalimides react with free sulfhydryl groups to form
disulfides.
[0251] 3. Carboxyl-Reactive Residue
[0252] In another embodiment, carbodiimides soluble in both water
and organic solvent, are used as carboxyl-reactive reagents. These
compounds react with free carboxyl groups forming a pseudourea that
can then couple to available amines yielding an amide linkage teach
how to modify a carboxyl group with carbodiimide (Yamada et al.,
Biochemistry 20: 4836-4842, 1981).
[0253] ii. Preferred Nonspecific Sites in Crosslinking Reagents
[0254] In addition to the use of site-specific reactive moieties,
the present invention contemplates the use of non-specific reactive
groups to link the sugar to the modifying group.
[0255] Exemplary non-specific cross-linkers include
photoactivatable groups, completely inert in the dark, which are
converted to reactive species upon absorption of a photon of
appropriate energy. In one preferred embodiment, photoactivatable
groups are selected from precursors of nitrenes generated upon
heating or photolysis of azides. Electron-deficient nitrenes are
extremely reactive and can react with a variety of chemical bonds
including N--H, O--H, C--H, and C.dbd.C. Although three types of
azides (aryl, alkyl, and acyl derivatives) may be employed,
arylazides are presently preferred. The reactivity of arylazides
upon photolysis is better with N--H and O--H than C--H bonds.
Electron-deficient arylnitrenes rapidly ring-expand to form
dehydroazepines, which tend to react with nucleophiles, rather than
form C--H insertion products. The reactivity of arylazides can be
increased by the presence of electron-withdrawing substituents such
as nitro or hydroxyl groups in the ring. Such substituents push the
absorption maximum of arylazides to longer wavelength.
Unsubstituted arylazides have an absorption maximum in the range of
260-280 nm, while hydroxy and nitroarylazides absorb significant
light beyond 305 nm. Therefore, hydroxy and nitroarylazides are
most preferable since they allow to employ less harmful photolysis
conditions for the affinity component than unsubstituted
arylazides.
[0256] In another preferred embodiment, photoactivatable groups are
selected from fluorinated arylazides. The photolysis products of
fluorinated arylazides are arylnitrenes, all of which undergo the
characteristic reactions of this group, including C--H bond
insertion, with high efficiency (Keana et al., J. Org. Chem. 55:
3640-3647, 1990).
[0257] In another embodiment, photoactivatable groups are selected
from benzophenone residues. Benzophenone reagents generally give
higher crosslinking yields than arylazide reagents.
[0258] In another embodiment, photoactivatable groups are selected
from diazo compounds, which form an electron-deficient carbene upon
photolysis. These carbenes undergo a variety of reactions including
insertion into C--H bonds, addition to double bonds (including
aromatic systems), hydrogen attraction and coordination to
nucleophilic centers to give carbon ions.
[0259] In still another embodiment, photoactivatable groups are
selected from diazopyruvates. For example, the p-nitrophenyl ester
of p-nitrophenyl diazopyruvate reacts with aliphatic amines to give
diazopyruvic acid amides that undergo ultraviolet photolysis to
form aldehydes. The photolyzed diazopyruvate-modified affinity
component will react like formaldehyde or glutaraldehyde forming
crosslinks.
[0260] iii. Homobifunctional Reagents
[0261] 1. Homobifunctional Crosslinkers Reactive with Primary
Amines
[0262] Synthesis, properties, and applications of amine-reactive
cross-linkers are commercially described in the literature (for
reviews of crosslinking procedures and reagents, see above). Many
reagents are available (e.g., Pierce Chemical Company, Rockford,
Ill.; Sigma Chemical Company, St. Louis, Mo.; Molecular Probes,
Inc., Eugene, Oreg.).
[0263] Preferred, non-limiting examples of homobifunctional NHS
esters include disuccinimidyl glutarate (DSG), disuccinimidyl
suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl
tartarate (DST), disulfosuccinimidyl tartarate (sulfo-DST),
bis-2-(succinimidooxycarbonyloxy)ethylsulfone (BSOCOES),
bis-2-(sulfosuccinimidooxy-carbonyloxy)ethylsulfone
(sulfo-BSOCOES), ethylene glycolbis(succinimidylsuccinate) (EGS),
ethylene glycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS),
dithiobis(succinimidyl-propionate (DSP), and
dithiobis(sulfosuccinimidylpropionate (sulfo-DSP). Preferred,
non-limiting examples of homobifunctional imidoesters include
dimethyl malonimidate (DMM), dimethyl succinimidate (DMSC),
dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl
suberimidate (DMS), dimethyl-3,3'-oxydipropionimidate (DODP),
dimethyl-3,3'-(methylenedioxy)dipropionimidate (DMDP),
dimethyl-3'-(dimethylenedioxy)dipropionimidate (DDDP),
dimethyl-3,3'-(tetramethylenedioxy)-dipropionimidate (DTDP), and
dimethyl-3,3'-dithiobispropionimidate (DTBP).
[0264] Preferred, non-limiting examples of homobifunctional
isothiocyanates include: p-phenylenediisothiocyanate (DITC), and
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS).
[0265] Preferred, non-limiting examples of homobifunctional
isocyanates include xylene-diisocyanate, toluene-2,4-diisocyanate,
toluene-2-isocyanate-4-isothiocyanate,
3-methoxydiphenylmethane-4,4'-diisocyanate,
2,2'-dicarboxy-4,4'-azophenyldiisocyanate, and
hexamethylenediisocyanate.
[0266] Preferred, non-limiting examples of homobifunctional
arylhalides include 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and
4,4'-difluoro-3,3'-dinitrophenyl-sulfone.
[0267] Preferred, non-limiting examples of homobifunctional
aliphatic aldehyde reagents include glyoxal, malondialdehyde, and
glutaraldehyde.
[0268] Preferred, non-limiting examples of homobifunctional
acylating reagents include nitrophenyl esters of dicarboxylic
acids.
[0269] Preferred, non-limiting examples of homobifunctional
aromatic sulfonyl chlorides include phenol-2,4-disulfonyl chloride,
and .alpha.-naphthol-2,4-disulfonyl chloride.
[0270] Preferred, non-limiting examples of additional
amino-reactive homobifunctional reagents include
erythritolbiscarbonate which reacts with amines to give
biscarbamates.
[0271] 2. Homobifunctional Crosslinkers Reactive with Free
Sulfhydryl Groups
[0272] Synthesis, properties, and applications of such reagents are
described in the literature (for reviews of crosslinking procedures
and reagents, see above). Many of the reagents are commercially
available (e.g., Pierce Chemical Company, Rockford, Ill.; Sigma
Chemical Company, St. Louis, Mo.; Molecular Probes, Inc., Eugene,
Oreg.).
[0273] Preferred, non-limiting examples of homobifunctional
maleimides include bismaleimidohexane (BMH), N,N'-(1,3-phenylene)
bismaleimide, N,N'-(1,2-phenylene)bismaleimide,
azophenyldimaleimide, and bis(N-maleimidomethyl)ether.
[0274] Preferred, non-limiting examples of homobifunctional pyridyl
disulfides include 1,4-di-3'-(2'-pyridyldithio)propionamidobutane
(DPDPB).
[0275] Preferred, non-limiting examples of homobifunctional alkyl
halides include 2,2'-dicarboxy-4,4'-diiodoacetamidoazobenzene,
.alpha.,.alpha.'-diiodo-p-xylenesulfonic acid,
.alpha.,.alpha.'-dibromo-p-xylenesulfonic acid,
N,N'-bis(b-bromoethyl)benzylamine,
N,N'-di(bromoacetyl)phenylhydrazine, and
1,2-di(bromoacetyl)amino-3-phenylpropane.
[0276] 3. Homobifunctional Photoactivatable Crosslinkers
[0277] Synthesis, properties, and applications of such reagents are
described in the literature (for reviews of crosslinking procedures
and reagents, see above). Some of the reagents are commercially
available (e.g., Pierce Chemical Company, Rockford, Ill.; Sigma
Chemical Company, St. Louis, Mo.; Molecular Probes, Inc., Eugene,
Oreg.).
[0278] Preferred, non-limiting examples of homobifunctional
photoactivatable crosslinker include
bis-.beta.-(4-azidosalicylamido)ethyldisulfide (BASED),
di-N-(2-nitro-4-azidophenyl)-cystamine-S,S-dioxide (DNCO), and
4,4'-dithiobisphenylazide.
[0279] iv. HeteroBifunctional Reagents
[0280] 1. Amino-Reactive HeteroBifunctional Reagents with a Pyridyl
Disulfide Moiety
[0281] Synthesis, properties, and applications of such reagents are
described in the literature (for reviews of crosslinking procedures
and reagents, see above). Many of the reagents are commercially
available (e.g., Pierce Chemical Company, Rockford, Ill.; Sigma
Chemical Company, St. Louis, Mo.; Molecular Probes, Inc., Eugene,
Oreg.).
[0282] Preferred, non-limiting examples of hetero-bifunctional
reagents with a pyridyl disulfide moiety and an amino-reactive NHS
ester include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl 6-3-(2-pyridyldithio)propionamidohexanoate (LC-SPDP),
sulfosuccinimidyl 6-3-(2-pyridyldithio)propionamidohexanoate
(sulfo-LCSPDP),
4-succinimidyloxycarbonyl-.alpha.-methyl-.alpha.-(2-pyridyldithio)toluene
(SMPT), and sulfosuccinimidyl
6-.alpha.-methyl-.alpha.-(2-pyridyldithio)toluamidohexanoate
(sulfo-LC-SMPT).
[0283] 2. Amino-Reactive HeteroBifunctional Reagents with a
Maleimide Moiety
[0284] Synthesis, properties, and applications of such reagents are
described in the literature. Preferred, non-limiting examples of
hetero-bifunctional reagents with a maleimide moiety and an
amino-reactive NHS ester include succinimidyl maleimidylacetate
(AMAS), succinimidyl 3-maleimidylpropionate (BMPS),
N-.gamma.-maleimidobutyryloxysuccinimide ester
(GMBS)N-.gamma.-maleimidobutyryloxysulfo succinimide ester
(sulfo-GMBS) succinimidyl 6-maleimidylhexanoate (EMCS),
succinimidyl 3-maleimidylbenzoate (SMB),
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS),
succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate
(SMCC), sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC),
succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), and
sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).
[0285] 3. Amino-Reactive HeteroBifunctional Reagents with an Alkyl
Halide Moiety
[0286] Synthesis, properties, and applications of such reagents are
described in the literature Preferred, non-limiting examples of
hetero-bifunctional reagents with an alkyl halide moiety and an
amino-reactive NHS ester include
N-succinimidyl-(4-iodoacetyl)aminobenzoate (SIAB),
sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate (sulfo-SIAB),
succinimidyl-6-(iodoacetyl)aminohexanoate (SIAX),
succinimidyl-6-(6-((iodoacetyl)-amino)hexanoylamino)hexanoate
(SIAXX),
succinimidyl-6-(((4-(iodoacetyl)-amino)-methyl)-cyclohexane-1-carbonyl)am-
inohexanoate (SIACX), and
succinimidyl-4((iodoacetyl)-amino)methylcyclohexane-1-carboxylate
(SIAC).
[0287] A preferred example of a hetero-bifunctional reagent with an
amino-reactive NHS ester and an alkyl dihalide moiety is
N-hydroxysuccinimidyl 2,3-dibromopropionate (SDBP). SDBP introduces
intramolecular crosslinks to the affinity component by conjugating
its amino groups. The reactivity of the dibromopropionyl moiety
towards primary amine groups is controlled by the reaction
temperature (McKenzie et al., Protein Chem. 7: 581-592 (1988)).
[0288] Preferred, non-limiting examples of hetero-bifunctional
reagents with an alkyl halide moiety and an amino-reactive
p-nitrophenyl ester moiety include p-nitrophenyl iodoacetate
(NPIA).
[0289] Other cross-linking agents are known to those of skill in
the art. See, for example, Pomato et al., U.S. Pat. No. 5,965,106.
It is within the abilities of one of skill in the art to choose an
appropriate cross-linking agent for a particular application.
[0290] v. Cleavable Linker Groups
[0291] In yet a further embodiment, the linker group is provided
with a group that can be cleaved to release the modifying group
from the sugar residue. Many cleaveable groups are known in the
art. See, for example, Jung et al., Biochem. Biophys. Acta 761:
152-162 (1983); Joshi et al., J. Biol. Chem. 265: 14518-14525
(1990); Zarling et al., J. Immunol. 124: 913-920 (1980); Bouizar et
al., Eur. J. Biochem. 155: 141-147 (1986); Park et al., J. Biol.
Chem. 261: 205-210 (1986); Browning et al., J. Immunol. 143:
1859-1867 (1989). Moreover a broad range of cleavable, bifunctional
(both homo- and hetero-bifunctional) linker groups is commercially
available from suppliers such as Pierce.
[0292] Exemplary cleaveable moieties can be cleaved using light,
heat or reagents such as thiols, hydroxylamine, bases, periodate
and the like. Moreover, certain preferred groups are cleaved in
vivo in response to being endocytized (e.g., cis-aconityl; see,
Shen et al., Biochem. Biophys. Res. Commun. 102: 1048 (1991)).
Preferred cleaveable groups comprise a cleaveable moiety which is a
member selected from the group consisting of disulfide, ester,
imide, carbonate, nitrobenzyl, phenacyl and benzoin groups.
Conjugation of Modified Sugars to Peptides
[0293] 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.
[0294] A number of methods of using glycosyltransferases to
synthesize desired oligosaccharide structures are known and are
generally applicable to the instant invention. Exemplary methods
are described, for instance, WO 96/32491, Ito et al., Pure Appl.
Chem. 65: 753 (1993), U.S. Pat. Nos. 5,352,670, 5,374,541,
5,545,553, and commonly owned U.S. Pat. Nos. 6,399,336, and
6,440,703 which are incorporated herein by reference.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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 yet further embodiments, the GlcNAc residue
on the glycosyl donor molecule is modified. For example, the GlcNAc
residue may comprise a 1,2 oxazoline moiety.
[0299] 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.
[0300] 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 30.degree. C. In another exemplary
embodiment, one or more components of the present method are
conducted at an elevated temperature using a thermophilic
enzyme.
[0301] The reaction mixture is maintained for a period of time
sufficient for the acceptor to be glycosylated, thereby forming the
desired conjugate. Some of the conjugate can often be detected
after a few hours, with recoverable amounts usually being obtained
within 24 hours or less. Those of skill in the art understand that
the rate of reaction is dependent on a number of variable factors
(e.g, enzyme concentration, donor concentration, acceptor
concentration, temperature, solvent volume), which are optimized
for a selected system.
[0302] 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.
[0303] 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.
[0304] 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.
[0305] An acceptor for the sialyltransferase is present on the
peptide to be modified by the methods of the present invention
either as a naturally occurring structure or one placed there
recombinantly, enzymatically or chemically. Suitable acceptors,
include, for example, galactosyl acceptors such as
Gal.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)).
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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.
[0310] 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.
[0311] 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.
[0312] In yet a further example, a PEG moiety is added onto a Gal
residue using a modified sialic acid.
[0313] 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.
[0314] 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.
[0315] High mannose may also be trimmed back to the first GlcNAc
attached to the Asn of the peptide. In one example, the GlcNAc of
the GlcNAc-(Fuc).sub.a residue is conjugated with ha GlcNAc bearing
a water soluble polymer. In another example, the GlcNAc of the
GlcNAc-(Fuc).sub.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.
[0316] 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.
[0317] 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.
[0318] 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. The approach is summarized in Scheme 2. ##STR58##
[0319] In yet a further approach, summarized in Scheme 3, 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 erythropoietin. 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. ##STR59##
[0320] Any modified sugar can be used with its appropriate
glycosyltransferase, depending on the terminal sugars of the
oligosaccharide side chains of the glycopeptide (Table 2). 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). TABLE-US-00004 TABLE
2 ##STR60## ##STR61## ##STR62## ##STR63## ##STR64## ##STR65## X =
O, NH, S, CH.sub.2, N--(R.sub.1-5).sub.2. Y = X; Z = X; A = X; B =
X. Q = H.sub.2, O, S, NH, N--R. R, R.sub.1-4 = H, Linker-M, M. M =
PEG, e.g., m-PEG
[0321] In a further exemplary embodiment, UDP-galactose-PEG is
reacted with bovine milk .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.
[0322] In another exemplary embodiment, a GlcNAc transferase, such
as GNT1-5, is utilized to transfer PEGylated-GlcN 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.
[0323] 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. This exemplary
embodiment is set forth in Scheme 4. 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.
##STR66##
[0324] 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.
[0325] i. Enzymes
Sugar Transfer
[0326] In addition to the enzymes discussed above in the context of
forming the acyl-linked conjugate, the glycosylation pattern of the
conjugate and the starting substrates (e.g., peptides, lipids) can
be elaborated, trimmed back or otherwise modified by methods
utilizing other enzymes. The methods of remodeling peptides and
lipids using enzymes that transfer a sugar donor to an acceptor are
discussed in great detail in DeFrees, WO 03/031464 A2, published
Apr. 17, 2003. A brief summary of selected enzymes of use in the
present method is set forth below.
Glycosyltransferases
[0327] Glycosyltransferases catalyze the addition of activated
sugars (donor NDP-sugars), in a step-wise fashion, to a protein,
glycopeptide, lipid or glycolipid or to the non-reducing end of a
growing oligosaccharide. N-linked glycopeptides are synthesized via
a transferase and a lipid-linked oligosaccharide donor
Dol-PP-NAG.sub.2Glc.sub.3Man.sub.9 in an en block transfer followed
by trimming of the core. In this case the nature of the "core"
saccharide is somewhat different from subsequent attachments. A
very large number of glycosyltransferases are known in the art.
[0328] The glycosyltransferase to be used in the present invention
may be any as long as it can utilize the modified sugar as a sugar
donor. Examples of such enzymes include Leloir pathway
glycosyltransferase, such as galactosyltransferase,
N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase,
fucosyltransferase, sialyltransferase, mannosyltransferase,
xylosyltransferase, glucurononyltransferase and the like.
[0329] For enzymatic saccharide syntheses that involve
glycosyltransferase reactions, glycosyltransferase can be cloned,
or isolated from any source. Many cloned glycosyltransferases are
known, as are their polynucleotide sequences. See, e.g., "The WWW
Guide To Cloned Glycosyltransferases,"
(http://www.vei.co.uk/TGN/gt_guide.htm). Glycosyltransferase amino
acid sequences and nucleotide sequences encoding
glycosyltransferases from which the amino acid sequences can be
deduced are also found in various publicly available databases,
including GenBank, Swiss-Prot, EMBL, and others.
[0330] Glycosyltransferases that can be employed in the methods of
the invention include, but are not limited to,
galactosyltransferases, fucosyltransferases, glucosyltransferases,
N-acetylgalactosaminyltransferases,
N-acetylglucosaminyltransferases, glucuronyltransferases,
sialyltransferases, mannosyltransferases, glucuronic acid
transferases, galacturonic acid transferases, and
oligosaccharyltransferases. Suitable glycosyltransferases include
those obtained from eukaryotes, as well as from prokaryotes.
[0331] DNA encoding glycosyltransferases may be obtained by
chemical synthesis, by screening reverse transcripts of mRNA from
appropriate cells or cell line cultures, by screening genomic
libraries from appropriate cells, or by combinations of these
procedures. Screening of mRNA or genomic DNA may be carried out
with oligonucleotide probes generated from the glycosyltransferases
gene sequence. Probes may be labeled with a detectable group such
as a fluorescent group, a radioactive atom or a chemiluminescent
group in accordance with known procedures and used in conventional
hybridization assays. In the alternative, glycosyltransferases gene
sequences may be obtained by use of the polymerase chain reaction
(PCR) procedure, with the PCR oligonucleotide primers being
produced from the glycosyltransferases gene sequence. See, U.S.
Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to
Mullis.
[0332] The glycosyltransferase may be synthesized in host cells
transformed with vectors containing DNA encoding the
glycosyltransferases enzyme. Vectors are used either to amplify DNA
encoding the glycosyltransferases enzyme and/or to express DNA
which encodes the glycosyltransferases enzyme. An expression vector
is a replicable DNA construct in which a DNA sequence encoding the
glycosyltransferases enzyme is operably linked to suitable control
sequences capable of effecting the expression of the
glycosyltransferases enzyme in a suitable host. The need for such
control sequences will vary depending upon the host selected and
the transformation method chosen. Generally, control sequences
include a transcriptional promoter, an optional operator sequence
to control transcription, a sequence encoding suitable mRNA
ribosomal binding sites, and sequences which control the
termination of transcription and translation. Amplification vectors
do not require expression control domains. All that is needed is
the ability to replicate in a host, usually conferred by an origin
of replication, and a selection gene to facilitate recognition of
transformants.
[0333] In an exemplary embodiment, the invention utilizes a
prokaryotic enzyme. Such glycosyltransferases include enzymes
involved in synthesis of lipooligosaccharides (LOS), which are
produced by many gram negative bacteria (Preston et al., Critical
Reviews in Microbiology 23(3): 139-180 (1996)). Such enzymes
include, but are not limited to, the proteins of the rfa operons of
species such as E. coli and Salmonella typhimurium, which include a
.beta.1,6 galactosyltransferase and a .beta.1,3
galactosyltransferase (see, e.g., EMBL Accession Nos. M80599 and
M86935 (E. coli); EMBL Accession No. S56361 (S. typhimurium)), a
glucosyltransferase (Swiss-Prot Accession No. P25740 (E. coli), an
.beta.1,2-glucosyltransferase (rfaJ) (Swiss-Prot Accession No.
P27129 (E. coli) and Swiss-Prot Accession No. P19817 (S.
typhimurium)), and an .beta.1,2-N-acetylglucosaminyltransferase
(rfaK) (EMBL Accession No. U00039 (E. coli). Other
glycosyltransferases for which amino acid sequences are known
include those that are encoded by operons such as rfaB, which have
been characterized in organisms such as Klebsiella pneumoniae, E.
coli, Salmonella typhimurium, Salmonella enterica, Yersinia
enterocolitica, Mycobacterium leprosum, and the rh1 operon of
Pseudomonas aeruginosa.
[0334] Also suitable for use in the present invention are
glycosyltransferases that are involved in producing structures
containing lacto-N-neotetraose,
D-galactosyl-.beta.-1,4-N-acetyl-D-glucosaminyl-.beta.-1,3-D-galactosyl-.-
beta.-1,4-D-glucose, and the P.sup.k blood group trisaccharide
sequence,
D-galactosyl-.alpha.-1,4-D-galactosyl-.beta.0-1,4-D-glucose, which
have been identified in the LOS of the mucosal pathogens Neisseria
gonnorhoeae and N. meningitidis (Scholten et al., J. Med.
Microbiol. 41: 236-243 (1994)). The genes from N. meningitidis and
N. gonorrhoeae that encode the glycosyltransferases involved in the
biosynthesis of these structures have been identified from N.
meningitidis immunotypes L3 and L1 (Jennings et al., Mol.
Microbiol. 18: 729-740 (1995)) and the N. gonorrhoeae mutant F62
(Gotshlich, J. Exp. Med. 180: 2181-2190 (1994)). In N.
meningitidis, a locus consisting of three genes, lgtA, lgtB and lg
E, encodes the glycosyltransferase enzymes required for addition of
the last three of the sugars in the lacto-N-neotetraose chain
(Wakarchuk et al., J. Biol. Chem. 271: 19166-73 (1996)). Recently
the enzymatic activity of the lgtB and lgtA gene product was
demonstrated, providing the first direct evidence for their
proposed glycosyltransferase function (Wakarchuk et al., J. Biol.
Chem. 271(45): 28271-276 (1996)). In N. gonorrhoeae, there are two
additional genes, lgtD which adds .beta.-D-GalNAc to the 3 position
of the terminal galactose of the lacto-N-neotetraose structure and
lgtC which adds a terminal .alpha.-D-Gal to the lactose element of
a truncated LOS, thus creating the P.sup.k blood group antigen
structure (Gotshlich (1994), supra.). In N. meningitidis, a
separate immunotype L1 also expresses the P.sup.k blood group
antigen and has been shown to carry an lgtC gene (Jennings et al.,
(1995), supra.). Neisseria glycosyltransferases and associated
genes are also described in U.S. Pat. No. 5,545,553 (Gotschlich).
Genes for .alpha.1,2-fucosyltransferase and
.alpha.1,3-fucosyltransferase from Helicobacter pylori has also
been characterized (Martin et al., J. Biol. Chem. 272: 21349-21356
(1997)). Also of use in the present invention are the
glycosyltransferases of Campylobacter jejuni (see, for example,
http://afmb.cnrs-mrs.fr/.about.pedro/CAZY/gtf.sub.--42.html).
Fucosyltransferases
[0335] In some embodiments, a glycosyltransferase used in the
method of the invention is a fucosyltransferase.
Fucosyltransferases are known to those of skill in the art.
Exemplary fucosyltransferases include enzymes, which transfer
L-fucose from GDP-fucose to a hydroxy position of an acceptor
sugar. Fucosyltransferases that transfer non-nucleotide sugars to
an acceptor are also of use in the present invention.
[0336] In some embodiments, the acceptor sugar is, for example, the
GlcNAc in a Gal.beta.(1.fwdarw.3,4)GlcNAc.beta.-group in an
oligosaccharide glycoside. Suitable fucosyltransferases for this
reaction include the
Gal.beta.(1.fwdarw.3,4)GlcNAc.beta.1-.alpha.(1.fwdarw.3,4)fucosyltransfer-
ase (FTIII E.C. No. 2.4.1.65), which was first characterized from
human milk (see, Palcic, et al., Carbohydrate Res. 190:1-11 (1989);
Prieels, et al., J. Biol. Chem. 256: 10456-10463 (1981); and Nunez,
et al., Can. J. Chem. 59: 2086-2095 (1981)) and the
Gal.beta.(1.fwdarw.4)GlcNAc.beta.-.alpha.fucosyltransferases (FTIV,
FTV, FTVI) which are found in human serum. FTVII (E.C. No.
2.4.1.65), a sialyl
.alpha.(2.fwdarw.3)Gal.beta.((1.fwdarw.3)GlcNAc.beta.
fucosyltransferase, has also been characterized. A recombinant form
of the
Gal.beta.(1.fwdarw.3,4)GlcNAc.beta.-.alpha.(1.fwdarw.3,4)fucosyltransfera-
se has also been characterized (see, Dumas, et al., Bioorg. Med.
Letters 1: 425-428 (1991) and Kukowska-Latallo, et al., Genes and
Development 4: 1288-1303 (1990)). Other exemplary
fucosyltransferases include, for example, .alpha.1,2
fucosyltransferase (E.C. No. 2.4.1.69). Enzymatic fucosylation can
be carried out by the methods described in Mollicone, et al., Eur.
J. Biochem. 191: 169-176 (1990) or U.S. Pat. No. 5,374,655. Cells
that are used to produce a fucosyltransferase will also include an
enzymatic system for synthesizing GDP-fucose.
Galactosyltransferases
[0337] In another group of embodiments, the glycosyltransferase is
a galactosyltransferase. Exemplary galactosyltransferases include
.alpha.(1,3) galactosyltransferases (E.C. No. 2.4.1.151, see, e.g.,
Dabkowski et al., Transplant Proc. 25:2921 (1993) and Yamamoto et
al. Nature 345: 229-233 (1990), bovine (GenBank j04989, Joziasse et
al., J. Biol. Chem. 264: 14290-14297 (1989)), murine (GenBank
m26925; Larsen et al., Proc. Nat'l. Acad. Sci. USA 86: 8227-8231
(1989)), porcine (GenBank L36152; Strahan et al., Immunogenetics
41: 101-105 (1995)). Another suitable .alpha.1,3
galactosyltransferase is that which is involved in synthesis of the
blood group B antigen (EC 2.4.1.37, Yamamoto et al., J. Biol. Chem.
265: 1146-1151 (1990) (human)). Yet a further exemplary
galactosyltransferase is core Gal-T1.
[0338] Also suitable for use in the methods of the invention are
.beta.(1,4) galactosyltransferases, which include, for example, EC
2.4.1.90 (LacNAc synthetase) and EC 2.4.1.22 (lactose synthetase)
(bovine (D'Agostaro et al., Eur. J. Biochem. 183: 211-217 (1989)),
human (Masri et al., Biochem. Biophys. Res. Commun. 157: 657-663
(1988)), murine (Nakazawa et al., J. Biochem. 104: 165-168 (1988)),
as well as E.C. 2.4.1.38 and the ceramide galactosyltransferase (EC
2.4.1.45, Stahl et al., J. Neurosci. Res. 38: 234-242 (1994)).
Other suitable galactosyltransferases include, for example,
.alpha.1,2 galactosyltransferases (from e.g., Schizosaccharomyces
pombe, Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)).
Sialyltransferases
[0339] Sialyltransferases are another type of glycosyltransferase
that is useful in the recombinant cells and reaction mixtures of
the invention. Cells that produce recombinant sialyltransferases
will also produce CMP-sialic acid, which is a sialic acid donor for
sialyltransferases. Examples of sialyltransferases that are
suitable for use in the present invention include ST3Gal III (e.g.,
a rat or human ST3Gal III), ST3Gal IV, ST3Gal I, ST6Gal 1, ST3Gal
V, ST6Gal 11, ST6GalNAc I, ST6GalNAc II, and ST6GalNAc III (the
sialyltransferase nomenclature used herein is as described in Tsuji
et al., Glycobiology 6: v-xiv (1996)). An exemplary
.alpha.(2,3)sialyltransferase referred to as
.alpha.(2,3)sialyltransferase (EC 2.4.99.6) transfers sialic acid
to the non-reducing terminal Gal of a Gal.beta.1.fwdarw.3Glc
disaccharide or glycoside. See, Van den Eijnden et al., J. Biol.
Chem. 256: 3159 (1981), Weinstein et al., J. Biol. Chem. 257: 13845
(1982) and Wen et al., J. Biol. Chem. 267: 21011 (1992). Another
exemplary .alpha.2,3-sialyltransferase (EC 2.4.99.4) transfers
sialic acid to the non-reducing terminal Gal of the disaccharide or
glycoside. see, Rearick et al., J. Biol. Chem. 254: 4444 (1979) and
Gillespie et al., J. Biol. Chem. 267: 21004 (1992). Further
exemplary enzymes include Gal-.beta.-1,4-GlcNAc .alpha.-2,6
sialyltransferase (See, Kurosawa et al. Eur. J. Biochem. 219:
375-381 (1994)).
[0340] Preferably, for glycosylation of carbohydrates of
glycopeptides the sialyltransferase will be able to transfer sialic
acid to the sequence Gal.beta.1,4GlcNAc-, the most common
penultimate sequence underlying the terminal sialic acid on fully
sialylated carbohydrate structures (see, Table 2). TABLE-US-00005
TABLE 2 Sialyltransferases which use the Gal.beta.1,4GlcNAc
sequence as an acceptor substrate Sialyltrans- ferase Source
Sequence(s) formed Ref. ST6Gal I Mammalian
NeuAc.quadrature.2,6Gal.beta.1,4GlCNAc-- 1 ST3Gal III Mammalian
NeuAc.quadrature.2,3Gal.beta.1,4GlCNAc-- 1
NeuAc.quadrature.2,3Gal.beta.1,3GlCNAc-- STSGal IV Mammalian
NeuAc.quadrature.2,3Gal.beta.1,4GlCNAc-- 1
NeuAc.quadrature.2,3Gal.beta.1,3GlCNAc-- ST6Gal II Mammalian
NeuAc.quadrature.2,6Gal.beta.1,4GlCNA ST6Gal II photobacterium
NeuAc.quadrature.2,6Gal.beta.1,4GlCNAc-- 2 ST3Gal V N. meningitides
NeuAc.quadrature.2,3Gal.beta.1,4GlCNAc-- 3 N. gonorrhoeae 1 Goochee
et al., Bio/Technology 9: 1347-1355 (1991) 2 Yamamoto et al., J.
Biochem. 120: 104-110 (1996) 3 Gilbert et al., J. Biol. Chem. 271:
28271-28276 (1996)
[0341] An example of a sialyltransferase that is useful in the
claimed methods is ST3Gal III, which is also referred to as
.alpha.(2,3)sialyltransferase (EC 2.4.99.6). This enzyme catalyzes
the transfer of sialic acid to the Gal of a Gal.beta.1,3GlcNAc or
Gal.beta.1,4GlcNAc glycoside (see, e.g., Wen et al., J. Biol. Chem.
267: 21011 (1992); Van den Eijnden et al., J. Biol. Chem. 256: 3159
(1991)) and is responsible for sialylation of asparagine-linked
oligosaccharides in glycopeptides. The sialic acid is linked to a
Gal with the formation of an .alpha.-linkage between the two
saccharides. Bonding (linkage) between the saccharides is between
the 2-position of NeuAc and the 3-position of Gal. This particular
enzyme can be isolated from rat liver (Weinstein et al., J. Biol.
Chem. 257: 13845 (1982)); the human cDNA (Sasaki et al. (1993) J.
Biol. Chem. 268: 22782-22787; Kitagawa & Paulson (1994) J.
Biol. Chem. 269: 1394-1401) and genomic (Kitagawa et al. (1996) J.
Biol. Chem. 271: 931-938) DNA sequences are known, facilitating
production of this enzyme by recombinant expression. In a preferred
embodiment, the claimed sialylation methods use a rat ST3Gal
III.
[0342] Other exemplary sialyltransferases of use in the present
invention include those isolated from Campylobacter jejuni,
including the .alpha.(2,3). See, e.g, WO99/49051.
[0343] Sialyltransferases other those listed in Table 2, are also
useful in an economic and efficient large-scale process for
sialylation of commercially important glycopeptides. As a simple
test to find out the utility of these other enzymes, various
amounts of each enzyme (1-100 mU/mg protein) are reacted with
asialo-.alpha..sub.1 AGP (at 1-10 mg/ml) to compare the ability of
the sialyltransferase of interest to sialylate glycopeptides
relative to either bovine ST6Gal I, ST3Gal III or both
sialyltransferases. Alternatively, other glycopeptides or
glycopeptides, or N-linked oligosaccharides enzymatically released
from the peptide backbone can be used in place of
asialo-.alpha..sub.1 AGP for this evaluation. Sialyltransferases
with the ability to sialylate N-linked oligosaccharides of
glycopeptides more efficiently than ST6Gal I are useful in a
practical large-scale process for peptide sialylation.
GalVAc Transferases
[0344] N-acetylgalactosaminyltransferases are of use in practicing
the present invention, particularly for binding a GalNAc moiety to
an amino acid of the O-linked glycosylation site of the peptide.
Suitable N-acetylgalactosaminyltransferases include, but are not
limited to, .alpha.(1,3) N-acetylgalactosaminyltransferase,
.beta.(1,4) N-acetylgalactosaminyltransferases (Nagata et al., J.
Biol. Chem. 267: 12082-12089 (1992) and Smith et al., J. Biol.
Chem. 269: 15162 (1994)) and polypeptide
N-acetylgalactosaminyltransferase (Homa et al., J. Biol. Chem. 268:
12609 (1993)).
[0345] Production of proteins such as the enzyme GalNAc T.sub.I-XX
from cloned genes by genetic engineering is well known. See, eg.,
U.S. Pat. No. 4,761,371. One method involves collection of
sufficient samples, then the amino acid sequence of the enzyme is
determined by N-terminal sequencing. This information is then used
to isolate a cDNA clone encoding a full-length (membrane bound)
transferase which upon expression in the insect cell line Sf9
resulted in the synthesis of a fully active enzyme. The acceptor
specificity of the enzyme is then determined using a
semiquantitative analysis of the amino acids surrounding known
glycosylation sites in 16 different proteins followed by in vitro
glycosylation studies of synthetic peptides. This work has
demonstrated that certain amino acid residues are overrepresented
in glycosylated peptide segments and that residues in specific
positions surrounding glycosylated serine and threonine residues
may have a more marked influence on acceptor efficiency than other
amino acid moieties.
Cell-Bound Glycosyltransferases
[0346] In another embodiment, the enzymes utilized in the method of
the invention are cell-bound glycosyltransferases. Although many
soluble glycosyltransferases are known (see, for example, U.S. Pat.
No. 5,032,519), glycosyltransferases are generally in
membrane-bound form when associated with cells. Many of the
membrane-bound enzymes studied thus far are considered to be
intrinsic proteins; that is, they are not released from the
membranes by sonication and require detergents for solubilization.
Surface glycosyltransferases have been identified on the surfaces
of vertebrate and invertebrate cells, and it has also been
recognized that these surface transferases maintain catalytic
activity under physiological conditions. However, the more
recognized function of cell surface glycosyltransferases is for
intercellular recognition (Roth, MOLECULAR APPROACHES to
SUPRACELLULAR PHENOMENA, 1990).
[0347] Methods have been developed to alter the
glycosyltransferases expressed by cells. For example, Larsen et
al., Proc. Natl. Acad. Sci. USA 86: 8227-8231 (1989), report a
genetic approach to isolate cloned cDNA sequences that determine
expression of cell surface oligosaccharide structures and their
cognate glycosyltransferases. A cDNA library generated from mRNA
isolated from a murine cell line known to express UDP-galactose:
..beta..-D-galactosyl-1,4-N-acetyl-D-glucosaminide
.alpha.-1,3-galactosyltransferase was transfected into COS-1 cells.
The transfected cells were then cultured and assayed for .alpha.1-3
galactosyltransferase activity.
[0348] Francisco et al., Proc. Natl. Acad. Sci. USA 89: 2713-2717
(1992), disclose a method of anchoring .beta.-lactamase to the
external surface of Escherichia coli. A tripartite fusion
consisting of (i) a signal sequence of an outer membrane protein,
(ii) a membrane-spanning section of an outer membrane protein, and
(iii) a complete mature .beta.-lactamase sequence is produced
resulting in an active surface bound .beta.-lactamase molecule.
However, the Francisco method is limited only to procaryotic cell
systems and as recognized by the authors, requires the complete
tripartite fusion for proper functioning.
Sulfotransferases
[0349] The invention also provides methods for producing peptides
that include sulfated molecules, including, for example sulfated
polysaccharides such as heparin, heparan sulfate, carragenen, and
related compounds. Suitable sulfotransferases include, for example,
chondroitin-6-sulphotransferase (chicken cDNA described by Fukuta
et al., J. Biol. Chem. 270: 18575-18580 (1995); GenBank Accession
No. D49915), glycosaminoglycan N-acetylglucosamine
N-deacetylase/N-sulphotransferase 1 (Dixon et al., Genomics 26:
239-241 (1995); UL18918), and glycosaminoglycan N-acetylglucosamine
N-deacetylase/N-sulphotransferase 2 (murine cDNA described in
Orellana et al., J. Biol. Chem. 269: 2270-2276 (1994) and Eriksson
et al., J. Biol. Chem. 269: 10438-10443 (1994); human cDNA
described in GenBank Accession No. U2304).
Glycosidases
[0350] This invention also encompasses the use of wild-type and
mutant glycosidases. Mutant .beta.-galactosidase enzymes have been
demonstrated to catalyze the formation of disaccharides through the
coupling of an .alpha.-glycosyl fluoride to a galactosyl acceptor
molecule. (Withers, U.S. Pat. No. 6,284,494; issued Sep. 4, 2001).
Other glycosidases of use in this invention include, for example,
.beta.-glucosidases, .beta.-galactosidases, .beta.-mannosidases,
.beta.-acetyl glucosaminidases, .beta.-N-acetyl galactosaminidases,
.beta.-xylosidases, .beta.-fucosidases, cellulases, xylanases,
galactanases, mannanases, hemicellulases, amylases, glucoamylases,
.alpha.-glucosidases, .alpha.-galactosidases, .alpha.-mannosidases,
.alpha.-N-acetyl glucosaminidases, .alpha.-N-acetyl
galactose-aminidases, .alpha.-xylosidases, .alpha.-fucosidases, and
neuraminidases/sialidases.
Immobilized Enzymes
[0351] The present invention also provides for the use of enzymes
that are immobilized on a solid and/or soluble support. In an
exemplary embodiment, there is provided a glycosyltransferase that
is conjugated to a PEG via an intact glycosyl linker according to
the methods of the invention. The PEG-linker-enzyme conjugate is
optionally attached to solid support. The use of solid supported
enzymes in the methods of the invention simplifies the work up of
the reaction mixture and purification of the reaction product, and
also enables the facile recovery of the enzyme. The
glycosyltransferase conjugate is utilized in the methods of the
invention. Other combinations of enzymes and supports will be
apparent to those of skill in the art.
Fusion Proteins
[0352] In other exemplary embodiments, the methods of the invention
utilize fusion proteins that have more than one enzymatic activity
that is involved in synthesis of a desired glycopeptide conjugate.
The fusion polypeptides can be composed of, for example, a
catalytically active domain of a glycosyltransferase that is joined
to a catalytically active domain of an accessory enzyme. The
accessory enzyme catalytic domain can, for example, catalyze a step
in the formation of a nucleotide sugar that is a donor for the
glycosyltransferase, or catalyze a reaction involved in a
glycosyltransferase cycle. For example, a polynucleotide that
encodes a glycosyltransferase can be joined, in-frame, to a
polynucleotide that encodes an enzyme involved in nucleotide sugar
synthesis. The resulting fusion protein can then catalyze not only
the synthesis of the nucleotide sugar, but also the transfer of the
sugar moiety to the acceptor molecule. The fusion protein can be
two or more cycle enzymes linked into one expressible nucleotide
sequence. In other embodiments the fusion protein includes the
catalytically active domains of two or more glycosyltransferases.
See, for example, U.S. Pat. No. 5,641,668. The modified
glycopeptides of the present invention can be readily designed and
manufactured utilizing various suitable fusion proteins (see, for
example, PCT Patent Application PCT/CA98/01180, which was published
as WO 99/31224 on Jun. 24, 1999.)
Purification of Erythropoietin Conjugates
[0353] The products produced by the above processes can be used
without purification. However, it is usually preferred to recover
the product. Standard, well-known techniques for recovery of
glycosylated saccharides such as thin or thick layer
chromatography, column chromatography, ion exchange chromatography,
or membrane filtration can be used. It is preferred to use membrane
filtration, more preferably utilizing a reverse osmotic membrane,
or one or more column chromatographic techniques for the recovery
as is discussed hereinafter and in the literature cited herein. For
instance, membrane filtration wherein the membranes have molecular
weight cutoff of about 3000 to about 10,000 can be used to remove
proteins such as glycosyl transferases. Nanofiltration or reverse
osmosis can then be used to remove salts and/or purify the product
saccharides (see, e.g., WO 98/15581). Nanofilter membranes are a
class of reverse osmosis membranes that pass monovalent salts but
retain polyvalent salts and uncharged solutes larger than about 100
to about 2,000 Daltons, depending upon the membrane used. Thus, in
a typical application, saccharides prepared by the methods of the
present invention will be retained in the membrane and
contaminating salts will pass through.
[0354] If the modified glycoprotein is produced intracellularly, as
a first step, the particulate debris, either host cells or lysed
fragments, is removed, for example, by centrifugation or
ultrafiltration; optionally, the protein may be concentrated with a
commercially available protein concentration filter, followed by
separating the polypeptide variant from other impurities by one or
more steps selected from immunoaffinity chromatography,
ion-exchange column fractionation (e.g., on diethylaminoethyl
(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.
[0355] 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.
[0356] A protease inhibitor, e.g., methylsulfonylfluoride (PMSF)
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0357] 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.
[0358] Finally, one or more RP-HPLC steps employing hydrophobic
RP-HPLC media, e.g., silica gel having pendant methyl or other
aliphatic groups, may be employed to further purify a polypeptide
variant composition. Some or all of the foregoing purification
steps, in various combinations, can also be employed to provide a
homogeneous modified glycoprotein.
[0359] The modified glycopeptide of the invention resulting from a
large-scale fermentation may be purified by methods analogous to
those disclosed by Urdal et al., J. Chromatog. 296: 171 (1984).
This reference describes two sequential, RP-HPLC steps for
purification of recombinant human IL-2 on a preparative HPLC
column. Alternatively, techniques such as affinity chromatography
may be utilized to purify the modified glycoprotein.
Pharmaceutical Compositions
[0360] 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.
[0361] 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).
[0362] 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.
[0363] Commonly, the pharmaceutical compositions are administered
parenterally, e.g., intravenously. Thus, the invention provides
compositions for parenteral administration which comprise the
compound dissolved or suspended in an acceptable carrier,
preferably an aqueous carrier, e.g., water, buffered water, saline,
PBS and the like. The compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting agents, wetting agents, detergents and
the like.
[0364] 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.
[0365] 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).
[0366] 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.
[0367] 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.
[0368] 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.
[0369] The active ingredient used in the pharmaceutical
compositions of the present invention is glycopegylated
erythropoietin and its derivatives having the biological properties
of causing bone marrow cells to increase production of
reticulocytes and red blood cells. The liposomal dispersion of the
present invention is useful as a parenteral formulation in treating
blood disorders characterized by low or defective red blood cell
production such as various forms of anemia, including anemias
associated with chronic renal failure, zidovidine treated HIV
infected patients, and cancer patients on chemotherapy. It may also
have application in the treatment of a variety of disease states,
disorders and states of hematologic irregularity such as sickle
cell disease, beta-thalassemia, cystic fibrosis, pregnancy and
menstrual disorders, early anemia of prematurity, spinal cord
injury, space flight, acute blood loss, aging and the like.
Preferably, the EPO composition of the present invention is
administered parenterally (e.g. IV, IM, SC or IP). Effective
dosages are expected to vary considerably depending on the
condition being treated and the route of administration but are
expected to be in the range of about 0.1 (.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 an erythropoietin with an enhanced in vivo
residence time, the stated dosages are optionally lowered when a
composition of the invention is administered.
[0370] The following examples are provided to illustrate the
conjugates, and methods and of the present invention, but not to
limit the claimed invention.
EXAMPLES
Example 1
Preparation of UDP-GalNAc-6'-CHO
[0371] UDP-GalNAc (200 mg, 0.30 mmoles) was dissolved in a 1 mM
CuSO.sub.4 solution (20 mL) and a 25 mM NaH.sub.2PO.sub.4 solution
(pH 6.0; 20 mL). Galactose oxidase (240 U; 240 .mu.L) and catalase
(13000 U; 130 .mu.L) were then added, the reaction system equipped
with a balloon filled with oxygen and stirred at room temperature
for seven days. The reaction mixture was then filtered (spin
cartridge; MWCO 5K) and the filtrate (.about.40 mL) was stored at
4.degree. C. until required. TLC (silica; EtOH/water (7/2);
R.sub.f=0.77; visualized with anisaldehyde stain).
Example 2
Preparation of UDP-GalNAc-6'-NH.sub.2):
[0372] Ammonium acetate (15 mg, 0.194 mmoles) and NaBH.sub.3CN (1M
THF solution; 0.17 mL, 0.17 mmoles) were added to the
UDP-GalNAc-6'-CHO solution from above (2 mL or .about.20 mg) at
0.degree. C. and allowed to warm to room temperature overnight. The
reaction was filtered through a G-10 column with water and the
product collected. The appropriate fractions were freeze-dried and
stored frozen. TLC (silica; ethanol/water (7/2); R.sub.f=0.72;
visualized with ninhydrin reagent).
Example 3
Preparation of UDP-GalNAc-6-NHCO(CH.sub.2).sub.2--O-PEG-OMe (1
KDa).
[0373] The
galactosaminyl-1-phosphate-2-NHCO(CH.sub.2).sub.2--O-PEG-OMe (1
KDa) (58 mg, 0.045 mmoles) was dissolved in DMF (6 mL) and pyridine
(1.2 mL). UMP-morpholidate (60 mg, 0.15 mmoles) was then added and
the resulting mixture stirred at 70.degree. C. for 48 h. The
solvent was removed by bubbling nitrogen through the reaction
mixture and the residue purified by reversed phase chromatography
(C-18 silica, step gradient between 10 to 80%, methanol/water). The
desired fractions were collected and dried at reduced pressure to
yield 50 mg (70%) of a white solid. TLC (silica,
propanol/H.sub.2O/NH.sub.4OH, (30/20/2), R.sub.f=0.54). MS (MALDI):
Observed, 1485, 1529, 1618, 1706.
Example 4
[0374] Preparation of Cysteine-PEG.sub.2 (2) ##STR67##
[0375] 4.1 Synthesis of Compound 1
[0376] Potassium hydroxide (84.2 mg, 1.5 mmol, as a powder) was
added to a solution of L-cysteine (93.7 mg, 0.75 mmol) in anhydrous
methanol (20 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 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 453 mg (44%) of a white solid (1). Structural data for the
compound were as follows: .sup.1H-NMR (500 MHz; D.sub.2O) .delta.
2.83 (t, 2H, O--C--CH.sub.2--S), 3.05 (q, 1H, S--CHH--CHN), 3.18
(q, 1H, (q, 1H, S--CHH--CHN), 3.38 (s, 3H, CH.sub.3O), 3.7 (t,
OCH.sub.2CH.sub.2O), 3.95 (q, 1H, CHN). The purity of the product
was confirmed by SDS PAGE.
[0377] 4.2 Synthesis of Compound 2 (Cysteine-PEG2)
[0378] 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).
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 5
Preparation of UDP-GalNAc-6-NHCO(CH.sub.2).sub.2--O-PEG-OMe (1
KDa).
[0379] Galactosaminyl-1-phosphate-2-NHCO(CH.sub.2).sub.2--O-PEG-OMe
(1 kilodalton) (58 mg, 0.045 mmoles) was dissolved in DMF (6 mL)
and pyridine (1.2 mL). UMP-morpholidate (60 mg, 0.15 mmoles) was
then added and the resulting mixture stirred at 70.degree. C. for
48 h. The solvent was removed by bubbling nitrogen through the
reaction mixture and the residue purified by reversed phase
chromatography (C-18 silica, step gradient between 10 to 80%,
methanol/water). The desired fractions were collected and dried at
reduced pressure to yield 50 mg (70%) of a white solid. TLC
(silica, propanol/H.sub.2O/NH.sub.4OH, (30/20/2), R.sub.f=0.54). MS
(MALDI): Observed, 1485, 1529, 1618, 1706.
Example 6
GnT1 and GalT1 Reaction in One Pot
[0380] 6.1 Reaction in One Pot
[0381] The one pot GlcNAc transferase-1 and galactose transferase-1
reaction was carried out by incubating EPO (1 mg/mL) in 100 mM Tris
HCl pH 7.5 or MES pH 6.5 containing 150 mM NaCl, 5 mM UDP-GlcNAc, 5
mM UDP-Gal, 5 mM MnCl.sub.2, 0.02% sodium azide, 30 mU/mL of
purified GlcNAc transferase-1 and 200 mU/mL of purified galactose
transferase-1 at 32.degree. C. for 16 h.
[0382] 6.2 Purification of EPO on Superdex75
[0383] A Superdex 75 column was equilibrated in 100 mM MES buffer
pH 6.5 containing 150 mM NaCl at a flow rate of 5 mL/min. The EPO,
product from step 6.1 (above) was loaded on to the column and
eluted with the equilibration buffer. The eluate was monitored for
absorbance at 280 nm and conductivity. SDS-PAGE was used to
determine which pooled peak fractions contains the EPO and used in
further experiments.
[0384] 6.3 ST3Gal-III Reaction
[0385] The ST3GalIII reaction was carried out by incubating 1 mg/mL
EPO-Gal (from step 6.2, above) in 100 mM Tris HCl pH 7.5 or MES pH
6.5 containing 150 mM NaCl, 0.5 mM CMP-N-acetyl-neuraminic acid-20
kilodalton-PEG, 0.02% sodium azide, and 200 mU/mL of purified
ST3Gal-III at 32.degree. C. for 16 hours.
Example 7
GnT1, GalT1 and ST3Gal-III (Using CMP-NAN-20 KPEG) Reaction in One
Pot
[0386] EPO (1 mg/mL) was incubated with 30 mU/mL of GlcNAc
transferase-1, 200 mU/mL of Galactose transferase-1 and 500 mU/mL
of ST3GalIII with sugar nucleotides and CMP-N-acetyl-neuraminic
acid-20 Kd PEG in 100 mM MES buffer pH 6.5 and analyzed using
SDS-PAGE. Similar to the results obtained in the two-step enzyme
remodeling reactions, three bands of PEGylated EPO are seen in the
one-pot, three enzyme preparations.
Example 8
Production of Biantennary PEG-EPO
[0387] 8.1 Addition of GlcNAc to rEPO
[0388] Recombinant EPO, expressed in baculovirus (1 mg/mL) in 0.1 M
Tris, 0.15 M NaCl, 5 mM MnCl.sub.2 and 0.02% sodium azide at pH 7.2
was incubated with 3 mM USP-GlcNAc, 50 mU/mg GlcNAc transferase-1
and 50 mU/mg GlcNAc transferase-II at 32.degree. C. for 24
hours.
[0389] 8.2 Addition of Galactose
[0390] To the GlcNAc-labeled peptide of step 8.1 (above) was added
3 mM UDP-Gal and 0.2 U/mg Galactose transferase-1. The mixture was
incubated for 36 hours at 32.degree. C. The galactosylated product
was isolated by gel filtration chromatography on a Superdex 75
column in Tris-buffered saline. The purified product was
concentrated to 1 mg/mL.
[0391] 8.3 Addition of Sialic Acid or Sialic Acid PEG
[0392] The galactosylated product from step 8.2 (above) (1 mg/mL)
in 0.1 M Tris, 0.1M NaCl at pH 7.2 was incubated at 32.degree. C.
for 24 hours with 200 mU/mg ST3GalIII and 0.5 mM CMP-sialic acid or
CMP-sialic acid-PEG (where the PEG has a molecular mass of 5
kilodaltons, 10 kilodaltons or 20 kilodaltons).
Example 9
N-linked 30K PEGylation by CST-II
[0393] EPO glycosylated as expressed in CHO (Chinese Hamster Ovary)
cells (5 mg, 0.166 .mu.mol, 5 ml) was concentrated and buffer
exchanged with tris buffer (50 mM Tris, 0.15M NaCl, 0.001 M
CaCl.sub.2+0.005% NaN.sub.3) to a final volume of 5 ml. Then
CMP-sialic acid-PEG (30 kilodaltons, 25 mg, 0.833 .mu.mol; see FIG.
3B for structure of 30 Kdalton CMP-sialic acid-PEG), 0.25 mL 100 mM
MnCl.sub.2 0.25 ml, and a bifunctional sialyltransferase from
Campylobacter jejuni, CST-II (1.4 U/mL, 0.5 ml, 0.7 U), were added.
The resulting mixture was rocked at 32.degree. C. for 48 hours.
[0394] At the conclusion of the reaction, the mixture was
concentrated by ultrafiltration to 1 mL final volume, and was then
buffer exchanged with 25 mM NaOAc+0.005% Tween-80 (pH 6.0) to 2.5
ml. Q-Sepharose IEX chromatography was performed using 25 mM
NaOAc+2M NaCl+0.005% Tween-80 (pH 6.0) as eluent. Peak 2 was
collected and concentrated to 1.5 ml by ultrafiltration, then
subjected to superdex-200 purification (column: Superdex 200, 16/60
GL, Amersham) using 1.times.PBS (pH 5.5+0.005% Tween80) as eluent.
Peak 2 was collected and concentrated to 1.5 ml. This resulting
material was sterile filtered and formulated to a final volume of
2.5 mL using 10 mM NaOAc (0.75% NaCl, pH 5.5). Protein
concentration 264 .mu.g/ml; 660 .mu.g protein was obtained (BCA
determination).
Example 10
[0395] The following example illustrates a method for preparing
O-linked 40 kilodalton PEG linked EPO using ST3GalIII:
[0396] 10.1 Desialylation
[0397] In this step EPO grown in Chinese Hamster Ovary cells (CHO
cells), was desilylated. The GlcNAc-Gal linkage serves as an
acceptor for transfer of the modified sialic acid PEG in step 10.2,
below.
[0398] EPO solution 10 ml (10 mg, 0.33 .mu.mol) glycosylated as
expressed in CHO (Chinese Hamster Ovary) cells, was buffer
exchanged with Tris buffer (20 mM Tris, 50 mM NaCl, 5 mM
CaCl.sub.2, 0.02% NaN.sub.3, pH 7.2) to give a final volume of 10
ml. Then 750 mU 2,3,6,8-neuramidase, from Arthrobacter Ureafaciens,
was added to the solution. The resulting mixture was rocked at
32.degree. C. for 48 hours. The product of this step was used
directly in the next step of the protocol (see below).
[0399] 10.2 O-Linked 40K PEGylation
[0400] In this step O-sialyltransferase is used to transfer a
modified sialic acid-PEG moiety to the desialylated EPO from step
10.1, above.
[0401] CMP-sialic acid-PEG (40 kilodalton, 33 mg, 0.825 .mu.mol;
see FIG. 3A for the structure of 40 kilodalton CMP-SA-PEG),
O-sialyltransferase (1.4 U/ml, 300 mU), and 0.25 mL of 100 mM
MnCl.sub.2 were added to half of the above mixture. This mixture
was rocked at 32.degree. C. for 48 hours. After the 48 hour period,
the reaction mixture was concentrated by ultrafiltration (MWCO 5K)
to 2.8 ml, then buffer exchanged with 25 mM NaOAc+0.001% Tween-80,
pH 6.0) to a final volume of 3 ml. The final product was ion
exchange purified on SP (5 mL) three times (three injections, 1 ml
each). PEGylated EPO (Peak 2) was collected and concentrated by
ultrafiltration to a final volume of 2 ml for SEC purification.
Purification on superdex 200 provided resolution of the desired
protein: EPO-GlcNAc-Gal-SA-PEG (40K) for the final step of the
reaction.
[0402] 10.3 Complete Terminal Sialylation of
CHO-EPO-GalNAc-Gal-SA-PEG (40K)
[0403] In this step of the process sialic acid was added to the
termini of glycosyl structures not bearing a modified sialic acid
residue.
[0404] Combined PEGylated EPO (approximately 2 mg from the reaction
in step, b above) was concentrated by ultrafiltration (MWCO 5K) and
then buffer exchanged with tris buffer (0.05M Tris, 0.15 M NaCl,
0.001 M CaCl.sub.2+0.005% NaN.sub.3) to a final volume of 2 mL.
Then CMP-N-acetyl neuraminic acid (CMP-NANA; 1.5 mg, 2.4 .mu.mol),
ST3GalIII (8.9 U/mL, 10 .mu.l, 0.089 U) and 50 .mu.l of 100 mM
MnCl.sub.2 were added. The resulting mixture was rocked at
32.degree. C. for 24 h, then concentrated to 1 ml final volume.
This solution was directly subjected to Superdex 200 purification
using 1.times.PBS (pH 5.5+0.005% Tween 80) as eluent. Peak 1 was
collected and diluted to 10 ml. Protein concentration 52.8 ug/ml
(BCA). A total of 528 .mu.g protein was obtained. A schematic
representation of the final peptide product is shown in FIG.
4A.
Example 11
[0405] In this example the pharmacokinetic profiles of
intravenously-administered CHO-derived EPO (a schematic
representation is shown in FIG. 5) and glycopegylated variants of
the CHO-derived EPO were compared using an ELISA assay.
[0406] The pharmacokinetics of two non-PEGylated batches of
CHO-derived Erythropoietin, a 30K PEGylated CHO-derived
Erythropoietin (FIG. 4B) produced by methods of the invention, and
40K PEGylated CHO-derived Erythropoietin (FIG. 4A) produced by
methods of the invention, were compared by ELISA after a single 30
.mu.g/kg intravenous dose into rats.
[0407] 11.1 Preparing the ELISA Plate.
[0408] A capture antibody against human EPO was dispensed into all
wells of a 96-well plate at a 100 .mu.L per well. The plate was
covered with plate seal tape and incubated for 2 hours at
37.degree. C. The Capture antibody was removed from the plate by
washing 2 times with Tris-buffered saline containing 0.2% Tween-20
(TBST). After a third wash, a 3% milk blocking solution (TBST plus
3% milk) was added to the plate, the plate was covered with plate
seal tape and incubated overnight at 4.degree. C.
[0409] In the morning the blocking solution was removed by washing
3 times with TBST. The rat plasma samples and standard proteins
were appropriately diluted with rat plasma and dispensed into the
wells at 100 .mu.L/well. The plate was covered with plate seal tape
and incubated overnight at 4.degree. C.
[0410] The next morning standard proteins were used to generate a
standard linear regression for each of the EPO proteins whose
pharmacokinetic properties were tested. A reverse phase-HPLC
analysis of the standard proteins was completed and the
concentrations were determined by calculating the area under the
peak(s) corresponding to the protein detected.
[0411] 11.2 Preparing and Adding the Test Samples.
[0412] Each test sample was diluted and the diluted samples were
dispensed into an ELISA plate at 100 .mu.L/well. The plate was then
covered with plate seal tape and incubated overnight at 4.degree.
C.
[0413] 11.3 Measuring the Europium Counts.
[0414] In the morning the rat serum samples were removed and the
plates were washed 3 times with TBST. The detection antibody, mouse
anti-human EPO which was previously labeled with Europium and
purified through a gel filtration column, was applied to the ELISA
plates. The plates are incubated at room temperature for 1 hour
under 100 rpm agitation.
[0415] The detection antibody was removed by washing the plates 6
times with TBST. Enhancement solution was added to the plates at
200 .mu.L/well and the plates were incubated at room temperature
for 20 minutes. The fluorescence was read with a Wallac plate
reader using a Europium counting program.
[0416] 11.4 Results
[0417] 11.4a Generating the Standard Linear Regression
[0418] The Europium counts from the standard proteins from each
plate were used to generate a standard linear regression curve and
equation. The Equation was used to convert the Europium count into
the equivalent EPO quantity for each sample well.
[0419] 11.4b Pharmacokinetic Results
[0420] The Results are shown in FIG. 6. The limit of detection is
approximately 0.4 ng/mL for non-PEGylated EPO, and approximately
0.8 ng/mL for both 30 kilodalton and 40 kilodalton PEGylated
EPO.
[0421] The 30 kilodalton PEGylated CHO-derived EPO, and 40
kilodalton PEGylated CHO-derived EPO, clearly display far superior
intravenous clearance parameters relative to their non-PEGylated
counterparts. As can be seen in the Figure, the various EPO
isoforms were ranked 40 kilodalton PEGylated CHO-derived EPO
.about.30 kilodalton PEGylated CHO-derived
EPO>>>non-PEGylated counterparts.
Example 12
[0422] In this example the pharmacokinetic profiles of
subcutaneously-administered CHO-derived Erythropoietin (EPO), a
hyperglycosylated non-glycopegylated EPO, an insect cell grown
glycopegylated EPO, and a CHO cell derived glycopegylated EPO were
determined using an ELISA assay.
[0423] Pharmacokinetics of a non-glycopegylated CHO-derived EPO, a
non-PEGylated hyperglycosylated CHO derived EPO, a glycoPEGylated
insect cell derived EPO; a 10K N-linked PEGylated insect
cell-derived Erythropoietin (a schematic representation is shown in
FIG. 7), and 40 kilodalton O-linked PEGylated CHO-derived
Erythropoietin (see FIG. 4A) were compared by ELISA after rats were
given a single 10 .mu.g/kg subcutaneous dose.
[0424] The ELISA plates were prepared and blocked as described in
Example 10. Standard proteins were also prepared and Europium
counts were also determined as described above.
[0425] 12.1 Preparing and Adding the Rat Samples.
[0426] Following the subcutaneous (S.C.) injections the amount of
EPO in the circulation was reduced as compared to that seen in
equivalent I.V. injections. Plasma concentrations of the S.C.
injected EPO proteins were typically detected between 30 minutes to
48 hours after injection.
[0427] 12.2 Pharmacokinetic Results.
[0428] Results of these experiments are shown in FIG. 8. FIG. 8
shows the average quantity of EPO in ng/mL and the standard
deviations in the rat serum samples at different time points after
injection time=0 hour for each EPO variant group. The limit of
detection is approximately 0.3 ng/mL for non-PEGylated EPO and
PEGylated EPO.
[0429] In the case of the 10K PEGylated EPO grown in insect cells
and the 40 kilodalton PEGylated CHO-EPO, the absorption appears to
be gradual, creating a situation where much of these EPO variants
remain to be absorbed well beyond the peak serum levels
(C.sub.max).
[0430] The 10K PEGylated EPO variant grown in insect cells attains
C.sub.max a time range of 24-36 hours after injection. Whereas the
40 kilodalton PEGylated CHO-EPO variant attains C.sub.max at 40-60
hours post injection. In addition, appreciable levels of the
pegylated variants were present at 96 hours after injection with
the current injected dose.
[0431] The serum rank order t.sub.1/2 is as follows: 40 kilodalton
PEGylated CHO-EPO>10K PEGylated EPO variant grown in insect
cells>hyperglycosylated CHO-EPO>>non-pegylated
CHO-EPO.
Example 13
[0432] The relative activities of two non-pegylated EPO variants (A
and B) were compared to two glycoPEGylated variants (30 kilodalton
and 40 kilodalton PEG) and to a hyperglycosylated PEG in
stimulating proliferation of EPO receptor-bearing TF1 cells in
culture. The activities of the glycopegylated EPO peptides in this
assay are similar to the hyperglycosylated EPO variant.
Example 14
[0433] Inhibition of binding of isotope-labeled EPO to a
recombinant chimeric EPO receptor by various concentrations of
unpegylated EPO (A and B) and glycoPEGylated 30 kilodalton and 40
kilodalton PEG variants. Receptor affinities (Ki) are similar for
unpegylated EPO and the glycoPEGylated variants.
[0434] 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