U.S. patent application number 10/332696 was filed with the patent office on 2003-10-09 for synthetic erythropoiesis stimulating proteins.
Invention is credited to Botti, Paolo, Bradburne, James A., Chen, Shiah-yun, Cressman, Sonya, Hunter, Christine L., Kent, Stephen B.H., Kochendoerfer, Gerd G., Low, Donald W..
Application Number | 20030191291 10/332696 |
Document ID | / |
Family ID | 26925033 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030191291 |
Kind Code |
A1 |
Kochendoerfer, Gerd G. ; et
al. |
October 9, 2003 |
Synthetic erythropoiesis stimulating proteins
Abstract
Synthetic erythropoiesis stimulating proteins are provided. Also
provided are methods for synthesizing the proteins. The invention
further relates to derivatives of such synthetic erythropoiesis
stimulating proteins that are polymer-modified in a defined manner.
Methods and uses for such proteins and derivatized proteins are
also provided.
Inventors: |
Kochendoerfer, Gerd G.;
(Oakland, CA) ; Botti, Paolo; (Piacenza, IT)
; Bradburne, James A.; (Redwood City, CA) ; Chen,
Shiah-yun; (Mountain View, CA) ; Cressman, Sonya;
(Ladysmith, CA) ; Hunter, Christine L.; (San
Mateo, CA) ; Kent, Stephen B.H.; (San Francisco,
CA) ; Low, Donald W.; (Burlingame, CA) |
Correspondence
Address: |
LINIAK, BERENATO & WHITE, LLC
6550 ROCK SPRING DRIVE
SUITE 240
BETHESDA
MD
20817
US
|
Family ID: |
26925033 |
Appl. No.: |
10/332696 |
Filed: |
January 13, 2003 |
PCT Filed: |
July 12, 2001 |
PCT NO: |
PCT/US01/21928 |
Current U.S.
Class: |
530/397 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 7/06 20180101; A61P 19/02 20180101; A61P 7/00 20180101; A61K
38/00 20130101; A61P 29/00 20180101; C07K 14/505 20130101; A61P
31/12 20180101; A61P 9/10 20180101; A61P 11/02 20180101; A61P 11/06
20180101; A61P 37/06 20180101; A61P 37/08 20180101; A61P 37/00
20180101; A61P 43/00 20180101; A61P 17/02 20180101 |
Class at
Publication: |
530/397 |
International
Class: |
C07K 014/575 |
Claims
What is claimed is:
1. A synthetic erythropoiesis stimulating protein having one or
more water-soluble polymers attached thereto.
2. The synthetic erythropoiesis stimulating protein of claim 1
having the in vivo biological activity of increasing production of
red blood cells.
3. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said protein comprises a polypeptide chain having an amino
acid sequence of a ribosomally specified erythropoietin and one or
more non-overlapping peptide segments covalently bonded by one or
more chemical ligation sites.
4. The synthetic erythropoiesis stimulating protein of claim 3,
wherein one or more of said water-soluble polymers is attached to
said polypeptide chain at a site corresponding to a glycosylation
site of said ribosomally specified erythropoietin.
5. The synthetic erythropoiesis stimulating protein of claim 3,
wherein said ribosomally specified erythropoietin is human.
6. The synthetic erythropoiesis stimulating protein of claim 3,
wherein said ribosomally specified erythropoietin glycoprotein is
non-natural.
7. The synthetic erythropoiesis stimulating protein of claim 6,
wherein said non-natural ribosomally specified erythropoietin has
one or more non-natural glycosylation sites.
8. The synthetic erythropoiesis stimulating protein of claim 2,
wherein said water-soluble polymer is attached exclusively to said
polypeptide chain at one or more sites corresponding to a
glycosylation site of said ribosomally specified
erythropoietin.
9. The synthetic erythropoiesis stimulating protein of claim 1,
wherein one or more of said water-soluble polymers comprises a
repeat unit comprising a polyalkylene oxide, a polyamide alkylene
oxide, or derivatives thereof.
10. The synthetic erythropoiesis stimulating protein of claim 9,
wherein said polyalkylene oxide and polyamide alkylene oxide
comprise an ethylene oxide repeat unit of the formula
--(CH2-CH2-O)--.
11. The synthetic erythropoiesis stimulating protein of claim 1,
wherein one or more of said water-soluble polymers is branched.
12. The synthetic erythropoiesis stimulating protein of claim 1,
wherein one or more of said water-soluble polymers comprises a net
charge under physiological conditions that is negative.
13. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said synthetic erythropoiesis stimulating protein has an
isoelectric point between 3 and 7.
14. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said water-soluble polymer is mono-disperse.
15. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said synthetic erythropoiesis stimulating protein is
mono-disperse.
16. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said synthetic erythropoiesis stimulating protein comprises
a monomer molecular weight of greater than 25 kDa.
17. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said synthetic erythropoiesis stimulating protein comprises
a monomer molecular weight of greater than 40 kDa.
18. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said synthetic erythropoiesis stimulating protein comprises
a monomer molecular weight of greater than 50 kDa.
19. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said synthetic erythropoiesis stimulating protein comprises
a monomer molecular weight of greater than 60 kDa.
20. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said synthetic erythropoiesis stimulating protein comprises
a monomer molecular weight of greater than 70 kDa.
21. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said polypeptide chain comprises one or more irregular
amino acids.
22. The synthetic erythropoiesis stimulating protein of claim 21,
wherein said irregular amino acid comprises a non-natural side
chain.
23. The synthetic erythropoiesis stimulating protein of claim 21,
wherein said irregular amino acid comprises a pseudo amino
acid.
24. The synthetic erythropoiesis stimulating protein of claim 23,
wherein said pseudo amino acid is pseudo glutamate.
25. The synthetic erythropoiesis stimulating protein of claim 22,
wherein said non-natural side chain is covalently bonded to a
water-soluble polymer.
26. The synthetic erythropoiesis stimulating protein of claim 25,
wherein said water-soluble polymer is covalently bonded to said
side chain through a bond formed by chemical ligation.
27. The synthetic erythropoiesis stimulating protein of claim 26,
wherein said bond formed by chemical ligation is selected from the
group consisting of amide, oxime, hydrazone, thaizolidine,
oxazolidine and thioester.
28. The synthetic erythropoiesis stimulating protein of claim 3,
wherein one or more of said chemical ligation sites comprises an
amide bond.
29. The synthetic erythropoiesis stimulating protein of claim 1,
wherein said water-soluble polymer is attached to said polypeptide
chain through a side chain of an amino acid at a chemical ligation
site of said polypeptide chain.
30. A synthetic erythropoiesis stimulating protein according to
claim 1 which comprises an amino acid sequence of a synthetic
erythropoiesis stimulating protein selected from the group
consisting of SEP-1 and SEP-3.
31. A synthetic erythropoiesis stimulating protein selected from
the group consisting of SEP-0, SEP-1 and SEP-3, and analogs
thereof.
32. The synthetic erythropoiesis stimulating protein of claim 31,
wherein said analogs are selected from the group consisting of
SEP-1-L26, SEP-1-L30, SEP-1-B50, SEP-1-B51, and SEP-1-B52.
33. A pharmaceutical composition comprising a synthetic
erythropoiesis stimulating protein according to claim 1, or
pharmaceutically acceptable salts thereof.
34. The pharmaceutical composition according to claim 33 which
comprises one or more excipients selected from the group consisting
of a buffer, a carrier protein, an amino acid, a detergent, a
lipid, a water soluble polymer, and a preservative.
35. The pharmaceutical composition according to claim 33, which
comprises one or more additional bioactive agents other than said
synthetic erythropoiesis stimulating protein.
36. A method of increasing the hematocrit of a mammal, said method
comprising administering to said mammal an effective amount of a
synthetic erythropoiesis stimulating protein according to claim 1,
whereby the hematocrit in said mammal is increased.
37. A method of increasing production of red blood cells in a
mammal, said method comprising administering to said mammal an
effective amount of a polymer-modified synthetic erythropoiesis
stimulating protein according to claim 1, whereby the production of
red blood cells in said mammal is increased.
38. A method of increasing production of hemoglobin in a mammal,
said method comprising administering to said mammal an effective
amount of a polymer-modified synthetic erythropoiesis stimulating
protein according to claim 1, whereby the production of hemoglobin
in said mammal is increased.
39. A method of increasing reticulocyte count in a mammal, said
method comprising administering to said mammal an effective amount
of a synthetic erythropoiesis stimulating protein according to
claim 1, whereby the reticulocyte count in said mammal is
increased.
40. A method of producing a polypeptide chain comprising a
synthetic erythropoiesis stimulating protein, said method
comprising chemically ligating peptide segments comprising
non-overlapping amino acid sequences of a polypeptide chain of said
synthetic erythropoiesis stimulating protein whereby a polypeptide
chain comprising said synthetic erythropoiesis stimulating protein
is produced.
41. The method of claim 40, which further comprises folding said
polypeptide chain whereby a bioactive synthetic erythropoiesis
stimulating protein is produced.
42. The method of claim 40, wherein one or more of said peptide
segments are partially protected.
43. The method of claim 40, wherein one or more of said peptide
segments are unprotected.
44. The method of claim 40, wherein one or more of said peptide
segments comprises a water-soluble polymer attached thereto.
45. The method of claim 40, wherein said chemically ligating
comprises a chemoselective ligation chemistry selected from native
chemical ligation, extended native chemical ligation, pseudo native
chemical ligation, oxime forming chemical ligation, hydrazone
forming chemical ligation, oxazolidine forming chemical ligation,
thaizolidine forming chemical ligation, and thioester forming
chemical ligation.
46. The method of claim 40, wherein said polypeptide chain
comprises one or more water-soluble polymers attached thereto.
47. The method of claim 40, wherein said polypeptide chain
comprises an amino acid sequence of a ribosomally specified
erythropoietin.
48. The method of claim 46, wherein one or more of said
water-soluble polymers is attached to said polypeptide chain at a
site corresponding to a glycosylation site of said ribosomally
specified erythropoietin.
49. The method of claim 48, wherein said ribosomally specified
erythropoietin glycoprotein is recombinantly produced.
50. The method of claim 48, wherein said ribosomally specified
erythropoietin glycoprotein is non-natural.
51. The method of claim 50, wherein said non-natural ribosomally
specified erythropoietin glycoprotein has one or more non-natural
glycosylation sites.
52. The method of claim 47, wherein said ribosomally specified
erythropoietin is human.
53. The method of claim 48, wherein said water-soluble polymer is
attached exclusively to said polypeptide chain at a site
corresponding to a glycosylation site of said ribosomally specified
erythropoietin.
54. The method of claim 46, wherein one or more of said
water-soluble polymers comprises a repeat unit comprising a
polyalkylene oxide, a polyamide alkylene oxide, or derivatives
thereof.
55. The method of claim 54, wherein said polyalkylene oxide and
polyamide alkylene oxide comprise an ethylene oxide repeat unit of
the formula --(CH2-CH2-O)--.
56. The method of claim 46, wherein one or more of said
water-soluble polymers is branched.
57. The method of claim 46, wherein one or more of said
water-soluble polymers comprises a net charge under physiological
conditions that is negative.
58. The method of claim 41, wherein said synthetic erythropoiesis
stimulating protein has an isoelectric point between 3 and 7.
59. The method of claim 46, wherein said water-soluble polymer is
mono-disperse.
60. The method of claim 40, wherein said synthetic erythropoiesis
stimulating protein is mono-disperse.
61. The method of claim 46, wherein said synthetic erythropoiesis
stimulating protein comprises a monomer molecular weight of greater
than 25 kDa.
62. The method of claim 46, wherein said synthetic erythropoiesis
stimulating protein comprises a monomer molecular weight of greater
than 40 kDa.
63. The method of claim 46, wherein said synthetic erythropoiesis
stimulating protein comprises a monomer molecular weight of greater
than 50 kDa.
64. The method of claim 46, wherein said synthetic erythropoiesis
stimulating protein comprises a monomer molecular weight of greater
than 60 kDa.
65. The method of claim 46, wherein said synthetic erythropoiesis
stimulating protein comprises a monomer molecular weight of greater
than 70 kDa.
66. The method of claim 40, wherein said polypeptide chain
comprises one or more irregular amino acids.
67. The method of claim 66, wherein said irregular amino acid
comprises a non-natural side chain.
68. The method of claim 67, wherein said irregular amino acid
comprises a pseudo amino acid.
69. The method of claim 40, wherein said polypeptide chain
comprises a chemical ligation site having a bond selected from the
group consisting of amide, oxime, hydrazone, thaizolidine,
oxazolidine and thioester.
70. The method of claim 46, wherein said water-soluble polymer is
attached to an amino acid of said polypeptide chain having a
non-natural side chain.
71. The method of claim 46, wherein said water-soluble polymer is
covalently bonded to said side chain through a bond formed by
chemical ligation.
72. The method of claim 71, wherein said bond formed by chemical
ligation is selected from the group consisting of amide, oxime,
hydrazone, thaizolidine, oxazolidine and thioester.
73. The method of claim 46, wherein said water-soluble polymer is
attached to said polypeptide chain through a side chain of an amino
acid at a chemical ligation site of said polypeptide chain.
74. The method of claim 41, wherein said bioactive synthetic
erythropoiesis stimulating protein has the in vivo biological
activity of increasing production of red blood cells.
75. The method of claim 40, wherein said polypeptide chain
comprises an amino acid sequence of a synthetic erythropoiesis
stimulating protein selected from the group consisting of SEP-1 and
SEP-3.
76. The method of claim 40, wherein synthetic erythropoiesis
stimulating protein is selected from the group consisting of SEP-0,
SEP-1 and SEP-3, and analogs thereof.
77. The method of claim 76, wherein said analogs are selected from
the group consisting of SEP-1-L26, SEP-1-L30, SEP-1-B50, SEP-1-B51,
and SEP-1-B52.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation in part of U.S. patent
application Ser. Nos. 60/231,339 (filed Sep. 8, 2000) and
60/236,377 (filed Sep. 29, 2000), both of which are incorporated
herein by reference in their entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] The invention relates to synthetic erythropoiesis
stimulating proteins that are polymer-modified in a defined manner,
and methods of production and use.
BACKGROUND OF THE INVENTION
[0003] Erythropoiesis is the process for the production of red
blood cells that occurs to regulate turnover and loss of red blood
cells. Erythropoietin (EPO) is a protein hormone that stimulates
erythropoiesis. Naturally occurring human EPO is a glycoprotein
containing 165 amino acids that is produced primarily in the
kidney, secreted into the blood stream, and stimulates production
of red blood cells from precursor cells in bone marrow. Although
the gene encoding human EPO predicts a molecule with 166 amino acid
residues, the carboxy-terminal arginine is removed in a
post-translational modification in its mature form.
[0004] Glycosylated human EPO has three N-linked carbohydrate
chains attached at positions 24, 38 and 83. It also has one
O-linked carbohydrate moiety present at position 126. The effects
of glycosylation are complex. Non-glycosylated EPO is active in
vitro. However, studies have shown glycosylation to be necessary
for full activity in vivo. Studies also have shown that variable
glycosylation patterns exhibit variable effects. For example,
desialyated EPO exhibits both enhanced in vitro and decreased in
vivo activity, an effect attributed to the exposure of galactose
residues that are recognized, bound, and cleared by hepatocytes.
The branching pattern of fully sialyated EPO also has been shown to
make a difference in biological activity. Predominantly
tetra-antennary branched EPO shows activity equivalent to
"standard" EPO, while predominantly bi-anternnary branched EPO
shows three-fold more activity in vitro but only 15% of normal
activity in vivo. Moreover, various studies have shown that only
N-linked, and not O-linked sugars are important in EPO
functioning.
[0005] Several pharmaceutical products containing recombinantly
produced glycosylated EPO are available. However, currently
available EPO pharmaceutical products are limited by their short
plasma half-life and susceptibility to protease degradation. They
also contain complex mixtures of different glycoforms dues a high
degree of heterogeneity in the branching and sialic acid content
that occurs at each N-linked glycosylation site and between sites.
These shortcomings have prevented them from attaining maximum
clinical potency.
[0006] To improve circulating half-life and other properties of
such EPO proteins, water-soluble polymers such as PEG (polyethylene
glycol) have been attached, but with mixed results given the
difficulty of attaching them in a controlled manner and with
user-defined precision. For example, a variety of means have been
used to attach polymer moieties such as PEG and related polymers to
reactive groups found on proteins (see, U.S. Pat. No. 4,179,337
(Davis et al.), and U.S. Pat. No. 4,002,531 (Royer). For a review,
see Abuchowski et al., in "Enzymes as Drugs," (J. S. Holcerberg and
J. Roberts, eds. pp. 367-383 (1981) and Zalipsky, S. (Bioconjugate
Chemistry (1995) 6:150-165. The use of PEG and other polymers to
modify proteins also is discussed by Cheng, T. -L. et al.,
Bioconjugate Chem. (1999) 10:520-528; Belcheva, N. et al.,
Bioconjugate Chem. (1999) 10: 932-937; Bettinger, T. et al.,
Bioconjugate Chem. (1998) 9:842-846; Huang, S. -Y. et al.,
Bioconjugate Chem. (1998) 9:612-617; Xu, B. et al. Langmuir (1997)
13:2447-2456; Schwarz, J. B. et al., J. Amer. Chem. Soc. (1999)
121:2662-2673; Reuter, J. D. et al., Bioconjugate Chem. (1999)
10:271-278; Chan, T. -H. et al., J. Org. Chem. (1997) 62:3500-3504.
Typical attachment sites in proteins include primary amino groups,
such as those on lysine residues or at the N-terminus, thiol
groups, such as those on cysteine side-chains, and carboxyl groups,
such as those on glutamate or aspartate residues or at the
C-terminus. Some of the most common sites of attachment are to the
sugar residues of glycoproteins, cysteines or to the N-terminus and
lysines of the target proteins.
[0007] Although many different approaches have been described for
conjugation of polymers to EPO, the conjugation process is not
without complications. Care must be taken to limit the loss of
biological activity caused by the conjugation reaction. For
example, folded or re-folded proteins are typically used to
minimize the number of sites of attachment. However, if too much of
the activated polymer is attached to the target protein or
polypeptide, biological activity can be severely reduced or lost.
Likewise, if the wrong linker joining the polymer to the protein is
used or an insufficient amount of polymer is attached to the
target, the therapeutic value of the resultant conjugate may be
limited, and may not demonstrate enough of an increase in
circulating life to compensate for the loss in its bioactivity.
Problems can also result due to a blockage of the protein's active
site by the modifying polymer. This problem can be difficult to
avoid since the polymer and protein are typically joined in
solution-based reactions. Pre-blocking the active sites with
materials such as pyridoxal phosphate has been suggested, but the
results have been inconsistent (see, U.S. Pat. No. 4,179,337 (Davis
et al.)). These problems are particularly acute with lower
molecular weight proteins and peptides, which often have few
attachment sites not associated with bioactivity.
[0008] For instance, a common technique has been the attachment of
water-soluble polymers to the primary amines in the target protein
(e.g., the N-terminal amino group and the epsilon amino groups of
lysines). Thiol-reactive polymer conjugates also have been used to
attach polymers to the thiol side-chains of cysteines. Both
approaches represent significant problems as most proteins contain
multiple copies of such reactive groups spread out over various
regions of the polypeptide backbone that play an important role in
defining the activity, folding, re-folding and stability of a
protein. Thus although widely used, such approaches suffer from
more or less incomplete or unwanted reduction in bioactivity of a
protein, and usually complex mixtures that are difficult to
separate and characterize (Delgada et al., Pharmaceutical Sciences
(1997) 3:59-66).
[0009] In an attempt to reduce random attachment, proteins have
been made in which the natural lysines are removed in conjunction
with adding lysines at the desired sites of polymer attachment
(U.S. Pat. No 4,904,584). For example G-CSF and IL-2 have been
modified in this manner. Other attempts to avoid random attachment
has been to make proteins in which the natural cysteines are
removed in conjunction with adding cysteines at the desired sites
of polymer attachment, or cysteines are added without removing the
natural cysteines if present (EP 0 668 353). For example, EPO has
been made this way. While such cysteine or lysine variants
theoretically allow site-specific polymer attachment, there still
is no guarantee that all selected sites can be modified in a
controlled manner.
[0010] Given the inability to site-specifically modify proteins
containing multiple amino acids with side-chains bearing the same
or similar reactive functional groups, recent efforts have focused
on the modification of the amino or carboxy terminus of proteins.
Modification of the amino or carboxy terminus has relied on the
ability of some chemical conjugation techniques to uniquely modify
these sites (WO 90/02136 and WO 90/02135). For example, this
technique was utilized for the attachment of PEG chains to the
N-terminal residue of G-CSF and the chemokine IL-8 (Gaertner et
al., Bioconjug. Chem. (1996) 7(1):38-44; and WO 96/41813). However,
modification of the N- or C-termini typically reduces a protein's
activity (See, e.g., U.S. Pat. No. 5,985,265 discussing attachment
of PEG to the N-terminus and lysine side chains of G-CSF). Despite
the drawbacks with modification of proteins with water-soluble
polymers, PEGylation and attachment of other water-soluble polymers
to proteins continues to be pursued. For example, attachment of PEG
to sugar chains of erythropoietin (EPO) and internal amino acids
such as lysines (EP 0 605 963 and WO 00/32772), and the N-terminus
(U.S. Pat. No. 6,077,939 and WO 00/32772) also has been described.
Unfortunately, such PEGylated EPOs result in complex mixtures that
are difficult to separate and characterize.
[0011] Another problem is that the above-discussed polymers are
highly non-homogeneous, which render analytical characterization
and purification of the mixtures of polymer-modified proteins
difficult (Delgada et al., Pharmaceutical Sciences (1997) 3:59-66).
For example, techniques used to prepare PEG or PEG-based chains,
even those of fairly low relative molecular mass such as 3400,
involve a poorly controlled polymerization step which leads to
preparations having a spread of chain lengths about a mean value;
that is, they involve polymer preparations of
(CH.sub.2CH.sub.2O).sub.n where n does not have a discrete value
but rather has a range of values about a mean. The resulting
heterogeneity of derivatized proteins is often associated with a
range of properties that one cannot easily identify much less
separate.
[0012] Unfortunately, however, the problem of identifying and
employing a suitable polymer is exacerbated by the fact that all
EPO proteins currently employed for therapeutic use are derived
from recombinant DNA technologies. The use of recombinant DNA
technologies puts severe limitations on the types and specificity
of linkages that can be formed between a performance-enhancing
moiety and the recombinantly produced EPO protein. This is because
firstly there are only a very limited number of functional groups
suitable for linkage, and secondly there will generally be several
copies of the reactive functional group in the protein being
modified, thus precluding any specificity of modification. For
example, it has been shown that in the case of nonselective
conjugation of superoxide dismutase with PEG, several fractions of
the modified enzyme were completely inactive (P. McGoff et al.,
Chem. Pharm. Bull. (1988) 36:3079-3091). Also, if differing numbers
of such moieties are randomly attached, the pharmacokinetics of the
therapeutic protein cannot be precisely predictable, making dosing
a large problem. The lack of control of attachment furthermore may
lead to (a) reduced potency, (b) a need for elaborate purification
schemes to separate a vast mixture of derivatives and (c) possibly
unstable attachment of the modifying moiety. Several linkages, such
as tresylchloride-based linkages, known to the art are also known
to be immunogenic.
[0013] Thus to improve circulating half-life, reduce proteolysis
and immunogenicity and improve other properties of biologically
produced proteins, water-soluble polymers such as PEG can be
attached, but with mixed results given the difficulty of attaching
them in a controlled manner and with user-defined precision. Also,
because of the limited success in attaching polymers at precise
user-defined sites, very little is known about preferred sites of
attachment that could be applicable to proteins in general. In
addition, because of the stochastic nature of attachment, and the
hetero-disperse nature of PEG and other water-soluble polymers
currently employed for such purposes, purification and analytical
characterization of PEG-protein conjugates has been difficult. Thus
the combined problem of poor control over reproducible attachment
and polymer heterogeneity has severely hampered the routine
approval of polymer-modified proteins as therapeutics (only a few
approved to date for therapeutic use despite its introduction in
the early 1970's).
[0014] Accordingly, a need exists for methods of forming bioactive
erythropoiesis stimulating proteins that are distinct from
recombinant DNA technologies and polymer technologies used to
modify natural and recombinantly produced EPO. Also needed are
erythropoiesis stimulating proteins that have improved clinical
properties. The present invention satisfies these and other
needs.
SUMMARY OF THE INVENTION
[0015] The present invention concerns synthetic erythropoiesis
stimulating proteins, methods for their manufacture and uses. In
particular, the invention is directed to synthetic erythropoiesis
stimulating proteins having one or more water-soluble polymers
attached thereto. Also provided are pharmaceutical compositions
comprising the synthetic erythropoiesis stimulating proteins of the
invention.
[0016] The invention is further directed to methods of treating a
mammal with polymer-modified synthetic erythropoiesis stimulating
proteins of the invention. In one embodiment, a method is provided
for increasing the hematocrit of a mammal. In another embodiment, a
method is provided for increasing production of red blood cells in
a mammal. Another embodiment is directed to a method for increasing
hemoglobin in a mammal. A further embodiment is directed to a
method for increasing reticulocyte count in a mammal. These methods
comprise administering to the mammal an effective amount of a
polymer-modified synthetic erythropoiesis stimulating protein of
the invention to achieve the desired effect.
[0017] Also provided are methods for the manufacture of the
synthetic erythropoiesis stimulating proteins of the invention,
including preferred polymer-modified forms, and intermediates. The
method comprises chemically ligating peptide segments comprising
non-overlapping amino acid sequences of a polypeptide chain of a
synthetic erythropoiesis stimulating protein, whereby a polypeptide
chain comprising the synthetic erythropoiesis stimulating protein
is produced.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIGS. 1A-1E depict schematics of processes for preparing the
polymer-modified synthetic erythropoiesis stimulating proteins of
the invention.
[0019] FIGS. 2A-2C depict schematics of processes for preparing
synthetic erythropoiesis stimulating proteins of the invention.
[0020] FIGS. 3A-3B depict schematics of processes for multi-segment
ligations that involve the chemical ligation of three or more
non-overlapping peptide segments, i.e., at least one segment is a
middle segment corresponding to the final full-length ligation
product.
[0021] FIGS. 4A-4C illustrate native chemical ligation and chemical
modification of the resulting side-chain thiol.
[0022] FIGS. 5A-5B depict solid phase process for generating the
branching core (B) and unique chemoselective functional group (U)
of the water-soluble polymer U--B-Polymer-J* of the invention.
[0023] FIGS. 6A-6D depict a solid phase process for generating
preferred substantially non-antigenic water-soluble polyamide
Polymer-J* components of the invention for subsequent attachment to
the U--B core.
[0024] FIG. 7 depicts process for coupling the U--B component to
Polymer-J* component to generate the preferred synthetic polymer
constructs of the invention of the formula U--B-Polymer-J*.
[0025] FIG. 8 depicts an alternative route for precision attachment
of a water-soluble polymer to a peptide segment employable for
ligation and production of erythropoiesis stimulating synthetic
proteins of the invention.
[0026] FIG. 9 illustrates the overall process of preparing the
synthetic erythropoiesis stimulating proteins of the present
invention.
[0027] FIG. 10 depicts the basic structure of a preferred type of
synthetic erythropoiesis stimulating proteins. pPEG="precision
PEG"
[0028] FIG. 11 shows the general structure of circularly permuted
SEP analogs having a relocated amino and carboxy terminus.
[0029] FIG. 12 shows a structure of a preferred water-soluble
polymer, and various linear and branched constructs thereof.
[0030] FIG. 13 depicts schematically the formation of
branched-chain pPEG polymers.
[0031] FIG. 14 depicts the synthesis of a synthetic cytokine
designated SEP1-L30, which is a precision polymer-modified
synthetic analog of human EPO prepared as described in Example
2.
[0032] FIG. 15 depicts a synthetic cytokine designated SEP1-L30,
which is a precision polymer-modified synthetic analog of human EPO
prepared as described in Example 2.
[0033] FIG. 16 depicts a synthetic cytokine designated SEP1-L26,
which is a precision polymer-modified synthetic analog of human EPO
prepared as described in Example 3.
[0034] FIG. 17 depicts schematically the formation of a preferred
branched-chain water-soluble polymer that is attached to a
synthetic cytokine designated SEP1-B50, which is a precision
polymer-modified synthetic analog of human EPO prepared as
described in Example 4.
[0035] FIG. 18 depicts a synthetic cytokine designated SEP1-B50,
which is a precision polymer-modified synthetic analog of human EPO
prepared as described in Example 4.
[0036] FIG. 19 depicts the synthesis of a synthetic cytokine
designated SEP3-L42, which is a precision polymer-modified
synthetic analog of human EPO prepared as described in Example
5.
[0037] FIG. 20 depicts synthetic cytokine designated SEP3-L42,
which is a precision polymer-modified synthetic analog of human EPO
prepared as described in Example 5.
[0038] FIG. 21 depicts the synthesis of a preferred water-soluble
polymer for attaching to a synthetic cytokine designated SEP1-B51,
which is a precision polymer-modified synthetic analog of human EPO
prepared as described in Example 7.
[0039] FIG. 22 depicts a synthetic cytokine designated SEP1-B51,
which is a precision polymer-modified synthetic analog of human EPO
prepared as described in Example 7.
[0040] FIG. 23 depicts the synthesis of a preferred water-soluble
polymer for attaching to a synthetic cytokine designated SEP1-B52,
which is a precision polymer-modified synthetic analog of human EPO
prepared as described in Example 8.
[0041] FIG. 24 depicts a synthetic cytokine designated SEP1-B52,
which is a precision polymer-modified synthetic analog of human EPO
prepared as described in Example 8.
[0042] FIG. 25 shows representative analytical data for the
precision polymer-modified synthetic analogs of human EPO prepared
as described in Examples 2-5 and 7-8. As shown, a representative
Isoelectric Focusing Gel (IEF) and non-reducing SDS-PAGE gel
demonstrate the relative monomer molecular weight of the folded,
purified SEP1-B51.
[0043] FIG. 26 shows the in vitro activity in a factor-dependent
cell line of SEP compounds SEP0, SEP1-L26, SEP1-L30, SEP1-B50,
SEP1-B51 and SEP3-L42.
[0044] FIG. 27 shows a representative pharmacokinetic profile
comparing plasma concentration in nanograms per milliliter (ng/ml)
of SEP3-L42 and SEP1-B50 versus time in hours.
[0045] FIG. 28 shows linear regression analyses of the in vivo
activity as measured by the 72-hour red blood cell (RBC)--.sup.59Fe
uptake (as a % of dose) for SEP 1-B51 and recombinant glycosylated
human erythropoietin produced in CHO-cells ("rhEPO") in a hypoxic
rat model.
[0046] FIG. 29 shows a representative pharmacokinetic profile for
clearance in rats comparing plasma concentration in ng/ml of
SEP1-B51 and rhEPO versus time in hours following a single IV dose
of 5 .mu.g/kg for each compound.
[0047] FIG. 30 shows a circularly-permuted, polymer-modified SEP
construct.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0048] The present invention relates to synthetic erythropoiesis
stimulating proteins, methods of production and use.
[0049] I. Synthetic Erythropoiesis Stimulating Proteins of the
Invention
[0050] In a preferred embodiment, the invention is directed to a
synthetic erythropoiesis stimulating protein having one or more
water-soluble polymers attached thereto. By "erythropoiesis
stimulating protein" is intended a protein that has the in vitro
biological activity of promoting growth of an
erythropoietin-dependent cell line. In a preferred embodiment, the
synthetic erythropoiesis stimulating proteins of the invention have
the in vivo biological activity of promoting production of red
blood cells. Erythropoiesis stimulating activity of a protein may
be determined by any of a variety of means, such as by following
erythropoiesis after in vivo administration, by assaying the
capacity of the protein to mediate the proliferation of
EPO-dependent cell lines, etc.
[0051] By "water-soluble polymer" is intended a substantially
non-antigenic polymer that is soluble in water.
[0052] As used herein, a protein is said to be "synthetic" if
non-recombinant technology has been employed to polymerize some,
and most preferably all, of its amino acid residues. The term
"non-recombinant technology" is intended to distinguish
technologies that involve organic chemistry and other synthetic
polymerization approaches from technologies involving the
translation of RNA into protein, either in vivo or in vitro.
Synthetic proteins include totally synthetic and semi-synthetic
proteins. A totally synthetic protein is produced where all
ligation components are man-made by chemical synthesis, i.e.,
ribosomal-free synthesis. A semi-synthetic protein is produced
where at least part of a ligation component is made by biological
synthesis, i.e., ribosomally in a cell or cell-free translation
system, and another part is made by chemical synthesis.
[0053] In a preferred embodiment, the synthetic erythropoiesis
stimulating proteins of the invention comprise a polypeptide chain
having an amino acid sequence of a ribosomally specified
erythropoietin and one or more non-overlapping peptide segments
covalently bonded by one or more chemical ligation sites. Most
preferably the erythropoietin is mammalian, and more preferably
human or derivatives thereof. For example, the amino acid sequence
of many erythropoietins is known, and for human erythropoietins
many active variants thereof have been made (See, e.g., U.S Pat.
Nos. 5,856,298; 5,955,422; 8,888,772; 5,858,347; 5,614,184;
5,457,089; and 6,077,939; and W00/32772), and thus a synthetic
erythropoiesis stimulating protein of the invention can comprise a
polypeptide chain having a such amino acid sequences.
[0054] In a preferred embodiment, the ribosomally specified
erythropoietin comprises one or more glycosylation sites, and a
water-soluble polymer is attached to the polypeptide chain of the
synthetic erythropoiesis stimulating protein at one or more sites
corresponding to one or more the glycosylation sites of the
ribosomally specified erythropoietin. In a more preferred
embodiment, the water-soluble polymer is attached to the
polypeptide chain exclusively at one or more sites corresponding to
one or more such glycosylation sites. This aspect of the invention
includes synthetic erythropoiesis stimulating proteins where the
ribosomally specified erythropoietin is recombinantly produced.
Accordingly, the ribosomally specified erythropoietin can be a
natural erythropoietin or a non-natural erythropoietin, the latter
of which can further include one or more non-natural glycosylation
sites.
[0055] By "glycosylation site" is intended an amino acid sequence
of a protein that encodes for the enzymatic attachment of an
oligosaccharide (carbohydrate) chain to the side-chain of an amino
acid residue of the amino acid sequence of the protein; exemplified
by N-linked and O-linked glycosylation sites. It will be
appreciated that erythropoietins that have been mutated to
eliminate one or more of such glycosylation sites are embodied in
the definition of `glycosylation site`, as the residual site for
polymer modification will be positionally the same. By "naturally
occurring glycosylation site" is intended a glycosylation site of a
erythropoietin glycoprotein found in nature. By "non-naturally
occurring glycosylation site" is intended a glycosylation site that
has been engineered into an erythropoietin. For instance,
recombinant human EPO has been engineered in this manner (see,
e.g., U.S. Pat. No. 5,856,298).
[0056] This embodiment of the invention is based in part on the
finding that when a synthetic erythropoiesis stimulating protein
having a polypeptide chain of human erythropoietin is modified at
one or more glycosylation sites thereof with a water-soluble
polymer, such as the polymers described herein, the water-soluble
polymer can be utilized to take advantage of one or more of the
biological effects attributed to a carbohydrate chain normally
found at that position in the counterpart naturally occurring
erythropoietin glycoprotein. Such biological effects include
modulation of protease resistance, immunogenicity,
receptor-specificity, specific activity, potency and
pharmacokinetics. However, by eliminating the presence of a
carbohydrate chain, and replacing it with preferred water-soluble
polymers of the invention, significant benefits are obtained
including the avoidance of enzymatic degradation and clearance such
as when pendant sialic acid residues of the sugar chains are
removed, instability, limited circulation half-life, distribution,
poor handling properties etc. Significantly, its also has been
found that such polymer-modified synthetic erythropoiesis
stimulating proteins of the invention retain such advantageous
biological properties in the substantive absence of loss of
biological activity when compared to a non-modified counterpart
synthetic protein, including increased bioactivity, even when the
monomer molecular weight of the synthetic protein is greater than
25 kDa.
[0057] Thus, in another preferred embodiment, the invention is
directed to a synthetic erythropoiesis stimulating protein
comprising a polypeptide chain comprising an amino acid sequence of
a ribosomally specified erythropoietin, where the polypeptide chain
has one or more water-soluble polymers attached thereto and a
monomer molecular weight of greater than 25 kDa, and where the
synthetic erythropoiesis stimulating protein comprises a
bioactivity that is equal to or better than a corresponding
synthetic protein having a polypeptide chain that is devoid of said
water-soluble polymer. The bioactivity can be in vitro, in vivo or
both. In a preferred embodiment, the bioactivity is in vivo. In
another preferred embodiment, the invention is directed to a
synthetic erythropoiesis stimulating protein comprising a
polypeptide chain comprising an amino acid sequence of a
ribosomally specified erythropoietin, where the polypeptide chain
has one or more water-soluble polymers attached thereto and a
monomer molecular weight of greater than 25 kDa, and where the
synthetic erythropoiesis stimulating protein comprises a
bioactivity that is equal to or better than the ribosomally
specified erythropoietin. Here again, the bioactivity can be in
vitro, in vivo or both. In a preferred embodiment, the bioactivity
is in vivo. Most preferably, such polymer modified synthetic
erythropoiesis stimulating proteins will have a discrete number of
water-soluble polymers attached thereto.
[0058] By "chemical ligation site" is intended the N-terminal amino
acid of a first peptide or polypeptide and the C-terminal amino
acid of a second peptide or polypeptide that form or are capable of
forming a non-reversible covalent bond therein between by chemical
ligation. As used herein, "chemical ligation" refers to a
chemoselective reaction involving the covalent joining of two
chemical moieties, each of which moieties bears a mutually reactive
functional group that is uniquely capable of forming a
non-reversible covalent bond with the other. Chemical ligation
includes covalent ligation of (1) a first peptide or polypeptide
bearing a uniquely reactive C-terminal group with (2) a second
peptide or polypeptide bearing a uniquely reactive N-terminal
group, where the C-terminal and N-terminal reactive groups form a
non-reversible covalent bond therein between. In particular,
chemical ligation includes any chemoselective reaction chemistry
that can be applied to ligation of unprotected peptide
segments.
[0059] With respect to the water-soluble polymer, preferred
water-soluble polymers can be represented by the formula:
Un-s1-B-s2-Polymer-s3-J*
[0060] U is a residue of a unique functional group covalently
bonded to the polypeptide chain of the synthetic erythropoiesis
stimulating protein. In particular, the U group is bonded to a
mutually reactive unique functional group of a side chain n of one
or more amino acids of one or more of the non-overlapping peptide
segments of the synthetic erythropoiesis stimulating protein, and
where n is a discrete integer from 1 to 6. More preferably side
chain n is a discrete integer from 1 to 4, and most preferably from
1 to 2. As used herein the term "amino acid" is intended to include
the 20 genetically coded amino acids, rare or unusual amino acids
that are found in nature, and any of the non-naturally occurring
amino acids, such as irregular amino acids; sometimes referred to
as amino acid residues when in the context of a peptide,
polypeptide or protein. Preferred embodiments of U and side chain n
are covalent bonds formed from unique mutually reactive groups,
where such bonds are selected from oxime, amide, amine, urethane,
ether, thioether, ester, hydrazide, oxazolidine, thaizolidine,
thioether, ether, and ester. The most preferred U and n bond is one
where U is covalently bonded to side chain n through a bond formed
by chemical ligation selected from the group consisting of amide,
oxime, thioester, hydrazone, thaizolidine, oxazolidine.
[0061] B is a branching core having three or more arms that may be
the same or different and may be present or absent. Most preferably
B is present and comprises three or more arms, and even more
preferably four or more arms. In particular, one arm of B is joined
to U (optionally through a spacer or linker s1), and a second arm
of B is joined to Polymer (optionally through a spacer or linker
s2). A favored polymer-modified synthetic erythropoiesis
stimulating protein is one in which at least one of the branching
arms of moiety B comprises a residue of bond selected from the
group consisting of oxime, amide, amine, urethane, thioether,
ester, hydrazide, oxazolidine, and thaizolidine. A preferred
branching group B of a polymer-modified synthetic erythropoiesis
stimulating protein of the invention comprises a branching core
selected from the group consisting of amino, carboxylate and mixed
amino-carboxylate. Preferred amino branching core comprise lysine,
preferred carboxylate branching core comprise glutamic or aspartic
acid, and preferred mixed amino-carboxylate branching core
comprises gamma-glutamic acid, or derivatives thereof.
[0062] The Polymer component is a substantially non-antigenic
water-soluble polymer that may be the same or different where B is
present. By "water-soluble polymer" is intended a substantially
non-antigenic polymer that is soluble in water and has an atomic
molecular weight greater than about 1,000 Daltons. The Polymer will
preferably have an effective hydrodynamic molecular weight of
greater than 10,000 Da, and more preferably about 20,000 to 500,000
Da, and most preferably about 40,000 to 300,000 Da. By "effective
hydrodynamic molecular weight" is intended the effective
water-solvated size of a polymer chain as determined by
aqueous-based size exclusion chromatography (SEC). When the
water-soluble polymer contains polymer chains having polyalkylene
oxide repeat units, such as ethylene oxide repeat units, it is
preferred that each chain have an atomic molecular weight of
between about 200 and about 80,000 Da and preferably between about
1,500 and about 42,000 Da, with 2,000 to about 20,000 Da being most
preferred. Unless referred to specifically, molecular weight is
intended to refer to atomic molecular weight.
[0063] The Polymer component can have a wide range of molecular
weight, and polymer subunits. These subunits may include a
biological polymer, a synthetic polymer, or a combination thereof.
Examples of such water-soluble polymers include: dextran and
dextran derivatives, including dextran sulfate, P-amino cross
linked dextrin, and carboxymethyl dextrin, cellulose and cellulose
derivatives, including methylcellulose and carboxymethyl cellulose,
starch and dextrines, and derivatives and hydroylactes of starch,
polyalklyene glycol and derivatives thereof, including polyethylene
glycol, methoxypolyethylene glycol, polyethylene glycol
homopolymers, polypropylene glycol homopolymers, copolymers of
ethylene glycol with propylene glycol, wherein said homopolymers
and copolymers are unsubstituted or substituted at one end with an
alkyl group, heparin and fragments of heparin, polyvinyl alcohol
and polyvinyl ethyl ethers, polyvinylpyrrolidone, aspartamide, and
polyoxyethylated polyols, with the dextran and dextran derivatives,
dextrine and dextrine derivatives. It will be appreciated that
various derivatives of the specifically recited water-soluble
polymers are also contemplated.
[0064] Water-soluble polymers such as those described above are
well known, particularly the polyalkylene oxide based polymers such
as polyethylene glycol "PEG" (See. e.g., "Poly(ethylene glycol)
Chemistry: Biotechnical and Biomedical Applications", J. M. Harris,
Ed., Plenum Press, New York, N.Y. (1992); and "Poly(ethylene
glycol) Chemistry and Biological Applications", J. M. Harris and S.
Zalipsky, Eds., ACS (1997); and International Patent Applications:
WO 90/13540, WO 92/00748, WO 92/16555, WO 94/04193,WO 94/14758, WO
94/17039, WO 94/18247, WO 94/28937, WO 95/11924, WO 96/00080, WO
96/23794, WO 98/07713, WO 98/41562, WO 98/48837, WO 99/30727, WO
99/32134, WO 99/33483, WO 99/53951, WO 01/26692, WO 95/13312, WO
96/21469, WO 97/03106, WO 99/45964, and U.S. Pat. Nos. 4,179,337;
5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264;
5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808;
5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877; 5,455,027;
5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460;
5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388;
5,681,567; 5,686,110; 5,730,990; 5,739,208; 5,756,593; 5,808,096;
5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; 5,900,461;
5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566;
5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939; 6,113,906;
6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966).
[0065] The more preferred Polymer component comprises a
polyalkylene oxide, polyamide alkylene oxide, or derivatives
thereof. A more favored polyalkylene oxide and polyamide alkylene
oxide comprise an ethylene oxide repeat unit of the formula
--(CH2-CH2-O)--. An even more preferred Polymer component is a
polyamide having a molecular weight greater than about 1,000
Daltons of the formula --[C(O)--X--C(O)--NH--Y--NH]n- or
--[NH--Y--NH--C(O)--X--C(O)]n-, where X and Y are divalent radicals
that may be the same or different and may be branched or linear,
and n is a discrete integer from 2-100, and more preferably from 2
to 50, and where either or both of X and Y comprises a
biocompatible, substantially non-antigenic water-soluble repeat
unit that may be linear or branched. The most preferred
water-soluble repeat unit comprises an ethylene oxide of the
formula --(CH.sub.2--CH.sub.2--O)-- or --(CH.sub.2--CH.sub.2--O)---
. The number of such water-soluble repeat units can vary
significantly, but the more preferred number of such units is from
2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, and most
preferably 2 to 50. An example of a more preferred embodiment is
where one or both of X and Y is selected from:
--((CH.sub.2).sub.n1--(CH.sub.2--CH.sub.2--O).sub.n2--(CH.sub.2)--
or
--((CH.sub.2).sub.n1--(O--CH.sub.2--CH.sub.2).sub.n2--(CH.sub.2).sub.n-
1--), where n1 is 1 to 6, 1 to 5, 1 to 4 and most preferably 1 to
3, and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8, and
most preferably 2 to 5. An example of a highly preferred embodiment
is where X is --(CH.sub.2--CH.sub.2)--, and where Y is
--(CH.sub.2--(CH.sub.2--CH.su-
b.2--O).sub.3--CH.sub.2--CH.sub.2--CH.sub.2)-- or
--(CH.sub.2--CH.sub.2--C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.3--CH.sub.2)--.
[0066] The Polymer component or one or more of the spacers or
linkers, when present, may include polymer chains or units that are
biostable or biodegradable. For example, Polymers with repeat
linkages have varying degrees of stability under physiological
conditions depending on bond lability. Polymers with such bonds can
be categorized by their relative rates of hydrolysis under
physiological conditions based on known hydrolysis rates of low
molecular weight analogs, e.g., from less stable to more stable
polycarbonates (--O--C(O)--O--)>polyesters
(--C(O)--O--)>polyurethanes (--NH--C(O)--O--)>polyorthoesters
(--O--C((OR)(R'))--O--)>polyamides (--C(O)--NH--). Similarly,
the linkage systems attaching a water-soluble polymer to a target
molecule may be biostable or biodegradable, e.g., from less stable
to more stable carbonate (--O--C(O)--O--)>ester
(--C(O)--O--)>urethane (--NH--C(O)--O--)>orthoester
(--O--C((OR)(R'))--O--)>amide (--C(O)--NH--). These bonds are
provided by way of example, and are not intended to limit the types
of bonds employable in the polymer chains or linkage systems of the
water-soluble polymers of the invention.
[0067] Component J* is a residue of pendant group having a net
charge under physiological conditions selected from the group
consisting of negative, positive and neutral. This includes alkyl,
aryl, heteroalkyl, heteroaryl, arylalkyl, acyl, alkoxy, alkenyl,
alkynyl, amideo, amino, carbonyl groups and the like, that are
substituted or unsubstituted, and as well as salts thereof. Neutral
groups preferably are alkyl or alkoxy groups, and can include, but
are not limited to moieties containing from 1 to 18 carbons, and
may be linear or branched. When provided as a charged group, J*
comprise an ionizable functional group. Examples of functional
groups include, but are not limited to, carboxylic acids, esters,
amides, nitriles, thiols, and hydroxyls. Such J* groups may be a
component of amino acids, nucleic acids, fatty acids,
carbohydrates, and derivatives thereof, and moieties such as
chitin, chitosan, heparin, heparan sulfate, chondroitin,
chondroitin sulfate, dermatan and dermatan sulfate, cyclodextrin,
dextran, hyaluronic acid, phospholipid, sialic acid and the like.
J* preferably comprises an ionizable moiety selected from carboxyl,
amino, thiol, hydroxyl, phosphoryl, guanidinium, imidazole and
salts thereof. The most preferred is where J* comprises an
ionizable carboxylate moiety and has a net negative charge under
physiological conditions. For instance, the preferred
polymer-modified synthetic erythropoiesis stimulating proteins of
the invention have an isoelectric point between 3 and 7, and thus
by J* groups with negative charges can be exploited for fine tuning
the pl.
[0068] The components s1, s2, and s3 are spacer or linker moieties
that may be the same or different, and may be individually present
or absent. Preferred spacers or linkers include linear or branched
moieties comprising one or more repeat units employed in a
water-soluble polymer, diamino and or diacid units, natural or
unnatural amino acids or derivatives thereof, as well as aliphatic
moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy,
and the like, which preferably contain up to 18 carbon atoms or
even an additional polymer chain. Most preferably the spacer or
linker comprises a polymer chain.
[0069] Alternatively, the above formula Un-s1-B-s2-Polymer-s3-J*
can be represented as "Un-B-Polymer-J*" where the s1, s2 and s3
groups may be present or absent.
[0070] The more preferred Polymer component is one where the
water-soluble polymer U-s1-B-s2-Polymer-s3-J* is produced in total
by stepwise synthesis. This permits one to construct polymers
having a precise molecular weight and defined structure. In
contrast, normal polymer synthesis, which is a polymerization
process, results in a mixture in which chains are of differing
lengths, and so there is a distribution of molecular weights and
sizes that are difficult if not impossible to separate. The ability
to control molecular purity is advantageous in that a synthetic
protein can be constructed that has a water-soluble polymer
attached thereto and that is monodisperse. This represents a
significant advantage in that variable properties resulting from
heterogeneous compounds can be avoided, and only those compounds
with the most preferred properties can be prepared and isolated
with relative ease.
[0071] In another preferred embodiment, the synthetic
erythropoiesis stimulating proteins of the present invention will
comprise one or more irregular amino acids. As used here,
"irregular amino acid" refers to an amino acid having a
non-genetically encoded side chain, a non-genetically encoded
backbone, a non-genetically encoded substituted N.alpha. or
.alpha.C(O) moiety, or a combination thereof, i.e., other than one
of the ribosomally installed 20 genetically encoded amino acids.
Examples of preferred irregular amino acids include amino acids
having side chains bearing a unique functional group other than a
genetically encoded functional group, as well as pseudo amino acids
and various amino acid derivatives. In this regard, the present
invention permits wide selectability and flexibility in the design
and/or construction of synthetic erythropoiesis stimulating
proteins. Examples of non-ribosomally installed amino acids that
may be used in accordance with a present invention include, but are
not limited to: D-amino acids, .beta.-amino acids,
pseudo-glutamate, .gamma.-aminobutyrate, ornithine, homocysteine,
N-substituted amino acids (R. Simon et al., Proc. Natl. Acad. Sci.
U.S.A. (1992) 89: 9367-71; WO 91/19735 (Bartlett et al.), U.S. Pat.
No. 5,646,285 (Baindur), .alpha.-aminomethyleneoxy acetic acids (an
amino acid-Gly dipeptide isostere), and .alpha.-aminooxy acids and
other amino acid derivatives having non-genetically non-encoded
side chain function groups etc. Peptide analogs containing
thioamide, vinylogous amide, hydrazino, methyleneoxy,
thiomethylene, phosphonamides, oxyamide, hydroxyethylene, reduced
amide and substituted reduced amide isosteres and
.beta.-sulfonamide(s) may be employed. By "pseudo amino acid" is
intended an amino acid having an identical backbone structure and
side-chain group as a genetically encoded amino acid, but differing
in the atomic composition of the side chain atoms.
[0072] In a preferred embodiment, synthetic erythropoiesis
stimulating proteins are provided that have a water-soluble polymer
attached to an irregular amino acid of a polypeptide chain thereof.
More preferred irregular amino acids for attachment of a
water-soluble polymer thereto employ chemoselective ligation
chemistry that can be used in the presence of genetically encoded
functional groups without reacting with them.
[0073] As noted above, water-soluble polymers that are made in
total by stepwise assembly can be made as monodisperse, for example
the preferred polyamide ethylene oxides of the invention. Thus
another preferred embodiment is one where the water-soluble polymer
is mono-disperse (i.e., a molecularly homogenous composition
containing a single and structurally defined molecular species of
interest). Also, as the synthetic erythropoiesis stimulating
proteins can be made in total by chemical synthesis they can be
made mono-disperse as well. Such compounds have the advantage of
being highly pure and avoid the problems of purification and
analytical characterization as when hetero-disperse polymers are
employed. Such compounds are advantageous in terms of reproducible
dosing and the like as well (for example, a single molecular
species as opposed to mixtures typical of glycoproteins and
PEGylated recombinantly produced proteins).
[0074] As noted above, the synthetic erythropoiesis stimulating
protein of the invention can have a monomer molecular weight of
greater than 25 kDa. For the purposes of the present invention,
such determinations of molecular weight are to be made by
denaturing SDS polyacrylamide electrophoresis. The term "monomer
molecular weight" is intended to refer to the molecular weight of a
monomer synthetic protein, as distinguished from synthetic proteins
that may possess multiple copies of a protein or polypeptide. The
term "monomer polypeptide molecular weight" is intended to refer to
the molecular weight of a monomer polypeptide, as distinguished
from synthetic proteins that may possess polymers attached thereto
and/or multiple copies of a protein or polypeptide. More
preferably, the synthetic erythropoiesis stimulating protein of the
invention will have a monomer molecular weight of above 40 kDa, and
more preferably 50 kDa and above, with the most preferred being
above 60 to 70. For example, a preferred synthetic erythropoiesis
stimulating protein of the invention, designated SEP-1-B51 has a
monomer molecular weight of about 73 kDa. However, much larger
constructs are considered to be well within the scope of the
present invention.
[0075] In another embodiment, the invention is directed to a
synthetic erythropoiesis stimulating protein comprising a pseudo
amino acid at a ligation site of the protein, and optionally, a
water-soluble polymer attached to the protein. These compounds are
produced by forming a ligation product having an unprotected
side-chain functional group at the ligation site by ligating a
first peptide or polypeptide segment having an N-terminal amino
acid comprising a chemoselective reactive functional group, such as
a cysteine, to a second peptide or polypeptide having a chemical
ligation compatible C-terminal functional group, such as a
.alpha.-carboxy thioester, and chemically converting the
unprotected side-chain functional group at the ligation site to a
pseudo amino acid, such as carboxymethylation of the cysteine thiol
side-chain to form a pseudo glutamate amino acid. Pseudo amino
acids formed by conversion of cysteines at native chemical ligation
sites is referred to herein as "Pseudo Native Chemical Ligation,"
which is described in detail below.
[0076] Also provided is a synthetic erythropoiesis stimulating
protein comprising a water-soluble polymer attached to a side-chain
of an amino acid at a ligation site of the protein. These compounds
are synthesized by forming a ligation product having an unprotected
side-chain functional group at the ligation site by ligating a
first peptide or polypeptide segment having an N-terminal amino
acid comprising a chemoselective reactive functional group, such as
a cysteine, to a second peptide or polypeptide having a ligation
compatible C-terminal functional group, such as a .alpha.-carboxy
thioester, and attaching a water-soluble polymer to the unprotected
side-chain functional group at the ligation site.
[0077] Utilization of pseudo amino acid chemical ligation and the
chemical ligation site polymer modification methods of the
invention afford several advantages over the prior art, including
the robust synthesis of a diverse range of erythropoiesis
stimulating proteins that are otherwise devoid of suitable ligation
sites, expansion of the sites (and attachment chemistries) to which
site-specific polymer modification can be exploited in a routine
and cost-effective manner, as well as the synthesis of synthetic
erythropoiesis stimulating proteins of substantial molecular
weight, among others. They also are particularly suited for high
throughput analoging for fine-tuning of desired biological
properties, including scanning individual or multiple sites for
water-soluble polymer attachment.
[0078] As noted above, the biological properties of the
polymer-modified synthetic erythropoiesis stimulating proteins of
the invention can be modified by precisely adjusting the sites and
linkage chemistries of polymer attachment in combination with the
precision adjustment of the molecular weight, the polymer
composition, the structure (e.g., linear versus branched, or
mixtures thereof), and the pendant group (e.g., charged versus
uncharged, or mixtures thereof) of the water-soluble polymer. In
particular, in addition to increasing the molecular weight of a
protein to improve half-life, and branching etc., the water-soluble
polymer attached thereto can render the synthetic erythropoiesis
stimulating protein to have a precise charge, as measured by
isoelectric point that is approximately equal to the charge of a
corresponding biologically produced protein on which the amino acid
sequence of the synthetic erythropoiesis stimulating protein is
based. This has the advantage of mimicking the natural charge of a
corresponding ribosomally specified protein. A preferred embodiment
of the invention is thus directed to synthetic erythropoiesis
stimulating proteins that combine the above features, as well as
the water-soluble polymers utilized therefor, particularly
structurally defined polymer adducts that are capable of being
attached at preselected positions.
[0079] In particular, preferred synthetic erythropoiesis
stimulating proteins ("SEPs") of the present invention are
preferably polymer-modified synthetic analogs of human urinary EPO.
In a preferred embodiment, they contain one or more water-soluble
polymers attached to one or more peptide residue(s) through a
thioether, oxime, amide or other linkage. In a highly preferred
embodiment, such linkages will be at one or more of the positions
that are naturally glycosylated in human urinary EPO (i.e.,
positions 24, 38, 83 and 126). Most preferably, the SEP molecules
will have polymer moieties at two or more of such positions. In an
alternative embodiment, other protein residues may be modified by
polymer moieties. The positions of modification for the synthetic
erythropoiesis stimulating proteins of the present invention
include residues located in a disordered loop, region or domain of
the protein, or at or near sites of potential protease cleavage.
For example, polymer modifications may be introduced at one or more
positions of 9, 69 and/or 125 of EPO. Moreover, if one or more of
the natural glycosylation sites are left unmodified with a polymer,
one or more amino acids in those regions can be converted to other
residues if desired, for example, lysines if a positive charge is
desired, or aspartic or glutamic acids where a negative charge is
desired, or alanine and the like. The total molecular weight of the
SEP molecules of the present invention may vary from about 25 to
about 150 kDa, and more preferably from about 30 to about 80 kDa.
The molecular weight can be controlled by increasing or decreasing
the number and structure of the water-soluble polymer utilized for
modification of a given analog.
[0080] For example, oxime-linked polymer SEP analogs are preferably
constructed by attaching an aminooxy, ketone or aldehyde
functionalized water-soluble polymer to the SEP protein at a
non-naturally encoded amino acid bearing a side chain aminooxy,
ketone or aldehyde functionality. For example, positions 89 and 117
of SEP-0 and 1 contain pseudo glutamates (a non-naturally encoded
amino acid bearing a side chain of the formula
--CH.alpha.-CH.sub.2--S--COOH (as compared to glutamate side chain
--CH.alpha.-CH.sub.2--CH.sub.2--COOH)). SEP analogs utilizing
thioether linkages are preferably constructed to contain a thiol
functionality provided by a cysteine or unnatural amino acid with a
side chain bearing the thiol. FIG. 10 depicts the basic structure
of one type of preferred synthetic erythropoiesis stimulating
protein.
[0081] In an alternative embodiment, the SEP molecules of the
present invention may comprise "circularly permuted" EPO analogs in
which the natural amino and carboxy terminus of EPO have been
relocated. Most preferably, such relocation will move the amino and
carboxy termini to positions of low structural constraint, such as
to disordered loops, etc. The disordered loop around positions 125
and 126 (relative to the native EPO residue numbering system) is an
example of such a relocation site. Most preferably, such SEPs will
be disulfide free, and will be chemically modified to contain
polymer moieties at preselected residues.
[0082] Alternatively, the SEP molecules may have amino and carboxy
termini relocated to a naturally occurring glycosylation site, or
to other sites amenable to glycosylation, such as position 126 and
125. The SEP molecules may also include modifications of the amino
and carboxy termini to eliminate or modify charge (such as by
carboxy amidation, etc.). In a preferred example of such circularly
permuted molecules, new N- and C-termini are provided by positions
126 and 125, respectively. The natural disulfide-forming cysteines
at positions 7, 29, 33 and 161 are preferably replaced by the
non-naturally encoded amino acid, L-.alpha.-N-butyric acid (Aba),
which cannot form disulfide bridges. Residues R166, E37 and V82 are
preferably replaced with alanines to improve production. Also, an
additional cysteine is preferably inserted between positions 1 and
166 relative to the native EPO number scheme, which is numbered
below as `0`. The resulting molecule contains four cysteines (at
positions 126, 0, 38, and 83 (as read in the N- to C-terminal
direction)), which are utilized as (1) ligation sites and (2)
thioether-forming pPEG attachment sites. Optionally, a cysteine may
replace A125 to provide an additional pPEG attachment site.
[0083] The total molecular weight of such SEP molecules of the
present invention may vary from about 25 to about 150 kDa, and more
preferably from about 30 to about 80 kDa. The molecular weight can
be controlled by increasing or decreasing the number and structure
of the polymer (such as pPEG) utilized for modification of a given
analog. The pPEG mediated hydrodynamic MW for the larger constructs
is greater than 100 kDa. Optional pPEG attachment sites are located
at positions 125, 9, and 24 (as read in the N- to C-terminal
direction). Additional SEP analog designs have alternative N- and
C-termini in the disordered loop region, and/or truncate residues
from the new N- and/or C-termini. The basic structure of preferred
circularly permuted molecules is shown in FIG. 11. FIG. 12 shows a
structure of a preferred water-soluble polymer, and various linear
and branched constructs.
[0084] For example, in addition to the specific SEP constructs
described in the Examples herein, a sequence was designed by
circularly permuting the native sequence of human EPO in the
following manner: the natural N-terminal Ala.sup.1 and the
C-terminal Arg.sup.166 were joined by an additional Cys residue,
thus giving a polypeptide of 167 amino acids; new N- and
C-terminals were created by disjoining the chain at residues
125-126 of the native sequence; all the native Cys residues were
replaced with L-.sup..alpha.amino-n-butyric acid residues; and the
following substitutions were made: Glu37Ala; Asn38Cys; Val82Ala;
Asn83Cys; Ser126Cys; Arg166Ala (all residue numbers based on the
native sequence of human EPO). Together, these design elements
result in the amino acid sequence of SEP-5 as shown. The SEP-5-L28
protein was polymer-modified at Cys residues 126, 0, 24, 38, and 83
(numbering based on the human EPO sequence) with a
maleimide-modified linear (TTD-Succ).sub.6 carboxylate polymer
construct, by a Michael addition reaction. These changes were
designed to improve synthesis and handling of the SEP-5-L28
protein. The designed sequence was made from four peptide segments,
each with an N-terminal Cys residue. The chemical ligation sites
were Cys.sup.0, Cys.sup.38, Cys.sup.83. Peptides were synthesized
where the C-terminal peptide was an .alpha.-carboxylate; the other
three peptides were .alpha.-thioesters, and the N-terminal Cys
residues were thus side chain Acm protected. Cys.sup.24 was side
chain unprotected. The segments were joined sequentially by native
chemical ligation, starting with the C-terminal two segments. After
the ligation, the free Cys side chain at the ligation site was
reacted with the maleimide-modified linear (TTD-Succ).sub.6
carboxylate polymer construct, by a Michael addition reaction, then
the Cys side chain Acm protecting group was removed, and ligation
of the next segment and polymer modification performed in similar
fashion. After removal of the Acm group, ligation of the next
(fourth) segment was performed, the final Acm group was removed and
polymer modification was performed in similar fashion giving the
circularly-permuted, polymer-modified SEP construct described above
and shown in FIG. 30.
[0085] II. Production of the Synthetic Erythropoiesis Stimulating
Proteins of the Invention
[0086] Although polymer-modified erythropoiesis stimulating
proteins have been previously described, the present invention
markedly differs from such prior efforts. For instance, the
synthetic erythropoiesis stimulating proteins of the present
invention are chemically synthesized, in whole or in part. In
addition, the synthetic erythropoiesis stimulating proteins of the
invention are modified with water-soluble polymers at one or more
particular, user-selected and user-defined sites.
[0087] Moreover, the synthetic production of the erythropoiesis
stimulating proteins in the present invention permits one to ensure
that such modifications are present at each of the user-selected
and user-defined sites of every molecule in a preparation. Such
uniformity and control of synthesis markedly distinguishes the
present invention from the random modifications permitted to the
use of the methods of the prior art. Significantly, the present
invention permits one to design a synthetic erythropoiesis
stimulating protein in which any non-critical residue may be
derivatized to contain a polymer adduct. Moreover, for each such
user-selected and user-defined site, the user may define the
precise linkage (amide, thioester, thioether, oxime, etc.) through
which such adducts will be bonded to the polypeptide or protein
backbone. Additionally, the particular polymer adducts desired to
be present at a particular site may be varied and controlled, such
that a final preparation is obtained in which every protein or
polypeptide present contains precisely the same derivatized adducts
at precisely the same derivatized sites. Thus, the present
invention permits the formation of homogeneous preparations of
polymer-modified erythropoiesis stimulating polypeptides and
proteins.
[0088] In addition to providing a means for varying the position
and number of attachment sites to which a water-soluble polymer can
be bound, the present invention permits one to vary the nature of
the bound polymer. The polymer adducts that may be incorporated
into any particular user-selected and user-defined site can be of
any defined length. Likewise, consistent with the methods of
present invention, it is possible to employ polymers of different
lengths at different sites. Thus, in one embodiment, the
polymer-modified synthetic erythropoiesis stimulating proteins of
the present invention may be either mono-modified, or
poly-modified, with a water-soluble polymer adduct. Where more than
one polymer adduct is introduced into a particular polypeptide or
protein, the employed polymers may be "mono-speciated,"
"poly-speciated," "uniformly speciated," or to "diversely speciated
in." As used herein, the term "mono-speciated" is intended to refer
to a polypeptide or protein that has been modified by a single
species of polymer. In contrast, the term "poly-speciated" is
intended to refer to a polypeptide or protein that has been
modified by more than a single polymer species. Such poly-speciated
polypeptides or proteins are said to be "uniformly-speciated" if,
at each modified site of the polypeptide or protein, the same,
single species of polymer is present. In contrast, a poly-speciated
polypeptide or protein is said to be "diversely-speciated" if, the
modified sites of the polypeptide or protein are modified with
different species of polymer.
[0089] Moreover, it is possible to vary the extent of linearity or
branchedness of the polymer adduct at each of the user-selected in
user-defined sites. Thus the polymer adducts may be linear,
branched, or uniformly branched. The term "uniformly branched" is
intended to mean that all branches of a polymer at a particular
site have the same structure and length. As we appreciate, the
present invention permits one to independently vary both the length
of any individual branch, as well as the structure of the polymer
present at such branch point.
[0090] In sum, the present invention permits one to define (1) the
location and (2) frequency of polymer-modified, user-selected and
user-defined sites in a polypeptide or protein backbone, as well as
to control the (3) length, (4) species, and (5) degree of branching
present at each such site. Additionally, with respect to
polypeptide and proteins having multiple polymer modifications, the
present invention permits one to independently define each of the
above-identified five variables for each site. Moreover, with
respect synthetic erythropoiesis stimulating polypeptides and
proteins having branched polymer modifications, the present
invention permits one to independently define each of the
above-identified five variables for each branch point. This allows
for the synthesis of homogenous compositions of precision
polymer-modified synthetic erythropoiesis stimulating proteins
having multiple copies of any or all of the 20 genetically encoded
amino acid side chains present and left unencumbered by unwanted
polymer attachment.
[0091] Thus the invention affords significant flexibility in the
design, synthesis and use of polymer-modified synthetic
erythropoiesis stimulating proteins. For instance, the amino acid
sequence of the synthetic erythropoiesis stimulating proteins of
the invention may comprise one or more deletions, insertions or
substitutions relative to the amino acid sequence of a genetically
encoded protein on which it is based. The amino acid sequence can
be substituted with one or more irregular amino acids, such as a
pseudo amino acid and or other amino acid bearing an unnatural side
chain the like, for example, one that is modified to bear a unique
chemoselective functional group for attaching a water-soluble
polymer adduct thereto. Thus, the water-soluble polymer can be
attached through an irregular amino acid, or a genetically encoded
amino acid. The water-soluble polymer can be linear or branched,
and it can have a terminal group comprising a chemical moiety such
as a carboxylic acid, aliphatic, amide or amine. Moreover, the
water-soluble polymer can be monodisperse, i.e., it can be made to
comprise a single molecular species of precisely defined structure
and composition. Thus, the water-soluble polymer can be designed to
contain features to precisely tune half-life, immunogenicity,
potency, storage stability, dosage, deliverability and the like of
the target protein modified therewith.
[0092] In particular, the production of the polymer-modified
synthetic erythropoiesis stimulating proteins of the invention can
be envisioned as having the following steps: design, peptide
synthesis, peptide ligation, folding of the full-length ligation
product to generate protein, and assessment of the protein's
bioactivity. We have found that polymer modification can be
performed at one or more of the peptide synthesis, ligation or on
the folded product steps. It is preferred, however, to attach
polymer to the peptides or to the ligation product prior to
folding. Proteins produced in this manner that exhibit a desired
bioactivity are selected and represent the synthetic erythropoiesis
stimulating proteins of the invention.
[0093] In a preferred method, a polypeptide chain comprising a
synthetic erythropoiesis stimulating protein is made chemically
ligating peptide segments comprising non-overlapping amino acid
sequences of a polypeptide chain of the synthetic erythropoiesis
stimulating protein. In particular, synthetic erythropoiesis
stimulating protein molecules of the invention can be made by
chemically ligating peptide segments comprising non-overlapping
amino acid sequences of a polypeptide chain of the synthetic
erythropoiesis stimulating protein molecule of interest, where one
or more of the peptide segments used for ligation has a
water-soluble polymer attached thereto at a user-defined and
pre-selected site. The polymer-modified polypeptide chain may then
be folded to produce a polymer-modified synthetic erythropoiesis
stimulating protein of the invention.
[0094] Another preferred method for producing the synthetic
erythropoiesis stimulating proteins of the invention comprises
chemically ligating peptide segments comprising non-overlapping
amino acid sequences of a polypeptide chain of a synthetic
erythropoiesis stimulating polymer-modified protein of the
invention, and attaching one or more water-soluble polymers to a
side-chain of an amino acid at one or more chemical ligation sites
thereof. The polymer-modified polypeptide chain may then be folded
to produce a polymer-modified synthetic erythropoiesis stimulating
protein of the invention.
[0095] Although less preferred, synthetic erythropoiesis
stimulating proteins can be made by (1) chemically ligating peptide
segments comprising non-overlapping amino acid sequences of a
polypeptide chain of a synthetic erythropoiesis stimulating protein
to form a full-length polypeptide chain corresponding to the
synthetic erythropoiesis stimulating protein, where at least one
peptide segment comprises an irregular amino acid having a first
chemoselective functional group, (2) folding the polypeptide chain,
and (3) attaching a water-soluble polymer thereto that comprises a
second chemoselective group that is uniquely and mutually reactive
with the first chemoselective group.
[0096] In particular, these various processes embodied in the
invention, and as exemplified in the Examples, can be illustrated
as shown in the Figures for synthetic erythropoiesis stimulating
proteins in general. For example, FIGS. 1A-1E depict ligation
schemes involving the attachment of a water-soluble polymer
(U--B-Polymer-J* as defined herein) to partially or fully
unprotected peptide segments () before or after ligation, or
combinations thereof. In FIGS. 1A-1D, Y.sub.aa represents the
C-terminal amino acid on a first peptide segment that bears a
unique chemoselective moiety (e.g., amino acid bearing
alpha-carboxyl thioester) for chemical ligation to a second peptide
segment bearing a unique and mutually reactive N-terminal amino
acid X.sub.aa (e.g., amino terminal cysteine) moiety that is
capable of chemoselective chemical ligation with Y.sub.aa.
Chemoselective reaction between Y.sub.aa and X.sub.aa generate a
covalent linkage therein between (e.g., amide bond). Thus Y.sub.aa
and X.sub.aa form a chemoselective ligation pairing. U.sub.n--, as
shown in the Figures, represents a second unique chemoselective
moiety that has been incorporated at a precise user-defined site on
the side chain of an amino acid and is chemoselective for, and
mutually reactive with group U-- of the water-soluble polymer
U--B-Polymer-J*. For example, when U.sub.n is a side chain that has
been modified to bear a ketone group, U--of the water-soluble
polymer U-Polymer-J* is a group chemoselective for reacting with
the ketone, e.g., an aminooxy group which yields an oxime bond
therein between. The subscript "n" of U.sub.n--represent the number
of amino acids and their side chains designed to bear
chemoselective group U, for example, where two specific and
user-defined sites are to be polymer modified n=2, which also can
be represented as U.sub.2 or U.sub.n=2. In all cases, n is a
positive integer that is precisely controlled by design. Thus
U.sub.n and U represent a second chemoselective ligation pairing
that is compatible and unreactive with chemoselective groups of
X.sub.aa and Y.sub.aa. FIGS. 1A and 1B illustrate two different
potential reactions. In the first depicted reaction, a polypeptide
chain bearing a U.sub.n functionality is ligated to a second
polypeptide, and is then reacted with a U--B-Polymer-J* moiety in
order to obtain a polymer-modified polypeptide. In the second
depicted reaction, the polypeptide chain bearing the U.sub.n
functionality is reacted with a U--B-Polymer-J* moiety in order to
obtain a polymer-modified polypeptide, and then ligated to a second
polypeptide, to obtain a larger polymer-modified polypeptide. The
figures differ in that in FIG. 1A, the polypeptide bearing the Xaa
residue is to receive the polymer modification, whereas in FIG. 1B,
the polypeptide bearing the Yaa residue receives the polymer
modification. FIG. 1C illustrates the ability of the present
invention to modify multiple polypeptide chains, either before or
after their ligation to form a larger polypeptide. In FIG. 1D, PG
and PG' represent protecting groups, where PG' depicts an
orthogonal protecting group, i.e., PG and PG' are removable under
different conditions, and are useful where different water-soluble
polymers are attached via same chemistry to U.sub.n groups, or
where U.sub.n groups represent side-chain functional groups that
one does not wish to modify with a polymer (e.g., side chains
bearing reactive --NH.sub.2 or --SH where U group of water-soluble
polymer is designed to react exclusively with primary amino or side
chain thiols). FIG. 1D shows that protecting groups can be employed
in order to protect desired side chains of the polypeptides being
ligated in accordance with the methods of the present
invention.
[0097] FIG. 1E illustrates the diversity of groups that may be
present or absent in the peptide segments employed in ligation and
polymer modification according to FIGS. 1A-1D.
[0098] FIGS. 2A-2C depict additional schematics of processes for
preparing synthetic erythropoiesis stimulating proteins of the
invention. In particular, FIGS. 2A-2B depict ligation scheme
involving the attachment of a water-soluble polymer U--B-Polymer-J*
to the side chain of amino terminal group Xaa at a ligation site
(e.g., side chain thiol of cysteine). FIG. 2C illustrates the
diversity of groups that may be present or absent in the peptide
segments employed in ligation and polymer modification according to
FIGS. 2A-2B.
[0099] FIGS. 3A-3B depict additional schematics of processes for
accomplishing multi-segment ligations that involve the chemical
ligation of three or more non-overlapping peptide segments, i.e.,
at least one segment is a middle segment corresponding to the final
full-length ligation product. Peptides prepared in this manner can
be used for preparing peptide segments involved in another ligation
reaction, for example, as shown in FIGS. 1A-1E, FIGS. 2A-2C, and
FIGS. 4A-4C. In general, for multi-segment ligations, the middle
segment(s) has either a protected Xaa group or a protected Yaa
group to avoid cyclization or concatomer formation of that peptide
depending on the ligation chemistry employed. For sequential or
serial ligations, the Xaa group of a middle segment is protected
(e.g., Cys(Acm)) while the Yaa group is unprotected (e.g.,
Yaa-COSR, where --COSR is an alpha-carboxyl thioester). Here the
Yaa group is free to react with a second peptide bearing an
unprotected Xaa group, where the second peptide is devoid of a free
Yaa group. Following ligation, the protecting group is removed to
regenerate the Xaa group for the next ligation reaction. This
process can be continued, as needed thereby generating an elongated
polypeptide chain. Protection of the Yaa group is particularly
useful for convergent chemical ligation involving the production of
a final ligation product composed of four or more segments. For
example, for a protein target generated from a four-segment
ligation (i.e., three ligation reactions), two segments
corresponding to one end of the protein and two segments
corresponding to the other end of the protein can be ligated in
parallel, as opposed to sequentially, and the two ends joined in a
final ligation reaction. Such a convergent chemical ligation
schemes also can employ orthogonal ligation chemistries. Here again
the diversity of groups that may be present or absent in such
peptide segments are illustrated in FIGS. 1E, 2C and 4C.
[0100] FIGS. 4A-4C illustrate how native chemical ligation and
chemical modification of the resulting side-chain thiol may be
accomplished in accordance with the principles of the present
invention. In particular, FIGS. 4A-4B depict the use of native
chemical ligation and chemical modification of the resulting
cysteine side-chain thiol at the ligation site(s) to form a "pseudo
amino acid" (depicted by .psi.Xaa) via thioalkylation and
generation of chemically modified side chain (depicted by .psi.)
comprising a thioether bond. In an alternative embodiment not
depicted, the side chain thiol can be converted to an alanine in a
desulfurization reaction (Liang et al, J. Amer. Chem. Soc. (2001)
123(4):526-533). An significant aspect for both reactions is that
any other side-chain thiols that one does not wish to convert or
modify be protected with a suitable protecting group (PG) or for
multi-segment ligations and orthogonal protecting group (PG') where
the segment bearing the carboxy terminal Yaa group comprises a
protected amino terminal Xaa group, i.e., PG-Xaa-peptide, which is
provided by way of example in FIG. 4B. FIG. 4C illustrates the
diversity of groups that may be present or absent in the peptide
segments employed in ligation and polymer modification according to
FIGS. 4A-4B, as well as FIGS. 1A-1E, FIGS. 2A-2C, and FIGS.
3A-3B.
[0101] In conjunction with the design, the peptides or polypeptide
segments utilized for synthesizing the polypeptide backbone are
constructed. This involves selection of suitable ligation sites
that are chosen based on the ligation chemistry selected for
assembling the various polypeptide backbone segments, the polymer
attachment chemistry chosen for a given target protein, and the
particular polymer attachment sites. When native chemical ligation
is employed, cysteine ligation sites are determined by scanning the
target polypeptide backbone amino acid sequence for suitable
naturally occurring cysteine residue. When Extended Native Chemical
Ligation" is employed, as described herein, ligation sites can be
selected by scanning the target polypeptide backbone amino acid
sequence for suitable naturally occurring ligation site junctions
that permit robust ligations, such as Xaa-Gly sites. Because
extended native chemical ligation is not limited to ligation at
cysteine residues, any number of residues may serve as the ligation
site junction. In some instances, a combination of native and
extended native chemical ligation may be part of the design.
[0102] In a preferred embodiment, native chemical ligation is used
to generate part or all of the full-length polypeptide chain.
Cysteines present in the naturally occurring protein on which the
synthetic erythropoiesis stimulating protein is based can be used
as the chemical ligation sites. However, where a preferred ligation
junction is devoid of a suitable cysteine, the non-cysteine amino
acid at that position can be replaced with a cysteine so as to
permit native chemical ligation at that site. If desired, the newly
introduced cysteine can be converted to a pseudo amino acid residue
corresponding to the original amino acid at that position, as
described herein. Alternatively, when the cysteine is introduced at
a site for polymer modification, the side chain thiol can be
exploited for the attachment of a thiol-reactive water-soluble
polymer construct, provided that all other cysteines in the target
polypeptide that one does not wish to modify are protected.
[0103] In another preferred embodiment, extended native chemical
ligation, as described herein, can be utilized to generate part or
all of the full-length polypeptide. For this method, N-terminal
N.alpha.-substituted 2 or 3 carbon chain alkyl or aryl thiol amino
acids may be employed. Such residues (where present at the
N-terminus of a peptide or polypeptide segment used for ligation)
can be advantageously used to ligate that polypeptide to a
polypeptide having a C-terminal .alpha.-carboxy thioester moiety,
in accordance with the methods of extended native chemical ligation
described herein.
[0104] Typically, the synthesis of peptides employs stepwise
standard Boc and/or Fmoc solid phase peptide synthesis using
standard automated peptide synthesizers, or manually following
standard protocols, or ordered and purchased from commercial
vendors. ("Synthetic Peptides, A User's Guide," G. A. Grant, Ed.,
W. H. Freeman & Company, New York, N.Y., 1992; "Priciples of
Peptide Synthesis, 2nd ed.," M. Bodanszky, Ed., Springer-Verlag,
1993; "The Practice of Peptide Synthesis, 2nd ed.," M. Bodanszky
and A. Bodanszky, Eds., Springer-Verlag, 1994; and "Protecting
Groups," P. J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart,
Germany, 1994; "Fmoc Solid Phase Peptide Synthesis, A Practical
Approach, Eds. W. C. Chan and P. D. White, Oxford University Press,
2000). For peptides utilized for thioester-mediated ligation, such
as for native chemical ligation, they can be made following
standard protocols as well. (see, e.g., Dawson et al., Science
(1994) 266:776-779; Canne et al. Tetrahedron Lett. (1995)
36:1217-1220; Kent, et al., WO 96/34878; Kent, et al., WO 98/28434;
Ingenito et al., JACS (1999) 121(49):11369-11374; and Hackeng et
al., Proc. Natl. Acad. Sci. U.S.A. (1999) 96:10068-10073); Amiato
et al., supra.).
[0105] For ligation and site-specific attachment of water-soluble
polymers, chemically orthogonal strategies are employed in the
synthesis of the peptides (See, e.g., FIGS. 1-4, and Examples) so
as to avoid side reactions that result in unwanted attachment. For
instance, depending on ligation design and the polymer attachment
approach, a variety of orthogonal synthesis strategies can be
exploited. In particular, the nature of the water-soluble polymer
to be attached, and in particular the functional group for joining
it to the polypeptide are considered, for instance as discussed
below for the preferred glyco-mimetic polymers of the
invention.
[0106] In particular, the water-soluble polymer is made to comprise
a unique functional group U that is selectively reactive with a
unique functional group on a target peptide employed for ligation,
full-length material or even the folded polypeptide. As chemical
synthesis is employed, the peptide is made to contain a mutually
reactive chemoselective group at a precise, user-defined site. This
aspect of the invention embodies the principles of peptide
synthesis (protecting group strategies) and chemical ligation
(partial or no protecting group strategies). For the protecting
group strategy, all potentially reactive functional groups except
for the desired functional group on the water-soluble polymer and
its mutually reactive functional group present on the target
molecule are blocked with suitable protecting groups. Many
protecting groups are known and suitable for this purpose (See,
e.g., "Protecting Groups in Organic Synthesis", 3rd Edition, T. W.
Greene and P. G. M. Wuts, Eds., John Wiley & Sons, Inc., 1999;
NovaBiochem Catalog 2000; "Synthetic Peptides, A User's Guide," G.
A. Grant, Ed., W. H. Freeman & Company, New York, N.Y., 1992;
"Advanced Chemtech Handbook of Combinatorial & Solid Phase
Organic Chemistry," W. D. Bennet, J. W. Christensen, L. K. Hamaker,
M. L. Peterson, M. R. Rhodes, and H. H. Saneii, Eds., Advanced
Chemtech, 1998; "Principles of Peptide Synthesis, 2nd ed.," M.
Bodanszky, Ed., Springer-Verlag, 1993; "The Practice of Peptide
Synthesis, 2nd ed.," M. Bodanszky and A. Bodanszky, Eds.,
Springer-Verlag, 1994; and "Protecting Groups," P. J. Kocienski,
Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994).
[0107] Thus, the water-soluble polymer can represent be made to
posses a wide range of functional groups, such as those described
above. For the partial or no protecting group strategy, the
functional group on the polymer and its mutually reactive
functional group present on the target peptide or polypeptide
employ a chemoselective reaction pair in which other functional
groups may be present in the reaction system but are unreactive.
This includes groups amenable to amine capture strategies (e.g.,
ligation by hemiaminal formation, by imine formation, and by
Michael addition), thiol capture strategies (e.g., ligation by
mercaptide formation, by disulfide exchange), native chemical
ligation strategies (e.g., ligation by thioester exchange involving
cysteine or thiol contain side-chain amino acid derivative), and
orthogonal ligation coupling strategies (e.g., ligation by
thiazolidine formation, by thioester exchange, by thioester
formation, by disulfide exchange, and by amide formation) (See,
e.g., Coltart, D M., Tetrahedron (2000) 56:3449-3491).
[0108] A preferred chemoselective U group for this embodiment will
comprise a residue of a unique functional group employed in an
aqueous compatible ligation chemistry such as native chemical
ligation (Dawson, et al., Science (1994) 266:776-779; Kent, et al.,
WO 96/34878), extended general chemical ligation (Kent, et al., WO
98/28434), oxime-forming chemical ligation (Rose, et al., J. Amer.
Chem. Soc. (1994) 116:30-33), thioester forming ligation
(Schnolzer, et al., Science (1992) 256:221-225), thioether forming
ligation (Englebretsen, et al., Tet. Letts. (1995)
36(48):8871-8874), hydrazone forming ligation (Gaertner, et al.,
Bioconj. Chem. (1994) 5(4):333-338), and thiazolidine forming
ligation and oxazolidine forming ligation (Zhang, et al., Proc.
Natl. Acad. Sci. (1998) 95(16):9184-9189; Tam, et al., WO 95/00846)
or by other methods (Yan, L. Z. and Dawson, P. E., "Synthesis of
Peptides and Proteins without Cysteine Residues by Native Chemical
Ligation Combined with Desulfurization," J. Am. Chem. Soc. 2001,
123, 526-533, herein incorporated by reference; Gieselnan et al.,
Org. Lett. 2001 3(9):1331-1334; Saxon, E. et al., "Traceless"
Staudinger Ligation for the Chemoselective Synthesis of Amide
Bonds. Org. Lett. 2000, 2, 2141-2143).
[0109] Given the various attachment chemistries described above,
the bond formed between the water-soluble polymer and a target
peptide or polypeptide can comprise a residue of a bond selected
from carbonate, ester, urethane, orthoester, amide, amine, oxime,
imide, urea, thiourea, thioether, thiourethane, thioester, ether,
thaizolidine, hydrazone, oxazolidine and the like. The most
preferred bonds are oxime and amide bonds.
[0110] The peptide ligation step, for example as shown in FIGS.
1-4, may employ solid or solution phase ligation strategies. FIG. 8
depicts another route for precision attachment of a water-soluble
polymer to a peptide segment employable for ligation and production
of polymer-modified synthetic erythropoiesis stimulating proteins
of the invention. The first step employs solid phase peptide
synthesis ("SPPS") (e.g., Fmoc or Boc SPPS), in which an amino acid
side chain targeted for polymer attachment is protected with an
orthogonal protecting group (e.g., if using Fmoc SPPS, a Boc group
can be used to protect the site of polymer attachment, or if using
Boc SPPS, an Fmoc group can be employed as the orthogonal
protecting group). Following peptide synthesis, the orthogonal
protecting group is selectively removed while the rest of the
peptide remains protected. This affords a single attachment site
for the next step--solid phase polymer synthesis. Once the
orthogonal protecting group is removed, the polymer chain is
attached as a precursor. More preferably, the polymer chain is
built through successive rounds of polymer synthesis using a
process depicted in FIGS. 6A-6D. Although a single polymer
attachment site is shown, more than one can be provided. FIG. 9
shows a general reaction scheme.
[0111] As noted above, chemical ligation involves the formation of
a selective covalent linkage between a first chemical component and
a second chemical component. Unique, mutually reactive, functional
groups present on the first and second components can be used to
render the ligation reaction chemoselective. For example, the
chemical ligation of peptides and polypeptides involves the
chemoselective reaction of peptide or polypeptide segments bearing
compatible unique, mutually reactive, C-terminal and N-terminal
amino acid residues. Several different chemistries have been
utilized for this purpose, examples of which include native
chemical ligation (Dawson, et al., Science (1994) 266:776-779;
Kent, et al., WO 96134878; Kent, et al., WO 98/28434), oxime
forming chemical ligation (Rose, et al., J. Amer. Chem. Soc. (1994)
116:30-34), thioester forming ligation (Schnolzer, et al., Science
(1992) 256:221-225), thioether forming ligation (Englebretsen, et
al., Tet. Letts. (1995) 36(48):8871-8874), hydrazone forming
ligation (Gaertner, et al., Bioconj. Chem. (1994) 5(4):333-338),
and thiazolidine forming ligation and oxazolidine forming ligation
(Zhang, et al., Proc. Natl. Acad. Sci. (1998) 95(16):9184-9189;
Tam, et al., WO 95/00846; U.S. Pat. No. 5,589,356); Gieselman et
al., Selenocysteine-mediated native chemical ligation (Org. Lett.
(2001) 3(9):1331-1334); and Staudinger amide forming chemical
ligation (Saxon et al., Org. Lett. (2000) 2:2141-2143). Thus, as
will be appreciated, any chemoselective reaction chemistry that can
be applied to the ligation of unprotected peptide segments that is
amenable for such purpose.
[0112] Reaction conditions for a given ligation chemistry are
selected to maintain the desired interaction of the peptide or
polypeptide segments employed for ligation. For example, pH and
temperature, water-solubility of the ligation label, ratio of the
first segment to the second segment, water content and composition
of the reaction mixture can be varied to optimize ligation.
Addition or exclusion of reagents that solubilize the ligation
segments to different extents may further be used to control the
specificity and rate of the desired ligation reaction, i.e.,
control exposure and presentation of reactive groups by
manipulating solubility of the peptide or polypeptide segments.
Reaction conditions are readily determined by assaying for the
desired chemoselective reaction product compared to one or more
internal and/or external controls.
[0113] Where the ligation involves the joining of a polypeptide
that possesses an N-terminal cysteine residue, the procedure of
native chemical ligation is preferably employed (Dawson, et al.,
Science (1994) 266:776-779; Kent, et al., WO 96/34878; Kent, et
al., WO 98/28434)). This methodology has proven a robust
methodology for generating a native amide bond at the ligation
site. Native chemical ligation involves a chemoselective reaction
between a first peptide or polypeptide segment having a C-terminal
.alpha.-carboxythioester moiety and a second peptide or polypeptide
having an N-terminal cysteine residue. A thiol exchange reaction
yields an initial thioester-linked intermediate, which
spontaneously rearranges to give a native amide bond at the
ligation site while regenerating the cysteine side chain thiol. In
many instances, the sequence of the natural protein will comprise
suitably placed cysteine residues such that polypeptide fragments
having an N-terminal cysteine residue may be synthesized and used
in a native chemical ligation reaction. In other instances, the
peptide synthesis can be conducted so as to introduce cysteine
residues into a polypeptide for this purpose. Thus in its standard
form, native chemical ligation involves thioester-mediated
chemoselective reaction at a cysteine residue in a target
polypeptide sequence; a peptide bond is formed at the ligation site
and the side chain of the Cys is regenerated in native form.
[0114] Alternatively, the proteins of the present invention may be
synthesized through the use of "Pseudo-Native Chemical Ligation,"
or "Extended Native Chemical Ligation. Pseudo-Native Chemical
Ligation involves the use of non-naturally occurring pseudo-amino
acid residues at preselected positions in the peptides employed in
the protein synthesis (e.g., See FIG. 4). The structures of such
pseudo-amino acids mimic both the structures of cysteine and the
structures of the amino acids that are naturally found at such
preselected positions in the protein being synthesized.
Pseudo-native chemical ligation is thus directed to the
thioalkylation of cysteine side chains generated at ligation sites
from native chemical ligation. A preferred aspect is thioalkylation
of cysteine ligation sites wherein at least one peptide contains a
native cysteine having its thiol side chain protected with a
suitable protecting group.
[0115] In a preferred embodiment of the invention, the thiol moiety
of a cysteine group is modified into a desired side chain, for
example, into the side chain of a ribosomally specified amino acid,
an analog of such an amino acid, or into a non-ribosomally
specified amino acid. As used herein, a ribosomally specified amino
acid is an amino acid that is recognized by ribosomes in the
process of protein translation and can be incorporated into a
ribosomally produced protein. Considerable published literature
exists describing chemical modifications of the cysteine side chain
thiol moiety (see, e.g., "Current Protocols in Protein Science,"
Edited by: John E. Coligan et al., John Wiley & Sons, NY
(2000)). Kaiser, E. T. has described the conversion of cysteine
residue side chains to mimic the chemical properties of a naturally
occurring amino acid side chain (see, e.g., Kaiser, E. T. et al.,
"Chemical Mutation Of Enzyme Active Sites," Science. Nov. 2,
1984;226(4674):505-11). Additionally, the use of a cysteine side
chain to introduce a label into a peptide or protein has been
described. Cysteine side chain modifications are reviewed in
Chemistry of Protein Conjugation and Crosslinking, S. S. Wong,
(1991, CRC Press); Chemical Modification of Proteins, Gary E. Means
et al., (1971, Holden-Day), Chemical Modification of Proteins:
Selected methods and analytical procedures, Glazer, A. N. et al.
(1975, Elsevier); Chemical Reagents for Protein Modification, R L
Lundblad (1991, CRC Press). Tam et al. (Biopolymers (1998)
46:319-327) have disclosed the use of homocysteine
(--CH.sub.2--CH.sub.2--SH) for non-cys native chemical ligation,
followed by thioalkylation using methyl p-nitrobenzenesulfonate
(methylating reagent) to convert the homocysteine side chain to a
native methionine side chain (--CH.sub.2--CH.sub.2--S--CH.sub.3).
The present invention also can be used for converting homocysteines
to pseudo amino acids as well, i.e., to amino acids other than
methionine. However, as with the conversion of cysteines described
herein, in accordance with the present invention it is necessary to
use protecting groups to avoid destruction of native cysteines
involved in disulfide pairing for peptides that contain at least
one native cysteine that one does not wish to convert. Suitable
protecting groups are described below.
[0116] While the method of pseudo-native chemical ligation does not
facilitate the mimicking of the side chains of certain
ribosomally-specified amino acids (e.g., the side chains of
glycine, alanine, valine, and proline) (alanine's side chain can,
however, be formed through a desulfurization reaction (Liang, Z. Y.
and Dawson, P. E., "Synthesis of Peptides and Proteins without
Cysteine Residues by Native Chemical Ligation Combined with
Desulfurization," J. Am. Chem. Soc. 2001, 123, 526-533, herein
incorporated by reference), it may be used to form side chains that
mimic many ribosomally-specified or non-encoded amino acids. Amino
acids produced in accordance with the pseudo-native chemical
ligation method of the present invention will contain a thioether
linkage, and will have no beta-branching (in that they will all
include a methyl group at the beta position, i.e.,
aa-CH.sub.2--S--. Thus, the pseudo-amino acid versions of the
beta-branched amino acids, isoleucine and threonine can be made to
have the pendant side chain structure, without having the beta
geometry and its attendant constraints.
[0117] Significantly, the methods of the present invention may be
used to form amino acid side chains that are the same length as
that of ribosomally specified amino acids, or are longer or shorter
than such length. Such alteration in side chain length can be used
to stabilize (or destabilize) the three-dimensional conformation to
increase protein stability (or to enhance the ability of the
protein to alter its conformation and thereby accept a different
range of substrates, inhibitors, receptors, ligands, etc. relative
to those accepted by the naturally occurring protein. For example,
Cys-CH.sub.2--SH+Br--CH.sub.2--- COOH yields
Cys-CH.sub.2--S--CH.sub.2--COOH (such "pseudo-glutamic acid" has
one additional side chain atom, namely the --S-- group;
alternatively, if used in the place of aspartic acid, it will
possess two additional side chain atoms, namely a --CH.sub.2--S--
group). Other side chains have the same number of atoms in the side
chain, but differ by inclusion of the thioether linkage (--S--).
For example, Cys-CH.sub.2--SH+Br--CH.sub.2--CH.sub.2--NH--PG,
followed by removal of PG yields
Cys-CH.sub.2--S--CH.sub.2--CH.sub.2--NH.sub.2. The resulting
structure has no additional atoms in the side chain, but one
--CH.sub.2-- group is replaced with --S--. Methionine is another
example here, Cys-CH.sub.2--SH+I--CH.sub.2--CH.sub.3 yields
Cys-CH.sub.2--S--CH.sub.2--- CH.sub.3 (versus native met structure
of Met-CH.sub.2--CH.sub.2--S--CH.sub- .3); thus the thioether is
relocated. Arginine also:
Cys-CH.sub.2--SH+Br--CH.sub.2--NH--CH((--NH.sub.2)(.dbd.NH.sub.2.sup.+))
yields
Cys-CH.sub.2--S--CH.sub.2--NH--CH((--NH.sub.2)(.dbd.NH.sub.2.sup.+-
)). Preferably, protection of reactive amino groups, particularly
for the constructing pseudo lysine can be employed to avoid
unwanted side reactions. Once the thioalkylation reaction is
performed, the protecting group can be removed.
[0118] In general, where the desire is to mimic a naturally
occurring protein as closely as possible, it is most preferred to
employ a pseudo amino acid molecule having a side chain length that
is the same length as that of the ribosomally-specified amino acid
normally present at such position in the protein; it is less
preferred to employ a pseudo amino acid molecule having a side
chain length that is one atom longer than that of the
ribosomally-specified amino acid, and still less preferred to
employ a pseudo amino acid molecule having a side chain length that
is two atoms longer than that of the ribosomally-specified amino
acid. Moreover, its is preferred to select a cysteine ligation site
that is in a location where genetic changes are not likely to
disrupt function or where amino acids at that site in related
proteins are conserved. Such sites can be identified by alanine
scanning, homology modeling, and other methods.
[0119] In pseudo-native chemical ligation, a peptide containing an
amino terminal cysteine residue is ligated to a peptide having a
carboxy terminal thioester, as in native chemical ligation. The
thiol side chain of the cysteine is then reacted with a compound of
the formula R.sub.aa--X, where X is a good leaving group, and
R.sub.aa is a group whose structure mimics the terminal portion of
the side chain of an ribosomally-specified or synthetic amino
acid.
[0120] Significantly, the reactions of pseudo-native chemical
ligation work with either the natural L-configuration of cysteine
side chain, or the D-configuration. Use of the D-configuration can
impart protease resistance at that ligation site, and thus may be
desired when increased stability to proteolysis is desired.
However, in using the D-cysteine, the backbone structure at that
site will be altered. Such alteration, in addition to protease
resistance, may be desired to alter bioactivity. However, to
minimize impact on bioactivity, it is preferred to locate the
D-cysteine at a site of high flexibility, such as a disorded
region, such as at a disorded loop that will be located on the
surface of the resulting folded molecule, on at a disorded terminus
of the molecule. Desirably, the reactions of pseudo-native chemical
ligation may be used to place large, charged side chains (e.g., the
side chains of Lys, Arg, Asp or Glu) on the surface of the
synthesized molecule.
[0121] Examples of suitable good leaving groups, X, include
halogens, especially Iodine and Bromine. Examples of R.sub.aa
groups include PO.sub.4, COOH, COO, CONH.sub.2, guanidinium, amine,
alkyl, substituted alkyl, aryl, substituted aryl, imidazole,
alkylated imidazole, indole, or alkylated indole groups.
[0122] The selection of which R.sub.aa to employ will depend upon
the amino acid side chain desired to be present at a particular
position. Thus, for example, a desired polypeptide or protein
having the amino acid sequence:
aa.sub.NH.sub..sub.2-Q-aa.sub.x-aa.sub.y-W-aa.sub.COOH
[0123] where Q and W each denote the optional presence or absence
of additional amino acid residues, and aa.sub.x and aa.sub.y denote
internal adjacent residues (having side chains x and y,
respectively), and aa.sub.NH.sub..sub.2 and respectively denote the
amino (N--) termimal residue and the carboxy (C--) terminal residue
of the polypeptide or protein an be synthesized by preparing two
peptide fragments:
aa.sub.NH.sub..sub.2-Q-aa.sub.x-COSR
[0124] and
Cys-W-aa.sub.COOH
[0125] where Cys denote the replacement of aa.sub.y with cysteine,
and R is any group compatible with the thioester group, including,
but not limited to, aryl, benzyl, and alkyl groups. Examples of R
include 3-carboxy-4-nitrophenyl thioesters, benzyl esters, and
mercaptoproprionic acid leucine esters (See, e.g., Dawson et al.,
Science (1994) 266:776-779; Canne et al. Tetrahedron Lett. (1995)
36:1217-1220; Kent, et al., WO 96/34878; Kent, et al., WO 98/28434;
Ingenito et al., JACS (1999) 121(49):11369-11374; and Hackeng et
al., Proc. Natl. Acad. Sci. U.S.A. (1999) 96:10068-10073). Other
examples include dithiothreitol, or alkyl or aryl thioesters, which
can be produced by intein-mediated biological techniques, which
also are well known (See, e.g., Chong et al., Gene (1997)
192:277-281; Chong et al., Nucl. Acids Res. (1998) 26:5109-5115;
Evans et al., Protein Science (1998) 7:2256-2264; and Cotton et
al., Chemistry & Biology (1999) 6(9):247-256); and then
ligating the fragments together to form:
aa.sub.NH.sub..sub.2-Q-aa.sub.x-Cys-W-aa.sub.COOH.
[0126] The ligated fragment is then reacted with R.sub.y--X, where
R.sub.y is a side group that mimics the structure of the y side
chain). The reaction is conducted under conditions sufficient to
convert the thiol group of the cysteine into a "pseudo-y" side
chain. For example, if R.sub.aa is selected to be CH.sub.2--COOH or
(CH.sub.2).sub.2--COOH, then the reaction will lead to the
formation an amino acid residue that mimics the structure and
function of aspartic acid ("pseudo-Asp") or glutamic acid
("pseudo-Glu"). As will be appreciated, in light of the above
description, more complicated synthesis can be conducted employing
more than two peptide fragments.
[0127] A significant feature of this approach is that cysteine
residues that one does not wish to modify in the reacting segments
be side chain protected, e.g. as Cys(Acm), to prevent chemical
modification of Cys residues other than the one(s) at the ligation
site(s), or that any other Cys residues in the reacting segments be
intended for the simultaneous formation of identical `pseudo amino
acids` by the chemical modification reaction after ligation.
[0128] As used herein, the symbol .phi. denotes a benzyl group; IM
denotes an imidazole group, and IN denotes an indole group; PG
denotes a protecting group. Below is a summary of R.sub.aa side
groups that may be used to synthesize peptides containing
pseudo-amino acid residues in accordance with the present invention
(where X is a halogen (I, Br, Cl, F, with I and Br preferred for
most, and F preferred for .phi. attachment):
[0129] Basic Amino Acids:
[0130] Lys (no extra atoms)
--CH.sub.2--SH+X--CH.sub.2--CH.sub.2--NH--PG, followed by
deprotection
gives --CH.sub.2--S--CH.sub.2--CH.sub.2--NH.sub.2
[0131] Arg (no extra atoms)
--CH.sub.2--SH+X--CH.sub.2--NH--C((NH.sub.2)(.dbd.NH.sub.2+))
gives
--CH.sub.2--S--CH.sub.2--NH--C((NH.sub.2)(.dbd.NH.sub.2+))
[0132] His (2 extra atoms)
--CH.sub.2--SH+X--CH.sub.2--IM gives
--CH.sub.2--S--CH.sub.2--IM
[0133] Acidic Amino Acids:
[0134] Asp (2 extra atoms)
--CH.sub.2--SH+X--CH.sub.2--COOH gives
--CH.sub.2--S--CH.sub.2--COOH
[0135] Glu (1 extra atoms)
--CH.sub.2--SH+X--CH.sub.2--COOH gives
--CH.sub.2--S--CH.sub.2--COOH
[0136] Uncharged Polar Amino Acids:
[0137] Tyr (no or 1 extra atom)
--CH.sub.2--SH+F-.phi.-pOH gives --CH.sub.2--S-.phi.-pOH)(no extra
atoms, same geometry
--CH.sub.2--SH+Br/I--CH.sub.2-.phi.-pOH gives
--CH.sub.2--S--CH.sub.2-.phi- .-pOH (1 extra atom)
[0138] Gln (1 extra atom)
--CH.sub.2--SH+X--CH.sub.2--C(O)(NH.sub.2) gives
--CH.sub.2--S--CH.sub.2--- C(O)(NH.sub.2)
[0139] Asn (2 extra atoms)
--CH.sub.2--SH+X--CH.sub.2--C(O)(NH.sub.2) gives
--CH.sub.2--S--CH.sub.2--- C(O)(NH.sub.2)
[0140] Ser (2 or 3 extra atoms)
--CH.sub.2--SH+X--CH.sub.2OH) gives --CH.sub.2--S--CH.sub.2OH (2
extra atoms)
--CH.sub.2--SH+X--CH.sub.2--CH.sub.2OH) gives
--CH.sub.2--S--CH.sub.2--CH.- sub.2OH (3 extra atoms)
[0141] Thr (2 or 3 extra atoms, missing beta branching)
--CH.sub.2--SH+X--CH((CH.sub.3)(O--PG)) followed by removal of
PG
gives --CH.sub.2--S--CH((CH.sub.3)(OH)) (2 extra atom)
--CH.sub.2--SH+X--CH.sub.2--CH((CH.sub.3)(O--PG)) followed by
removal of PG
gives --CH.sub.2--S--CH.sub.2--CH((CH.sub.3)(OH)) (3 extra
atoms)
[0142] Non-Polar Amino Acids:
[0143] Leu (1 or 2 extra atoms)
--CH.sub.2--SH+X--CH((CH.sub.3)(CH.sub.3)) gives
--CH.sub.2--S--CH((CH.sub- .3)(CH.sub.3)) (1 extra atom)
--CH.sub.2--SH+X--CH.sub.2--CH((CH.sub.3)(CH.sub.3)) gives
--CH.sub.2--S--CH.sub.2--CH((CH.sub.3)(CH.sub.3)) (2 extra
atoms)
[0144] Ile (2 or 3 extra atoms, missing beta branching)
--CH.sub.2--SH+X--CH(CH.sub.3)--CH.sub.2--CH.sub.3 gives
--CH.sub.2--S--CH(CH.sub.3)--CH.sub.2--CH.sub.3 (2 extra atoms)
--CH.sub.2--SH+X--CH.sub.2--CH(CH.sub.3)--CH.sub.2--CH.sub.3 gives
--CH.sub.2--S--CH.sub.2--CH(CH.sub.3)--CH.sub.2--CH.sub.3 (3 extra
atoms)
[0145] Phe (1 or 2 extra atoms)
--CH.sub.2--SH+F-.phi. gives --CH.sub.2--S-.phi. (1 extra atom)
--CH.sub.2--SH+Br/I--CH.sub.2-.phi. gives
--CH.sub.2--S--CH.sub.2-.phi. (2 extra atoms)
[0146] Met (no extra atoms)
--CH.sub.2--SH+I--CH.sub.2--CH.sub.3 gives
--CH.sub.2--S--CH.sub.2--CH.sub- .3
[0147] Trp (1 extra atom)
--CH.sub.2--SH+F--IN gives --CH.sub.2--S--IN
[0148] However, where it is either inconvenient or undesirable to
modify a protein sequence so as to introduce a cysteine or
homocysteine residue at a given N-terminus of a polypeptide
utilized for ligation, or utilize a pseudo amino acid at the
ligation site, the method of native chemical ligation may be
extended using polypeptides whose N-terminus has been modified to
contain an N-substituted, and preferably, N.alpha.-substituted, 2
or 3 carbon chain amino alkyl or aryl thiol, and thereby permit the
principles of native chemical ligation to be employed with
polypeptides lacking cysteine residues (see, U.S. patent
application Ser. No. 60/231,339, herein incorporated by
reference).
[0149] The method of "Extended Native Chemical Ligation" involves
ligating a first component comprising a carboxyl thioester, and
more preferably, an .alpha.-carboxyl thioester with a second
component comprising an acid stable N-substituted, and preferably,
N.alpha.-substituted, 2 or 3 carbon chain amino alkyl or aryl
thiol. Chemoselective reaction between the carboxythioester of the
first component and the thiol of the N-substituted 2 or 3 carbon
chain alkyl or aryl thiol of the second component proceeds through
a thioester-linked intermediate, and resolves into an initial
ligation product. More specifically, the thiol exchange occurring
between the COSR thioester component and the amino alkyl thiol
component generates a thioester-linked intermediate ligation
product that after spontaneous rearrangement generates an
amide-linked first ligation product through a 5-membered or
6-membered ring intermediate depending upon whether the amino alkyl
thiol component has formula I or II, respectively:
J1-C(O)--N(C1(R1)-C2-SH)-J2 I
J1-C(O)--N(C1(R1)-C2(R2)-C3(R3)-SH)-J2 II
[0150] where J1 is a peptide or polypeptide having one or more
optionally protected amino acid side chains, or a moiety of such
peptide or polypeptide, a polymer, a dye, a suitably functionalized
surface, a linker or detectable marker, or any other chemical
moiety compatible with chemical peptide synthesis or extended
native chemical ligation; R1, R2 and R3 are independently H or an
electron donating group conjugated to C1; with the proviso that at
least one of R1, R2 and R3 comprises an electron donating group
conjugated to C1; and J2 is a peptide or polypeptide having one or
more optionally protected amino acid side chains, or a moiety of
such peptide or polypeptide, a polymer, a dye, a suitably
functionalized surface, a linker or detectable marker; or any other
chemical moiety compatible with chemical peptide synthesis or
extended native chemical ligation.
[0151] The N-substituted 2 or 3 carbon chain alkyl or aryl thiol
[HS--C2-C1 (R1)-] or [HS--(C3(R3)-C2(R2)-C1(R1)-] at the ligation
site is amenable to being removed, under peptide-compatible
conditions, without damage to the product, to generate a final
ligation product of formula III, having a native amide bond at the
ligation site:
J1-C(O)--HN-J2 III
[0152] where J1, J2, R1, R2, and R3 are as defined above.
[0153] The R1, R2 and R3 groups are selected to facilitate cleavage
of the N--C1 bond under peptide compatible cleavage conditions. For
example, electron donating groups, particularly if conjugated to
C1, can be used to form a resonance stabilized cation at C1 that
facilitates cleavage. The chemical ligation reaction preferably
includes as an excipient a thiol catalyst, and is carried out
around neutral pH conditions in aqueous or mixed organic-aqueous
conditions. Chemical ligation of the first and second components
may proceed through a five or six member ring that undergoes
spontaneous rearrangement to yield an N-substituted amide linked
ligation product. Where the first and second components are
peptides or polypeptides, the N-substituted amide linked ligation
product has formula IV or V:
J1-C(O)--N.alpha.(C1(R1)-C2-HS)--CH(Z2)-C(O)-J2 IV
J1-C(O)--N.alpha.(C1(R1)-C2(R2)-C3(R3)-HS)--CH(Z2)-C(O)-J2 V
[0154] where J1, J2 and R1, R2, R3 and Z2 are as defined above
[0155] The conjugated electron donating groups R1, R2 or R3 of the
N-substituted amide bonded ligation product facilitate cleavage of
the N--C1 bond and removal of the 2 or 3 carbon chain alkyl or aryl
thiol from the N-substituted amide-linked ligation product. Removal
of the alkyl or aryl thiol chain of the N under peptide-compatible
cleavage conditions generates a ligation product having a native
amide bond at the ligation site. Where the first and second
components are peptides or polypeptides, the ligation product will
have the formula:
J1-CON.alpha.H--CH(Z2)-C(O)-J2 X
[0156] Exemplary R1 subtituents for Formula I are depicted in Table
1.
1TABLE I Formula I 1 R1 Substituent Groups for Formula I (C1
included for reference) 2
[0157] Exemplary R1, R2 and R3 substituents for Formula II are
depicted in Table 2.
2 Formula II 3 R1, R2 and R3 Substituents (C1 included for
reference) 4 5
[0158] As with the N-substituted 2 carbon chain compounds,
positioning of the benzyl and picolyl electron-donating
substituents R1', R3' and R5' in the ortho or para positions is
necessary to maintain electronic conjugation to the C1 carbon for
robust cleavage of the N.alpha.-C1 bond following ligation.
However, when R2 and R3 form a benzyl group with C2 and C3, at
least one of R1' and R3' comprises a strong electron donating
group, where R1' or R3' is selected from methoxy (--OCH3), thiol
(--SH), hydroxyl (--OH), and thiomethyl (--SCH3). For the
N-substituted 3 carbon chain thiols in which R2 and R3 are
hydrogens, R1 comprises a benzyl or picolyl group in which R1', R3'
and R5' include either strong or moderate electron-donating groups,
or a combination thereof. As with the N-substituted 2 carbon chain
alkyl or aryl thiols, the strong electron-donating groups enhance
the sensitivity of the 3 carbon chain alkyl or aryl thiol to
cleavage following ligation. Thus a particular electron-donating
group or combination thereof can be selected accordingly.
[0159] Similar to the N-substituted 2 carbon chain compounds, the
N-substituted 3 carbon chain compounds of the present invention may
include a thiol as a substituent of R1 in the R1' and R5' positions
when available for substitution in a construct of interest. Here
again the electron-donating thiol group is conjugated to C1 and its
introduction at these locations enables the compounds to have two
routes for the 6-member ring forming ligation event. It also
increases the local concentration of available thiols for reacting
with the .alpha.-carboxy thioester, and provides for additional
conformations in terms of structural constraints that can improve
ligation.
[0160] Synthesis of the N-terminal N-substituted 2 or 3 carbon
chain alkyl or aryl thiol amino acids of the invention can carried
out as described herein, for example, in Scheme I and Scheme II,
and in accordance with standard organic chemistry techniques known
in the art. See, e.g., "Advanced Organic Chemistry, Reactions,
Mechanisms, and Structure," 4.sup.th Edition, J. March (Ed.), John
Wiley & Sons, New York, N.Y., 1992; "Comprehensive Organic
Transformations, A Guide to Functional Group Preparations," R.
Larock (Ed.), VCH Publishers, New York, N.Y., 1989. They may be
synthesized in solution, by polymer-supported synthesis, or a
combination thereof. The preferred approach employs N alpha
protected N alkylated S-protected amino alkyl- or aryl-thiol amino
acid precursors. The reagents utilized for synthesis can be
obtained from any number of commercial sources. Also, it will be
well understood that the starting components and various
intermediates, such as the individual amino acid derivatives can be
stored for later use, provided in kits and the like.
[0161] In preparing the N-terminal N.alpha.-substituted 2 or 3
carbon chain alkyl or aryl thiol amino acids of the invention,
protecting group strategies are employed. The preferred protecting
groups (PG) utilized in the various synthesis strategies in general
are compatible with Solid Phase Peptide Synthesis ("SPPS"). In some
instances, it also is necessary to utilize orthogonal protecting
groups that are removable under different conditions. Many such
protecting groups are known and suitable for this purpose (See,
e.g., "Protecting Groups in Organic Synthesis", 3rd Edition, T. W.
Greene and P. G. M. Wuts, Eds., John Wiley & Sons, Inc., 1999;
NovaBiochem Catalog 2000; "Synthetic Peptides, A User's Guide," G.
A. Grant, Ed., W. H. Freeman & Company, New York, N.Y., 1992;
"Advanced Chemtech Handbook of Combinatorial & Solid Phase
Organic Chemistry," W. D.. Bennet, J. W. Christensen, L. K.
Hamaker, M. L. Peterson, M. R. Rhodes, and H. H. Saneii, Eds.,
Advanced Chemtech, 1998; "Priciples of Peptide Synthesis, 2nd ed.,"
M. Bodanszky, Ed., Springer-Verlag, 1993; "The Practice of Peptide
Synthesis, 2nd ed.," M. Bodanszky and A. Bodanszky, Eds.,
Springer-Verlag, 1994; and "Protecting Groups," P. J. Kocienski,
Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994). Examples
include benzyloxycarbonyl (Z), Boc, Bpoc, Trt, Nps, FmocCl-Z, Br-Z;
NSC; MSC, Dde, etc. For sulfur moieties, examples of suitable
protecting groups include, but are not limited to, benzyl,
4-methylbenzyl, 4-methoxybenzyl, trityl, ACM, TACAM, xanthyl,
disulfide derivatives, picolyl, and phenacyl.
[0162] More particularly, the N.alpha.-substituted 2 or 3 carbon
chain alkyl or aryl thiols can be prepared in accordance with
Scheme I (Solid-Phase preparation of the N.alpha.-substituted
precursor), Scheme II (Solution-Phase preparation of the
N.alpha.-substituted precursor ). In Scheme I, N.alpha.-substituted
2 or 3 carbon chain alkyl or aryl thiols are assembled directly on
the solid phase using standard methods of polymer-supported organic
synthesis, while the N.alpha.-protected, N-alkylated, S-protected,
aminoalkyl or arylthiol amino acid precursor of Scheme II are
coupled to the resin using standard coupling protocols. Where
racemic or diastereomeric products are produced, it may be
necessary to separate these by standard methods before use in
extended native chemical ligation. 6 7
[0163] The .alpha.-carboxythioesters can be generated by chemical
or biological methods following standard techniques known in the
art, such as those described herein, including the Examples. For
chemical synthesis, .alpha.-carboxythioester peptides can be
synthesized in solution or from thioester-generating resins, which
techniques are well known (See, e.g., Dawson et al., Science (1994)
266:776-779; Canne et al. Tetrahedron Lett. (1995) 36:1217-1220;
Kent, et al., WO 96/34878; Kent, et al., WO 98/28434; Ingenito et
al., JACS (1999) 121(49):11369-11374; and Hackeng et al., Proc.
Natl. Acad. Sci. U.S.A. (1999) 96:10068-10073); Amiato et al.,
supra.). For instance, chemically synthesized thioester peptides
can be made from the corresponding peptide .alpha.-thioacids, which
in turn, can be synthesized on a thioester-resin or in solution,
although the resin approach is preferred. The
peptide-.alpha.-thioacids can be converted to the corresponding
3-carboxy-4-nitrophenyl thioesters, to the corresponding benzyl
ester, or to any of a variety of alkyl thioesters. All of these
thioesters provide satisfactory leaving groups for the ligation
reactions, with the 3-carboxy-4-nitrophenyl thioesters
demonstrating a somewhat faster reaction rate than the
corresponding benzyl thioesters, which in turn may be more reactive
than the alkyl thioesters. As another example, a trityl-associated
mercaptoproprionic acid leucine thioester-generating resin can be
utilized for constructing C-terminal thioesters (Hackeng et al.,
supra). C-terminal thioester synthesis also can be accomplished
using a 3-carboxypropanesulfonamide safety-catch linker by
activation with diazomethane or iodoacetonitrile followed by
displacement with a suitable thiol (Ingenito et al., supra;
Bertozzi et al.).
[0164] C-terminal .alpha.-carboxythioester peptides also can be
made using biological processes, such as intein-mediated biological
techniques (See, e.g., Chong et al., Gene (1997) 192:277-281; Chong
et al., Nucl. Acids Res. (1998) 26:5109-5115; Evans et al., Protein
Science (1998) 7:2256-2264; and Cotton et al., Chemistry &
Biology (1999) 6(9):247-256). For instance, intein expression
systems, with or without labels such as affinity tags can be
utilized to exploit the inducible self-cleavage activity of an
`intein` protein-splicing element to generate a C-terminal
dithiothreitol (DTT) ester peptide or polypeptide segment. In
particular, the intein undergoes specific self-cleavage in the
presence of thiols such as DTT, b-mercaptoethanol or cysteine,
which generates a peptide segment bearing a C-terminal thioester.
See, e.g., Chong et al., (1997) supra; Chong et al., (1998) supra;
Evans et al., supra; and Cotton et al., supra. However, when using
a recombinantly produced segment, the chemically synthesized
portion of the final construct will typically contain the polymer
modification where included.
[0165] Ligation of the N-substituted 2 or 3 carbon chain alkyl or
aryl thiol components of the invention with the first
carboxythioester component generates a ligation product having an
N-substituted amide bond at the ligation site. The ligation
conditions of the reaction are chosen to maintain the selective
reactivity of the thioester with the N-substituted 2 or 3 carbon
chain alkyl or aryl thiol moiety. In a preferred embodiment, the
ligation reaction is carried out in a buffer solution having pH
6-8, with the preferred pH range being 6.5-7.5. The buffer solution
may be aqueous, organic or a mixture thereof. The ligation reaction
also may include one or more catalysts and/or one or more reducing
agents, lipids, detergents, other denaturants or solubilizing
reagents and the like. Examples of preferred catalysts are thiol
and phosphine containing moieties, such as thiophenol,
benzylmercaptan, TCEP and alkyl phosphines. Examples of denaturing
and/or solubilizing agents include guanidinium, urea in water or
organic solvents such as TFE, HFIP, DMF, NMP, acetonitrile admixed
with water, or with guanidinium and urea in water. The temperature
also may be utilized to regulate the rate of the ligation reaction,
which is usually between 5.degree. C. and 55.degree. C., with the
preferred temperature being between 15.degree. C. and 40.degree. C.
As an example, the ligation reactions proceed well in a reaction
system having 2% thiophenol in 6M guanidinium at a pH between 6.8
and 7.8.
[0166] For the N-substituted 2 carbon chain alkyl or aryl thiols,
the ligation event results from a thiol exchange that occurs
between the COSR thioester component and the amino alkyl thiol
component. The exchange generates a thioester-linked intermediate
ligation product that after spontaneous rearrangement through a
5-membered ring intermediate generates a first ligation product of
the formula J1-HN--CH(Z1)-C(O)--N.a- lpha.(C1(R1)-C2-SH)--CH(Z2)-J2
having a removable N-substituted 2 carbon chain alkyl or aryl thiol
[HS--C2-C1(R1)-] at the ligation site, where the substituents are
as defined above. The N-substituted 2 carbon chain alkyl or aryl
thiol [HS--C2-C1(R1)-] at the ligation site is amenable to being
removed, under peptide-compatible conditions, to generate a final
ligation product of the formula J1-HN--CH(Z1)-CO--NH--CH(Z2)-CO-J2
having a native amide bond at the ligation site.
[0167] For the N-substituted 3 carbon chain aryl or alkyl thiols,
the thiol exchange between the COSR thioester component and the
amino alkyl thiol component generates a thioester-linked
intermediate ligation product that after spontaneous rearrangement
through a 6-membered ring intermediate generates a first ligation
product of the formula
J1-HN--CH(Z1)-C(O)--N.alpha.(C1-C2(R2)-C3(R3)-SH)--CH(Z2)-J2 having
a removable N-substituted 3 carbon chain alkyl or aryl thiol
[HS--C3(R3)-C2(R2)-C1(R1)-] at the ligation site. The N-substituted
3 carbon chain aryl thiol [HS--C3(R3)-C2(R2)-C1(R1)-] at the
ligation site is amenable to being removed, under
peptide-compatible conditions, to generate a final ligation product
of the formula J1-HN--CH(Z1)-CO--NH--CH- (Z2)-CO-J2 having a native
amide bond at the ligation site.
[0168] Removal of the N-substituted alkyl or aryl thiol group is
preferably performed in acidic conditions to facilitate cleavage of
the N--C1 bond, yielding a stabilized, unsubstituted amide bond at
the ligation site. By "peptide-compatible cleavage conditions" is
intended physical-chemical conditions compatible with peptides and
suitable for cleavage of the alkyl or aryl thiol moiety from the
ligation product. Peptide-compatible cleavage conditions in general
are selected depending on the .alpha.-substituted compound
employed, which can be readily deduced through routine and well
known approaches (See, e.g., "Protecting Groups in Organic
Synthesis", 3rd Edition, T. W. Greene and P. G. M. Wuts, Eds., John
Wiley & Sons, Inc., 1999; NovaBiochem Catalog 2000; "Synthetic
Peptides, A User's Guide," G. A. Grant, Ed., W. H. Freeman &
Company, New York, N.Y.,1992; "Advanced Chemtech Handbook of
Combinatorial & Solid Phase Organic Chemistry," W. D. Bennet,
J. W. Christensen, L. K. Hamaker, M. L. Peterson, M. R. Rhodes, and
H. H. Saneii, Eds., Advanced Chemtech, 1998; "Priciples of Peptide
Synthesis, 2nd ed.," M. Bodanszky, Ed., Springer-Verlag, 1993; "The
Practice of Peptide Synthesis, 2nd ed.," M. Bodanszky and A.
Bodanszky, Eds., Springer-Verlag, 1994; and "Protecting Groups," P.
J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany,
1994).
[0169] For example, where the R1, R2 or R3 substituents comprises a
methoxy, hydroxyl, thiol or thiomethyl, methyl and the like, the
more universal method for removal involves acidic cleavage
conditions typical for peptide synthesis chemistries. This includes
cleavage of the N--C1 bond under strong acidic conditions or
water-acidic conditions, with or without reducing reagents and/or
scavenger systems (e.g., acid such as anhydrous hydrogen fluoride
(HF), triflouroacetic acid (TFA), or trimethylsulfonyl flouroacetic
acid (TMSFA) and the like). More specific acidic cleavage systems
can be chosen to optimize cleavage of the N.alpha.-C1 bond to
remove the aryl or alkyl thiol moiety for a given construct. Such
conditions are well known and compatible with maintaining the
integrity of peptides. A thiol scavenger may be included,
particularly where tryptophans are present in a peptide or
polypeptide sequence to avoid reaction of the tryptophan side chain
with the liberated aryl or alkyl thiol moiety. Examples of thiol
scavengers include ethanediol, cysteine, beta-mercaptoethanol and
thiocresol.
[0170] Other specialized cleavage conditions include light or
reductive-cleavage conditions when the picolyl group is the
substituent. As an example, when the R1, or R2 and R3 substituents
comprise a picolyl moiety, photolysis (e.g., ultraviolet light),
zinc/acetic acid or electrolytic reduction may be used for cleavage
following standard protocols. Where R1 of the N-substituted 2
carbon chain thiol comprises a thiomethane at R1, the mercury or HF
cleavages can be used. The cleavage system also can be used for
simultaneous cleavage from a solid support and/or as a deprotection
reagent when the first or second ligation components comprise other
protecting groups.
[0171] In one embodiment of the present invention
N.alpha.-substituted 2 or 3 chain alkyl or aryl thiols are employed
in the peptide synthesis step (particularly in automated peptide
synthesis and orthogonal and convergent ligation strategies) to
yield properly N-terminally derivatized polypeptides that can be
used as substrates for extended chemical ligation to yield
synthetic erythropoiesis stimulating proteins. Such compounds
comprise a fully protected, partially protected or fully
unprotected acid stable N.alpha.-substituted 2 or 3 carbon chain
amino alkyl or aryl thiol of the formula
(PG1)S--C2-C1(R1)-N.alpha.(PG2)-CH(Z2)- -C(O)-J2 or
(PG1)S--C3(R3)-C2(R2)-C1(R1)-N.alpha.(PG2)-CH(Z2)-C(O)-J2, which
are depicted below in Table III and Table IV. In particular, one or
more of R1, R2 and R3 comprises an electron donating group
conjugated to C1 that, following conversion of the
N.alpha.-substituted amino alkyl or aryl thiol to an
N.alpha.-substituted amide alkyl or aryl thiol, is capable of
forming a resonance stabilized cation at C1 that facilitates
cleavage of the N.alpha.-C1 bond under peptide compatible cleavage
conditions. PG1 and PG2 are protecting groups that are present
individually or in combination or are absent and can be the same or
different, where Z2 is any chemical moiety compatible with chemical
peptide synthesis or extended native chemical ligation, and where
J2 is any chemical moiety compatible with chemical peptide
synthesis or extended native chemical ligation. PG1 (or X1) is a
group for protecting the amine. PG2 (or X2) is a group for
protecting the thiol. Many such protecting groups are known and
suitable for this purpose (See, e.g., "Protecting Groups in Organic
Synthesis", 3rd Edition, T. W. Greene and P. G. M. Wuts, Eds., John
Wiley & Sons, Inc., 1999; NovaBiochem Catalog 2000; "Synthetic
Peptides, A User's Guide," G. A. Grant, Ed., W. H. Freeman &
Company, New York, N.Y., 1992; "Advanced Chemtech Handbook of
Combinatorial & Solid Phase Organic Chemistry," W. D. Bennet,
J. W. Christensen, L. K. Hamaker, M. L. Peterson, M. R. Rhodes, and
H. H. Saneii, Eds., Advanced Chemtech, 1998; "Priciples of Peptide
Synthesis, 2nd ed.," M. Bodanszky, Ed., Springer-Verlag, 1993; "The
Practice of Peptide Synthesis, 2nd ed.," M. Bodanszky and A.
Bodanszky, Eds., Springer-Verlag, 1994; and "Protecting Groups," P.
J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany,
1994).
3TABLE III 8 9
[0172] Examples of preferred protecting groups for PG1 and X1
include, but are not limited to [Boc(t-Butylcarbamate),
Troc(2,2,2,-Trichloroethylcarb- amate),
Fmoc(9-Fluorenylmethylcarbamate), Br-Z or Cl-Z(Br- or
Cl-Benzylcarbamate), Dde(4,4,-dimethyl-2,6-dioxocycloex1-ylidene),
MsZ(4-Methylsulfinylbenzyl-carbamate),
Msc(2-Methylsulfoethylcarbamate)
Nsc(4-nitrophenylethylsulfonyl-ethyloxycarbonyl]. Preferred PG1 and
X1 protecting groups are selected from "Protective Groups in
Organic Synthesis," Green and Wuts, Third
Edition,Wiley-interscience, (1999) with the most preferred being
Fmoc and Nsc. Examples of preferred protecting groups for PG2
include, but are not limited to [Acm(acetamidomethyl), MeoBzl or
Mob(p-Methoxybenzyl), Meb(p-Methylbenzyl), Trt(Trityl),
Xan(Xanthenyl), tButhio(s-t-butyl), Mmt(p-Methoxytrityl), 2 or 4
Picolyl(2 or 4 pyridyl)), Fm(9-Fluorenylmethyl), tbut(t-Butyl),
Tacam (Trimethylacetamidomethyl)]. Preferred protecting groups PG2
and X2 are selected from "Protective Groups in Organic Synthesis,"
Green and Wuts, Third Edition, Wiley-Interscience, (1999), with the
most preferred being Acm, Mob, MeB, Picolyl
4TABLE IV 10 11
[0173] The protected forms of the N.alpha.-substituted 2 or 3 chain
alkyl or aryl thiols of the invention can be prepared as in Schemes
I and II above.
[0174] The compounds of the present invention may be produced by
any of a variety of means, including halogen-mediated amino
alkylation, reductive amination, and by the preparation of
N.alpha.-protected, N-alkylated, S-protected, amino alkyl- or
aryl-thiol amino acid precursors compatible with solid phase amino
acid synthesis methods. When desirable, resolution of the racemates
or diastereomers produced to give compounds of acceptable chiral
purity can be carried out by standard methods.
[0175] III. Synthesis of Preferred Water-Soluble Polymers of the
Invention and Production
[0176] As used herein, the term "molecular heterogeneity" is
intended to refer to a variation in the number of polymer molecules
attached to each protein of a protein preparation. The term
"molecular diversity" is intended to refer to (a) a variation in
the site(s) of the protein that are modified by the polymer, (b) a
variation in the length of the polymer adducts of different sites
of the same protein, or of the same site(s) in different proteins
of a protein preparation, or (c) a variation in the extent and/or
nature of any branching of the polymer adducts of different sites
of the same protein, or of the same site(s) in different proteins
of a protein preparation. As used herein, the term "molecularly
homogeneous" is intended to refer to a preparation of a protein in
which all of the protein molecules contain the amino acid sequence
and the same polymer modifications at the same positions. A mixture
of two or more "molecularly homogeneous" protein preparations is
referred to herein as a "molecularly defined" preparation.
[0177] In accordance with this aspect of the invention, a solution
to the above-identified problems of polymer heterogeneity,
diversity, and unsuitability involves the production of a new class
of biocompatible polymers which combine the advantages of both
polypeptides (precise length, convenient synthesis) and "pPEG"
("precision PEG"), a flexible, amphiphilic, non-immunogenic,
polymer not susceptible to proteases) Rose, K. et at. (U.S. patent
application Ser. No. 09/379,297, herein incorporated by reference).
This new class of biocompatible polymer has the formula:
--[CO--X--CO--NH--Y--NH].sub.n--
[0178] n is an integer, preferably from 1-100 and more preferably
from 2-100, where X and Y are biocompatible repeat elements of
precise structure linked by an amide bond. Preferably, X and Y will
be divalent organic radicals lacking reactive functional groups or
are absent and are the same or different, and can vary
independently with each repeating unit (n). Preferably, when n=2,
at least one of X or Y will be selected from the group consisting
of a substituted, unsubstituted, branched and linear aliphatic and
aromatic group. More preferably, one of X or Y will be selected
from the group consisting of phenyl, a C.sub.1-C.sub.10 alkylene
moiety, a C.sub.1-C.sub.10 alkyl group, a heteroatom-containing
phenyl, a heteroatom-containing C.sub.1-C.sub.10 alkylene moiety, a
heteroatom-containing C.sub.1-C.sub.10 alkyl group, and a
combination thereof.
[0179] Particularly preferred pPEG moieties have the formulae:
--{CO--(CH.sub.2).sub.2--CO--NH--(CH.sub.2).sub.3--(OCH.sub.2CH.sub.2).sub-
.3--CH.sub.2--NH}.sub.n--
[0180] where n preferably varies from 1-100 and more preferably
from 2-100; or
--{CO--(CH.sub.2).sub.2--CO--NH--(CH.sub.2).sub.6--NH--CO--(CH.sub.2).sub.-
2--CO--NH--(CH.sub.2).sub.3--(OCH.sub.2CH.sub.2).sub.3--CH.sub.2--NH--}.su-
b.n--,
[0181] where n preferably varies from 1-50 and more preferably from
2-50.
[0182] Such pPEG moieties can be synthesized in any of a variety of
ways. Such moieties are, however, preferably produced using a solid
phase stepwise chain assembly of units, rather than a
polymerization process. The use of such an assembly process permits
the moieties of a preparation to have a defined and homogeneous
structure, as to their length, the nature of their X and Y
substituents, the position(s) (if any) of branch points, and the
length, X and Y substituents, and position(s) of any branches.
[0183] Preferably, such moieties will be synthesized by steps such
as:
[0184] (a) acylating the amino or hydroxyl group of a compound of
the formula Z-Q-support with a molar excess of a derivative of a
diacid having the formula, HOOC--X--COOH, where Z is H.sub.2N-- or
HO--; Q is a linker or a target molecule; and the support is a
solid phase, matrix or surface;
[0185] (b) activating the free carboxyl group of the product of
step (a);
[0186] (c) aminolysing the product of step (b) with a molar excess
of a diamine having the formula, NH.sub.2--Y--NH.sub.2; and
[0187] (d) optionally repeating steps (a)-(c) using HOOC--X--COOH
and NH2-Y--NH2, where said X and Y are divalent organic radicals or
are absent and are the same or different, and can vary
independently with each of said optionally repeated units, and are
the same or different from the X and Y substituents used in any of
the previous acylating and aminolysing steps.
[0188] In preferred embodiments, 6-mers, 12-mers, 18-mers and
32-mers of above repeat unit are employed. Where desired, the
repeat unit can be used, for example, in conjunction with the amino
group of lysine to form branched pPEG structures. The pPEG may be
attached to the synthetic proteins of the present invention by a
variety of chemistries, including thioether, oxime and amide
linkage formation.
[0189] In one embodiment, the solid phase stepwise chain assembly
of units comprises:
[0190] Step 1: Couple protected or unprotected diacid to amino on
resin to generate amide bond at linkage site (where PG is a
protecting group that is present or absent depending on diacid
employed):
PG-OOC--X--COOH+NH.sub.2--Y--NH-Resin
[0191] Step 2: Remove protecting group (PG) on resin, if
present
HOOC--X--CO--NH--Y--NH-Resin
[0192] Step 3: Couple protected or unprotected diamino to carboxy
on resin to generate amide bond at linkage site (where PG is
present or absent depending on diamino employed)
PG-NH--Y--NH+HOOC--X--CO--NH--Y--NH-Resin
[0193] Step 4: Remove protecting group (PG) on resin, if
present
--NH--Y--NH--OC--X--CO--NH--Y--NH-Resin
[0194] Step 5: Repeat steps 1-4 `n` times to add `n` units then
cleave from resin
--[CO--X--CO--NH--Y--NH]--[CO--X--CO--NH--Y--NH]--[CO--X--CO--NH--Y--NH]---
[CO--X--CO--NH--Y--NH]--
[0195] As discussed, linear and branched pPEG constructs are
preferred water-soluble polymers for attachment to the synthetic
erythropoiesis stimulating molecules of the invention. The pPEGs
employed bear pendant groups that are charged or neutral under
physiological conditions, and can be made to vary in attachment
chemistry, length, branching and solubility depending on the pPEG
structure one employs. As noted above, preferred pPEGs of the
invention comprise a water-soluble polyamide having the repeat unit
--CO--X--CO--NH--Y--NH--, where one or both of X and Y comprises a
water-soluble repeat unit, and most preferably a `PEG`-based repeat
unit. Although oligoureas are in principle accessible using a
simple two-step solid phase procedure, as shown below for the case
of an amino resin, NH.sub.2-Resin, and a symmetrical diamine,
NH.sub.2--Y--NH.sub.2:
Activation with carbonyldiimidazole.fwdarw.im-CO--NH-Resin
Aminolysis with diamine
NH.sub.2--Y--NH.sub.2.fwdarw.NH.sub.2--Y--NH--CO--- NH-Resin
[0196] where these two steps may be repeated a number of times to
give an oligourea with repeat unit --NH--Y--NH--CO--, this approach
is less preferred. This is because the yields of the above steps
may be non-quantitative at room temperature, even with very large
excesses of reagents and long reaction times. Accordingly, it is
preferable to use a three-step solid phase procedure, shown below
for the case of an amino resin, NH.sub.2-Resin, to form a
polyamide. The Reagents HO.sub.2C--X--CO.sub.2H and
NH.sub.2--Y--NH.sub.2 should be symmetrical in order to avoid
isomeric products.
Acylation with diacid
HO.sub.2C--X--CO.sub.2H.fwdarw.HO--CO--X--CO--NH-Res- in
Activation with
carbonyldiimidazole.fwdarw.im-CO--OCO--X--CO--NH-Resin
Aminolysis with diamine
NH.sub.2--Y--NH.sub.2.fwdarw.NH.sub.2--Y--NH--CO---
X--CO--NH-Resin
[0197] These three steps may be repeated in sequence a number of
times to give a polyamide with repeat unit
--NH--Y--NH--CO--X--CO--. The polymer contains a precise number of
monomer units, X and Y can be varied independently at each step,
and end-groups can be chosen at will. For example, by using
succinic anhydride ("Succ") for the acylation step and
4,7,10-trioxa-1,13-tridecanediamine (also referred to as "EDA" or
"TTD") for the aminolysis step, `PEG`-based polyamides are formed
wherein X is --CH.sub.2CH.sub.2--, Y is
--NH--CH.sub.2CH.sub.2CH.sub.2--(OCH.sub.2CH.s-
ub.2).sub.3--CH.sub.2--NH-- and the repeat unit is
--NH--CH.sub.2CH.sub.2C-
H.sub.2--(OCH.sub.2CH.sub.2).sub.3--CH.sub.2--NH--COCH.sub.2CH.sub.2CO--.
In spite of the fact that the procedure involves divalent reagents
with no protecting groups, cross-linking is not a problem when
standard commercial peptide synthesis resins are used (Rose et al.,
(U.S. patent application Ser. No. 09/379,297); and Rose et al., J.
Am. Chem. Soc. (1999) 121:7034), except as noted below for the case
of branched Lys cores for making branched constructs.
[0198] For example, branched pPEG constructs can be made in a
similar manner as for the "chemobody" constructs described in Rose
et al., (U.S. patent application Ser. No. 09/379,297) and Rose, K.
and Vizzavona, J. (J. Am. Chem. Soc. (1999) 121:7034). Thus,
branched pPEG constructs can be made to have a branching core such
as a lysine branching core, which is coupled through oxime linkers
to a preferred water-soluble polyamide such as
--(COCH.sub.2CH.sub.2CO--NH--CH.sub.2CH.sub.2CH.sub.2--(OCH.sub.2-
CH.sub.2).sub.3--CH.sub.2--NH).sub.n--. For instance, oxime bonds
can readily formed between an aminooxyacetyl group on a Lys side
chain at the other extremity of the polyamide, and glyoxylyl groups
on a lysine core, for example the tetrameric core
(O.dbd.CHCO).sub.4Lys.sub.2Lys-. Thus an oxime linker off of each
lysine branch point can be prepared as the structure
-Lys(COCH.sub.2ON.dbd.CHCO--)amide-. An alternative construction
can place the oxime bond between the polyamide and the free pendant
group of the polyamide, preferably using a monoprotected diamine
and deprotection after coupling the polyamide, to generate a
tetravalent branched constructed depicted below:
[O.dbd.CHCO--(NH--CH.sub.2CH.sub.2CH.sub.2--[OCH.sub.2CH.sub.2].sub.3--CH.-
sub.2--NH--COCH.sub.2CH.sub.2CO--).sub.n].sub.4Lys.sub.2Lys-
[0199] Oxime chemistry can be used in making not only dimeric and
tetrameric branched constructs, but it also suitable for assembling
octameric branched constructs (Rose, K., J. Am. Chem. Soc. (1994)
116:30; Rose, K. et al., Bioconj. Chem. (1996) 7:552). It is, of
course, possible to use other chemistries for such purposes when
using such pPEG polyamides (See, e.g., FIG. 13). Moreover,
polyamide formation may be incorporated into a synthetic scheme for
peptide synthesis involving Boc or Fmoc chemistry, but when
elaborating such schemes it must be borne in mind that the
aminolysis step will remove the Fmoc group, and will remove formyl
protection of indole if Boc chemistry is to be used.
[0200] Accordingly, such pPEGs can be made to have various
branching cores (e.g., Lysine branching core etc.) linked through a
bond of choice (e.g., amide, thioether, oxime etc.) to a linear
water-soluble polyamide (e.g., -(Succ-TTD).sub.n-, etc.), where the
free end of each linear water-soluble polyamide can each be capped
with a desired pendant group (e.g., carboxylate, amino, amide etc.)
to provide a desired charge. Moreover the linear and branched pPEGs
of the present invention can be made to comprise a unique
functional group for attachment to a synthetic protein bearing one
or more unique and mutually reactive functional groups, and the
pendant groups of the pPEGs will preferably be non-antigenic (See,
e.g., FIG. 13).
[0201] In a particularly preferred embodiment, the synthetic
erythropoiesis stimulating proteins of the invention are modified
with molecularly homogeneous glyco-mimetic water-soluble polymers
of the formula:
U-s1-B-s2-Polymer-s3-J*
[0202] where U is a residue of a unique functional group, B is a
branching core having three or more arms that may be the same or
different and may be present or absent, Polymer is a polyamide
having a molecular weight greater than about 5,000 Da of the
formula --[C(O)--X--C(O)--NH--Y--NH].s- ub.n-- or
--[NH--Y--NH--C(O)--X--C(O)].sub.n--, where X and Y are divalent
radicals that may be the same or different and may be branched or
linear, and n is a discrete integer from 2 to 50, and where either
or both of X and Y comprises a substantially non-antigenic
water-soluble repeat unit that may be linear or branched, J* is a
residue of a substantially non-antigenic pendant group having a net
charge under physiological conditions selected from the group
consisting of negative, positive and neutral, and where s1, s2, and
s3 are spacer or linker moieties that may be the same or different,
and may be individually present or absent. Such polymers include
those described in U.S. patent application Ser. No. 60/236,377,
which is incorporated herein in its entirety. Formula
U-s1-B-s2-Polymer-s3-J* also can be represented by the formula
U--B-Polymer-J*, where spacer or linker groups s1, s2, s3 may be
present or absent.
[0203] The preferred process of forming the preferred glycomimetic
polymers of the invention are illustrated in FIGS. 5-7. FIGS. 5A-5B
depict solid phase process for generating the branching core (B)
and unique chemoselective functional group (U) of the water-soluble
polymer U--B-Polymer-J* of the invention. The process may be
carried out in solution, although the solid phase approach as shown
is preferred. In particular, FIG. 5A shows orthogonally protected
U--B precursor moiety with reactive group () and the basic
geometric structure of such construct is depicted by dots linked
with bonds (.circle-solid.--.circle-- solid.); this basic geometric
structure is not intended to limit that types of chemical linkages
or groups employed, but merely illustrative of the relative points
of geometry for building structures and chemical elaboration points
suitable for generation of U--B moieties of the invention. The
orthogonally protected U--B precursor is coupled to a polymer
support/resin comprising a suitable cleavable linker and
co-reactive group 12
[0204] following activation that is capable of covalent linkage to
the U--B precursor: 13
[0205] This system employs the principles of polymer-supported
organic chemistry. Following coupling the branching core is
elaborated (only a first branch point shown, and additional branch
points may be present or absent) as illustrated to generate a
branching core that is suitable for subsequent attachment of a
desired Polymer component, such as a substantially non-antigenic,
water-soluble linear polymer. Also shown is the U group, which can
be provided at the outset of synthesis as part of the orthogonally
protected U--B precursor, or elaborated during or after elaboration
of the branching core B. While attachment of the Polymer component
can be achieved on-resin, i.e., prior to cleavage, a preferred
route is cleavage of the U--B moiety from the polymer support/resin
so as to generate a U--B core that can be purified for subsequent
attachment the Polymer component.
[0206] As illustrated, the pendant branch points of core group B
are built to comprise a functional group (Func), which can be the
same or different, and may be reversibly protected (PG, PG' or PG")
or unprotected. In each case, the final step for attachment of the
Polymer component involves the generation of a functional group
(Func) at the pendant branch points, and generation of group U (See
FIG. 7). FIG. 5B depicts an alternative process in which a
protected U--B precursor is employed in combination with a polymer
support bearing a linker, which upon cleavage generates the desired
protected or unprotected U-group. FIG. 5B also depicts attachment
of a pre-assembled branching core B to a polymer support, and use
of a U-group generating resin to make the U--B moiety for
subsequent attachment of the Polymer component.
[0207] FIGS. 6A-6D depict a solid phase process for generating
preferred substantially non-antigenic water-soluble polyamide
Polymer-J* components of the invention for subsequent attachment to
the U--B core. Although the solid phase process is illustrated,
which is the preferred process, a solution phase process can be
adapted to achieve the same end result. In FIGS. 6A and 6B, diacid
and diamino units are coupled using the principles of solid phase
organic chemistry. Optionally, protected amino-X'-acid and/or
amino-Y'-acid units can be incorporated for additional diversity of
the groups X and Y in the final cleavage product having a polyamide
structure of the formula --[NH--Y--NHCO--X--CO]--. FIG. 6A depicts
synthesis in the N- to C-terminal direction, whereas FIG. 6B
depicts synthesis in the C- to N-terminal direction. In FIGS. 6C
and 6D, protected amino-X'-acid and/or amino-Y'-acid units are
coupled using the principles of solid phase organic chemistry. FIG.
6C depicts synthesis in the N- to C-terminal direction, whereas
FIG. 6D depicts synthesis in the C- to N-terminal direction. As
apparent from FIGS. 6A-6D, the nature of the final polyamide
products can be precisely controlled, which depends on the number
of cycles one carries out for synthesis. Moreover, the pendant
group J* can be built to virtually any user-defined specification.
Where mono-disperse repeat units X, Y, X' and Y' are employed, the
exact molecular structure of the final polyamide product can be
precisely controlled.
[0208] A preferred process for coupling the U--B component to
Polymer-J* component to generate the preferred synthetic polymer
constructs of the invention of the formula U--B-Polymer-J* is
depicted in FIG. 7. As illustrated, various protecting groups can
be provided, and are optional depending on the intended end use of
a given construct. Also illustrated different routes to produce the
desired U--B-Polymer-J* constructs, including a solid phase
approach and a solution phase approach.
[0209] As noted above, preferred water-soluble repeat units
comprise a polyalkylene oxide, polyamide alkylene oxide, or
derivatives thereof. The most preferred water-soluble repeat unit
comprises an ethylene oxide of the formula
--(CH.sub.2--CH.sub.2--O)-- or --(CH.sub.2--CH.sub.2--O)--. The
number of such water-soluble repeat units can vary significantly,
but the more preferred number of such units is from 2 to 500, 2 to
400, 2 to 300, 2 to 200, 2 to 100, 2 to 50, and more preferably 2
to 32, with 3 to 8 being the most preferred. An example of a more
preferred embodiment is where one or both of X and Y is selected
from: --((CH.sub.2).sub.n1--(CH.-
sub.2--CH.sub.2--O).sub.n2--(CH.sub.2).sub.n1--)-- or
--((CH.sub.2).sub.n1--(O--CH.sub.2--CH.sub.2).sub.n2--(CH.sub.2).sub.n1---
), where n1 is 1 to 6, 1 to 5, 1 to 4 and most preferably 1 to 3,
and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8, and
most preferably 2 to 5. An example of a highly preferred embodiment
is where X is --(CH.sub.2--CH.sub.2)--, and where Y is
--(CH.sub.2--(CH.sub.2--CH.su-
b.2--O).sub.3--CH.sub.2--CH.sub.2--CH.sub.2)-- or
--(CH.sub.2--CH.sub.2--C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.3--CH.sub.2)--. Even more
preferred are mono-disperse compositions where each n of is a
discrete integer. In a highly preferred embodiment, X is
--(CH.sub.2--CH.sub.2)-- and Y is
--(CH.sub.2--(CH.sub.2--CH.sub.2--O).sub.3--CH.sub.2--CH.sub.2--CH.sub.2)-
n- or
--(CH.sub.2--CH.sub.2--(O--CH.sub.2--CH.sub.2).sub.3--CH.sub.2)-n
where n is 12 to 36.
[0210] In particular, the Polymer will preferably have when the
water-soluble polymer contains polymer chains having polyalkylene
oxide repeat units, such as ethylene oxide repeat units, it is
preferred that each chain have a molecular weight of between about
1500 and about 42,000 Da and preferably between about 2,000 to
about 20,000 Da being the most preferred.
[0211] A preferred U group comprises a moiety capable of chemical
ligation. Preferred polymers are branched where group B comprises
three or more arms. A preferred component J* comprises a group that
is ionizable under physiological conditions, and most preferably
one that is negatively charged. Either or both of U and J may
comprise a protecting group that is capable of being removed
without damaging the integrity of the construct.
[0212] Preferred spacers or linkers include linear or branched
moieties comprising one or more repeat units employed in a
water-soluble polymer, diamino and or diacid units, natural or
unnatural amino acids or derivatives thereof, as well as aliphatic
moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy,
and the like, which preferably contain up to 18 carbon atoms or
even an additional polymer chain.
[0213] As noted above, component U is a residue of a functional
group that is capable of being attached or is attached to a target
molecule, such as a peptide or polypeptide, or other surface of
interest. When U is a residue of a functional group for conjugation
to a target molecule, U comprises a nucleophilic group or
electrophilic group, and the target molecule comprises a mutually
reactive electrophilic group or nucleophilic group,
respectively.
[0214] Many such mutually reactive functional groups are known and
are capable of attachment to side chain functional groups common to
peptides and polypeptides, or derivatized side chain functional
groups (Zalipsky et al., Bioconjugate Chemistry (1995) 6:150-165;
"Perspectives in Bioconjugate Chemistry", C. F. Meares, Ed., ACS,
1993; "Chemistry of Protein Conjugation and Cross-Linking", S. S.
Wong, Ed., CRC Press, Inc. (1993)). Examples of functional groups
include groups capable of reacting with an amino group such as (a)
carbonates such as the p-nitrophenyl, or succinimidyl; (b) carbonyl
imidazole; (c) azlactones; (d) cyclic imide thiones; and (e)
isocyanates or isothiocyanates. Examples of functional groups
capable of reacting with carboxylic acid groups and reactive
carbonyl groups include (a) primary amines; or (b) hydrazine and
hydrazide functional groups such as the acyl hydrazides,
carbazates, semicarbamates, thiocarbazates, aminooxy etc.
Functional groups capable of reacting with mercapto or sulfhydryl
groups include phenyl glyoxals, maleimides, and halogens. Examples
of functional groups capable of reacting with hydroxyl groups such
as (carboxylic) acids, or other nucleophiles capable of reacting
with an electrophilic center, include hydroxyl, amino, carboxyl,
thiol groups, active methylene and the like.
[0215] For instance, the above polymer can be prepared to carry
component U as a functional group for attachment to a target
molecule, where the functional group is acrylate, aldehyde, ketone,
aminooxy, amine, carboxylic acid, ester, thioester, halogen, thiol,
cyanoacetate, dipalmitoyl phosphatidylethanolamine, distearoyl
phosphatidylethanolamine- , epoxide, hydrazide, azide, isocyanate,
maleimide, methacrylate, nitrophenyl carbonate, orthopyridyl
disulfide, silane, sulfhydryl, vinyl sulfones, succinimidyl
glutarate, succimidyl succinate, succinic acid, tresylate and the
like. U also may be provided in an activatable form, e.g., a
carboxylic acid that can be converted to an active ester thereof
that is capable of reacting with a nucleophile such as an
amine.
[0216] In a preferred embodiment, U is a residue of a unique
functional group that is selectively reactive with a unique
functional group on the target molecule. This aspect of the
invention embodies the principles of peptide synthesis (protecting
group strategies) and chemical ligation (partial or no protecting
group strategies) as discussed above in Section II. Thus, U can
represent a residue of a wide range of functional groups, such as
those described above. Preferred U groups are amenable to amine
capture strategies (e.g., ligation by hemiaminal formation, by
imine formation, and by Michael addition), thiol capture strategies
(e.g., ligation by mercaptide formation, by disulfide exchange),
native chemical ligation strategies (e.g., ligation by thioester
exchange involving cysteine or thiol contain side-chain amino acid
derivative), and orthogonal ligation coupling strategies (e.g.,
ligation by thiazolidine formation, by thioester exchange, by
thioester formation, by disulfide exchange, and by amide formation)
(See, e.g., Coltart, D M., Tetrahedron (2000) 56:3449-3491). A
particularly preferred U comprises a residue of a unique functional
group employed in an aqueous compatible chemical ligation chemistry
such as described above in Section II. Accordingly, U can be a
functional group capable of forming a bond selected from the group
consisting of carbonate, ester, urethane, orthoester, amide, amine,
oxime, imide, urea, thiourea, thioether, thiourethane, thioester,
ether, thaizolidine, hydrazone, oxazolidine and the like. Preferred
bonds are oxime and amide bonds.
[0217] As noted above, B is a branching core moiety having three or
more arms, and may be present or absent. When B is present, one arm
is joined to U or a spacer or linker attached to U, and each other
arm is joined to a Polymer or a spacer or linker attached to a
Polymer. Examples of branching cores B include, but are not limited
to, amino, amide, carboxylic, and combinations thereof. These
include oligoamides of lysine and the like, or oligomers prepared
from alkanediamines and acrylic acid ("polyamidoamines"), the later
providing a net positive charge in the branching core. (See, e.g.,
Zeng et al., J. Pept. Sci. (1996) 2:66-72; Rose et al.,
Bioconjugate Chem., (1996) 7(5):552-556; NovoBiochem Catalog and
Peptide Synthesis Handbook, Laufelfingen, 2001; Wells et al.,
Biopolymers (1998) 47:381-396; Mahajan et al., (1999)
40:4909-49-12; Judson et al. (1998) 39:1299-1302; Swali et al.,
(1997) 62:4902-4903; U.S. Pat. Nos. 5,932,462, 5,919,455,
5,643,575, 5,681,567). Many other different branching cores can be
used and are suitable for this purpose, including substituted
diamines, substituted diacids, alkyl acids such as glycerol and
other moieties having three or more functional or activatable
groups including multivalent alkyl, aryl, heteroalkyl, heteroaryl,
and alkoxy moities and the like, and oligosaccharides (e.g.,
Nierengarten et al., Tetrahedron Lett. (1999) 40:5681-5684;
Matthews et al., Prog. Polym. Sci. (1998) 1-56; Suner et al.,
Macromolecules (2000) 33:253; Fischer et al., Angew. Chem. Int. Ed.
(1999) 38:884; Sunder et al., Adv. Mater. (2000) 12:235; Mulders et
al., Tetrahedron Lett. (1997) 38:631-634; Mulders et al.,
Tetrahedron Lett. (1997) 38:30-3088; and Turnbull et al.,
Chembiocehm (2000) 1(1):70-74). The most preferred branching core
linked to the Polymer component is joined by amide bonds.
[0218] Preferred branching cores emanate from amino, carboxylic or
mixed amino and carboxylic functionalities. Examples of preferred
branching cores are lysine amino cores, aspartic or glutamic acid
branching cores, and gamma-glutamic acid branching cores,
respectively. Moreover, preferred branched polymer constructs are
those in which branching emanates from a single branching core,
such as an oligoamide or polyamidoamine core. However, other
classes of branched constructs can be employed, such as worm-like
structures in which the branching emanates from a sequence of
multiple branching cores distributed along a polymer backbone
(Schluter et al., Chem. Int. Ed. (2000) 39:864). Depending on the
chemical composition and/or the bulkiness of polymer backbone and
repeat units, the resulting worm-like structures can be designed to
be water soluble or prone to induce liquid crystalline organization
(Ouali et al., Macromolecules (2000) 33:6185), which can be
advantageous for delivery applications, stability and duration of
action. As with the other branched polymer constructs of the
invention, the worm-like constructs are made to contain a pendant
functional group comprising U.
[0219] The Polymer component, or one or more of the spacers or
linkers, may include polymer chains or interspaced linkers or bonds
that are biostable or biodegradable, for example as discussed above
in Section I. As also noted above in Section I, component J* is a
residue of pendant group having a net charge under physiological
conditions selected from the group consisting of negative, positive
and neutral. The most preferred is where J* comprises an ionizable
carboxylate moiety and has a net negative charge under
physiological conditions. The components s1, s2, and s3 are spacer
or linker moieties that may be the same or different, and may be
individually present or absent, such as those discussed in Section
I. Most preferably the spacer or linker comprises a polymer chain,
where the spacer or linker comprises one or more repeat units of
the formula --[CO--X--CO--NH--Y--NH].sub.n-.
[0220] IV. Pharmaceutics:
[0221] The polymer-modified synthetic erythropoiesis stimulating
proteins of the present invention may be employed as pharmaceutical
agents to effect the treatment of diseases and conditions.
Accordingly, in another embodiment, the invention is drawn to
pharmaceutical compositions comprising a synthetic erythropoiesis
stimulating protein of the invention, or pharmaceutically
acceptable salts thereof. Such pharmaceutical compositions can
comprise one or more excipients. Preferred excipients are selected
from the group consisting of a buffer, a carrier protein, an amino
acid, a detergent, a lipid, a water-soluble polymer, and a
preservative. The pharmaceutical preparations also can combine one
or more addition active pharmaceutical ingredients, such as one or
more additional bioactive agents (e.g., small-organic molecules,
other protein therapeutics etc.) other than an synthetic
erythropoiesis stimulating protein. Such pharmaceutical
compositions can be employed in preferred methods of the invention.
These include a method of increasing the production of red blood
cells in a mammal, hematocrit, hemoglobin, and reticulocyte count.
These methods comprise administering to the mammal an effective
amount of a synthetic erythropoiesis stimulating protein of the
invention so as to observe the desired effect.
[0222] Most preferably, when administered to a patient or
individual in need of therapy, such synthetic erythropoiesis
stimulating polypeptides and/or proteins will be administered using
a drug delivery system. The uses such a system enables a drug to be
presented to the patient in a manner that makes it acceptable for
them and enhances the effectiveness of the desired bioactivity. The
purposes of the present invention, preferred drug delivery systems
include systems capable of administering polypeptides or proteins
buyout oral, nasal, or inhalation routes, or intramuscularly,
subcutaneously, transdermally, intravenously, intraurally or
intraocularly.
[0223] Such drug delivery systems may include formulations that
provide site-specific release, or that enhance protection for the
intestinal mucosa. Suitable formulations include: dry powder
formulations, delivery via viral particle encapsulation, liposome
encapsulation, transdermal patches, electrically aided transport
(electroporation therapy) and polymer/niazone conjugation.
[0224] In a preferred embodiment, such drug delivery devices will
respond to changes in the biological environment and deliver--or
cease to deliver--drugs based on these changes. A range of
materials have been employed to control the release of drugs and
other active agents.: poly(urethanes), poly(siloxanes), poly(methyl
methacrylate), poly(vinyl alcohol) for hydrophilicity and strength,
poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxy ethyl
methacrylate), poly(n-vinyl pyrrolidone), poly(methyl
methacrylate), poly(vinyl alcohol), poly(acrylic acid),
polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene
glycol), poly(methacrylic acid), etc. In a further preferred
embodiment, biodegradeable polymers will be employed to facilitate
drug delivery. Such polymers incude polylactides (PLA),
polyglycolides (PGA), poly(lactide-co-glycolides) (PLGA),
polyanhydrides, and polyorthoesters. Drug delivery devices, and
methods for their use are described in U.S. Pat. Nos.; 6,072,041;
6,041,253; 6,018,678; 6,017,318; 6,002,961; 5,879,712; 5,849,331;
5,792,451; 5,783,212; 5,766,633; 5,759,566; 5,690,954; 5,681,811;
5,654,000; 5,641,511; 5,438,040; 4,810,499; and 4,659,558.
[0225] Accordingly, another aspect of the invention relates to
pharmaceutical compositions and methods of treating a mammal in
need thereof by administering therapeutically effective amounts of
compounds comprising the polymer-modified synthetic erythropoiesis
stimulating proteins of the invention, or pharmaceutically
acceptable salts thereof. By "pharmaceutically acceptable salt" is
intended to mean a salt that retains the biological effectiveness
and properties of the polypeptides of the invention and which are
not biologically or otherwise undesirable. Salts may be derived
from acids or bases. Acid addition salts are derived from inorganic
acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid
(giving the sulfate and bisulfate salts), nitric acid, phosphoric
acid and the like, and organic acids such as acetic acid, propionic
acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic
acid, succinic acid, maleic acid, fumaric acid, tartaric acid,
citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, salicylic acid,
p-toluenesulfonic acid, and the like. Base addition salts may be
derived from inorganic bases, and include sodium, potassium,
lithium, ammonium, calcium, magnesium salts, and the like. Salts
derived from organic bases include those formed from primary,
secondary and tertiary amines, substituted amines including
naturally-occurring substituted amines, and cyclic amines,
including isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, ethanolamine,
2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine,
caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,
glucosamine, N-alkylglucamines, theobromine, purines, piperazine,
piperidine, N-ethylpiperidine, and the like. Preferred organic
bases are isopropylamine, diethylamine, ethanolamine, piperidine,
tromethamine, and choline.
[0226] The term "treatment" as used herein covers any treatment of
a disease or condition (e.g., anemia) in a mammal, particularly a
human, and includes: (i) preventing the disease or condition from
occurring in a subject which may be predisposed to the disease but
has not yet been diagnosed as having it; (ii) inhibiting the
disease or condition, i.e. arresting its development; or (iii)
relieving the disease or condition, i.e. causing its regression or
the amelioration of its symptoms.
[0227] By the term "a disease state in mammals that is prevented or
alleviated by treatment with a erythropoiesis stimulating protein"
as used herein is intended to cover all disease states which are
generally acknowledged in the art to be usefully treated with
erythropoiesis stimulating proteins in general, and those disease
states which have been found to be usefully prevented or alleviated
by treatment with the specific compounds of the invention. These
include, by way of illustration and not limitation, anemia, beta
thalassemia, pernicious anemia, sickle cell disease, chronic
bacterial endocarditis, osteomyelitis, juvenile rheumatoid
arthritis, rheumatic fever, Crohn's disease, ulcerative colitis,
etc.
[0228] As used herein, the term "therapeutically effective amount"
refers to that amount of a polymer-modified synthetic
erythropoiesis stimulating protein which, when administered to a
mammal in need thereof, is sufficient to effect treatment (as
defined above), for example, as inducer of red cell production, an
anti-anemia agent, etc. The amount that constitutes a
"therapeutically effective amount" will vary depending on the
protein derivative, the condition or disease and its severity, and
the mammal to be treated, its weight, age, etc., but may be
determined routinely by one of ordinary skill in the art with
regard to contemporary knowledge and to this disclosure. As used
herein, the term "q.s." means adding a quantity sufficient to
achieve a stated function, e.g., to bring a solution to a desired
volume (e.g., 100 mL).
[0229] The polymer-modified synthetic erythropoiesis stimulating
proteins of this invention and their pharmaceutically acceptable
salts, i.e., the active ingredient, are administered at a
therapeutically effective dosage, i.e., that amount which, when
administered to a mammal in need thereof, is sufficient to effect
treatment, as described above. Administration of the synthetic
erythropoiesis stimulating proteins described herein can be via any
of the accepted modes of administration for agents that serve
similar utilities. As used herein, the terms "polymer-modified
synthetic erythropoiesis stimulating proteins of this invention",
"[pharmaceutically acceptable salts of the] polypeptides of the
invention" and "active ingredient" are used interchangeably.
[0230] The level of the polymer-modified synthetic erythropoiesis
stimulating proteins in a formulation can vary within the full
range employed by those skilled in the art, e.g., from about 0.01
percent weight (% w) to about 99.99% w of the protein antagonist or
agonist based on the total formulation and about 0.01% w to 99.99%
w excipient. More typically, the polymer-modified synthetic
erythropoiesis stimulating proteins will be present at a level of
about 0.5% w to about 80% w.
[0231] While human dosage levels have yet to be optimized for the
polymer-modified synthetic erythropoiesis stimulating proteins of
the invention, generally, the amount of compound administered will,
of course, be dependent on the subject and the disease state
targeted for prevention or alleviation, the nature or severity of
the affliction, the manner and schedule of administration and the
judgment of the prescribing physician. Such use optimization is
well within the ambit of those of ordinary skill in the art.
[0232] Administration can be via any accepted systemic or local
route, for example, via parenteral, oral (particularly for infant
formulations), intravenous, nasal, bronchial inhalation (i.e.,
aerosol formulation), transdermal or topical routes, in the form of
solid, semi-solid or liquid or aerosol dosage forms, such as, for
example, tablets, pills, capsules, powders, liquids, solutions,
emulsion, injectables, suspensions, suppositories, aerosols or the
like. The polymer-modified synthetic erythropoiesis stimulating
proteins of the invention can also be administered in sustained or
controlled release dosage forms, including depot injections,
osmotic pumps, pills, transdermal (including electrotransport)
patches, and the like, for the prolonged administration of the
polypeptide at a predetermined rate, preferably in unit dosage
forms suitable for single administration of precise dosages. The
compositions will include a conventional pharmaceutical carrier or
excipient and a protein antagonist or agonist of the invention and,
in addition, may include other medicinal agents, pharmaceutical
agents, carriers, adjuvants, etc. Carriers can be selected from the
various oils, including those of petroleum, animal, vegetable or
synthetic origin, for example, peanut oil, soybean oil, mineral
oil, sesame oil, and the like. Water, saline, aqueous dextrose, and
glycols are preferred liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical carriers include starch,
cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica gel, magnesium stearate, sodium stearate,
glycerol monostearate, sodium chloride, dried skim milk, glycerol,
propylene glycol, water, ethanol, and the like. Other suitable
pharmaceutical carriers and their formulations are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin.
[0233] If desired, the pharmaceutical composition to be
administered may also contain minor amounts of non-toxic auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents and the like, such as for example, sodium acetate, sorbitan
monolaurate, triethanolamine oleate, etc.
[0234] Although more of the active ingredient may be required, oral
administration can be used to deliver the polymer-modified
synthetic erythropoiesis stimulating proteins of the invention
using a convenient daily dosage regimen, which can be adjusted
according to the degree of prevention desired or in the alleviation
of the affliction. For such oral administration, a pharmaceutically
acceptable, non-toxic composition is formed by the incorporation of
any of the normally employed excipients, such as, for example,
pharmaceutical grades of mannitol, lactose, starch, povidone,
magnesium stearate, sodium saccharine, talcum, cellulose,
croscarmellose sodium, glucose, gelatin, sucrose, magnesium
carbonate, and the like. Such compositions take the form of
solutions, suspensions, dispersible tablets, pills, capsules,
powders, sustained release formulations and the like. Oral
formulations are particularly suited for treatment of
gastrointestinal disorders. Oral bioavailablity for general
systemic purposes can be adjusted by utilizing excipients that
improve uptake to systemic circulation, such as formulation
comprising acetylated amino acids. See, e.g., U.S. Pat. No.
5,935,601 and U.S. Pat. No. 5,629,020.
[0235] The compositions may take the form of a capsule, pill or
tablet and thus the composition will contain, along with the active
ingredient, a diluent such as lactose, sucrose, dicalcium
phosphate, and the like; a disintegrant such as croscarmellose
sodium, starch or derivatives thereof; a lubricant such as
magnesium stearate and the like; and a binder such as a starch,
polyvinylpyrrolidone, gum acacia, gelatin, cellulose and
derivatives thereof, and the like.
[0236] Liquid pharmaceutically administrable compositions can, for
example, be prepared by dissolving, dispersing, etc. a
polymer-modified synthetic erythropoiesis stimulating proteins of
the invention (about 0.5% to about 20%) and optional pharmaceutical
adjuvants in a carrier, such as, for example, water, saline,
aqueous dextrose, glycerol, glycols, ethanol, preservatives and the
like, to thereby form a solution or suspension. If desired, the
pharmaceutical composition to be administered may also contain
minor amounts of nontoxic auxiliary substances such as wetting
agents, suspending agents, emulsifying agents, or solubilizing
agents, pH buffering agents and the like, for example, sodium
acetate, sodium citrate, cyclodextrine derivatives,
polyoxyethylene, sorbitan monolaurate or stearate, etc. Actual
methods of preparing such dosage forms are known, or will be
apparent, to those skilled in this art; for example, see
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa. The composition or formulation to be administered will,
in any event, contain a quantity of the active ingredient in an
amount effective to prevent or alleviate the symptoms of the
subject being treated. For oral administration to infants, a liquid
formulation (such as a syrup or suspension) is preferred.
[0237] For a solid dosage form containing liquid, the solution or
suspension, in for example propylene carbonate, vegetable oils or
triglycerides, is preferably encapsulated in a gelatin capsule. For
a liquid dosage form, the solution, e.g. in a polyethylene glycol,
may be diluted with a sufficient quantity of a pharmaceutically
acceptable liquid carrier, e.g. water, to be easily measured for
administration.
[0238] Alternatively, liquid or semi-solid oral formulations may be
prepared by dissolving or dispersing the active ingredient in
vegetable oils, glycols, triglycerides, propylene glycol esters
(e.g. propylene carbonate) and the like, and encapsulating these
solutions or suspensions in hard or soft gelatin capsule
shells.
[0239] In applying the polymer-modified synthetic erythropoiesis
stimulating proteins of this invention to treatment of the above
conditions, administration of the active ingredients described
herein are preferably administered parenterally. Parenteral
administration is generally characterized by injection, either
subcutaneously, intramuscularly or intravenously, and can include
intradermal or intraperitoneal injections as well as intrasternal
injection or infusion techniques. Injectables can be prepared in
conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, as emulsions or in biocompatible polymer-based
microspheres (e.g., liposomes, polyethylene glycol derivatives,
poly(D,C)lactide and the like). Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol or the like. In
addition, if desired, the pharmaceutical compositions to be
administered may also contain minor amounts of non-toxic auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents, solubility enhancers, protein carriers and the like, such
as for example, sodium acetate, polyoxyethylene, sorbitan
monolaurate, triethanolamine oleate, cyclodextrins, serum albumin
etc.
[0240] The polymer-modified synthetic erythropoiesis stimulating
proteins of the present invention can be administered parenterally,
for example, by dissolving the compound in a suitable solvent (such
as water or saline) or incorporation in a liposomal formulation
followed, by dispersal into an acceptable infusion fluid. A typical
daily dose of a polypeptide of the invention can be administered by
one infusion, or by a series of infusions spaced over periodic
intervals. For parenteral administration there are especially
suitable aqueous solutions of an active ingredient in water-soluble
form, for example in the form of a water-soluble salt, or aqueous
injection suspensions that contain viscosity-increasing substances,
for example sodium carboxymethylcellulose, sorbitol and/or dextran,
and, if desired, stabilizers. The active ingredient, optionally
together with excipients, can also be in the form of a lyophilisate
and can be made into a solution prior to parenteral administration
by the addition of suitable solvents.
[0241] A more recently devised approach for parenteral
administration employs the implantation of a slow-release or
sustained-release system, such that a constant level of dosage is
maintained. See, e.g., U.S. Pat. No. 3,710,795, U.S. Pat. No.
5,714,166 and U.S. Pat. No. 5,041,292, which are hereby
incorporated by reference.
[0242] The percentage of the active ingredient contained in such
parental compositions is highly dependent on the specific nature
thereof, as well as the activity of the polypeptide and the needs
of the subject. However, percentages of active ingredient of 0.01%
to 10% in solution are employable, and will be higher if the
composition is a solid which will be subsequently diluted to the
above percentages. Preferably the composition will comprise 0.02-8%
of the active ingredient in solution.
[0243] Another method of administering the polymer-modified
synthetic erythropoiesis stimulating proteins of the invention
utilizes both a bolus injection and a continuous infusion.
[0244] Aerosol administration is an effective means for delivering
the polymer-modified synthetic erythropoiesis stimulating proteins
of the invention directly to the respiratory tract. Some of the
advantages of this method are: 1) it circumvents the effects of
enzymatic degradation, poor absorption from the gastrointestinal
tract, or loss of the therapeutic agent due to the hepatic
first-pass effect; 2) it administers active ingredients which would
otherwise fail to reach their target sites in the respiratory tract
due to their molecular size, charge or affinity to extra-pulmonary
sites; 3) it provides for fast absorption into the body via the
alveoli of the lungs; and 4) it avoids exposing other organ systems
to the active ingredient, which is important where exposure might
cause undesirable side effects. For these reasons, aerosol
administration is particularly advantageous for treatment of
asthma, local infections of the lung, and other diseases or disease
conditions of the lung and respiratory tract.
[0245] There are three types of pharmaceutical inhalation devices,
nebulizers inhalers, metered-dose inhalers and dry powder inhalers.
Nebulizer devices produce a stream of high velocity air that causes
the protein derivative (which has been formulated in a liquid form)
to spray as a mist that is carried into the patient's respiratory
tract. Metered-dose inhalers typically have the formulation
packaged with a compressed gas and, upon actuation, discharge a
measured amount of the polypeptide by compressed gas, thus
affording a reliable method of administering a set amount of agent.
Dry powder inhalers administer the polypeptide in the form of a
free flowing powder that can be dispersed in the patient's
air-stream during breathing by the device. In order to achieve a
free flowing powder, the protein derivative is formulated with an
excipient, such as lactose. A measured amount of the protein
derivative is stored in a capsule form and is dispensed to the
patient with each actuation. All of the above methods can be used
for administering the present invention.
[0246] Pharmaceutical formulations based on liposomes are also
suitable for use with the polymer-modified synthetic erythropoiesis
stimulating proteins of this invention. See, e.g., U.S. Pat. No.
5,631,018, U.S. Pat. Nos. 5,723,147, and 5,766,627. The benefits of
liposomes are believed to be related to favorable changes in tissue
distribution and pharmacokinetic parameters that result from
liposome entrapment of drugs, and may be applied to the
polypeptides of the present invention by those skilled in the art.
Controlled release liposomal liquid pharmaceutical formulations for
injection or oral administration can also be used.
[0247] For systemic administration via suppository, traditional
binders and carriers include, for example, polyethylene glycols or
triglycerides, for example PEG 1000 (96%) and PEG 4000 (4%). Such
suppositories may be formed from mixtures containing the active
ingredient in the range of from about 0.5 w/w % to about 10 w/w %;
preferably from about 1 w/w % to about 2 w/w %.
[0248] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference. Having now generally described the invention, the same
will be more readily understood through reference to the following
examples, which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLES
[0249] Abbreviations
[0250] Acm=acetamidomethyl thiol-protecting group [i.e.
--CH.sub.2NHCOCH.sub.3]
[0251] AoA=aminooxyacetyl
[0252] Arg(Tos)=L-arginine(side chain
N.sup.Gtoluenesulfonyl-protected)
[0253] ART=absolute reticulocyte count
[0254] Asp(cHex)=L-aspartic acid(side chain cyclohexyl
ester-protected)
[0255] AUC=area under the curve
[0256] Boc=tert.butoxycarbonyl
[0257] CD=circular dichroism
[0258] CDI=carbonyldiimidiazole
[0259] CHO=chinese hamster ovary
[0260] CL=clearance (mL/hr/kg)
[0261] Cmax=maximum concentration
[0262] Cys(4MeBzl)=L-cysteine(side chain
(4-methyl)benzyl-protected)
[0263] Cys(Acm)=L-cysteine(side chain acetamidomethyl [i.e.
--CH.sub.2NHCOCH.sub.3]-protected)
[0264] DCM=dichloromethane
[0265] DIC=diisopropylcarbodiimide
[0266] DIEA=diisopropylethylamine
[0267] DMF=dimethylformamide
[0268] DMSO=dimethylsulfoxide
[0269] DPC=dodecylphosphocholine
[0270] Dpr=L-1,2diaminopropionic acid
[0271] ED50=effective dose required to reach 50% maximum effect
[0272] EDA=(4,7,10)-trioxatridecane-1,13diamine
[0273] ELISA=enzyme-linked immunoassay
[0274] EPO=erythropoietin
[0275] ES-MS=electrospray ionization mass spectrometry
[0276] FBS=fetal bovine serum
[0277] Glu(cHex)=L-glutamic acid(side chain cyclohexyl
ester-protected)
[0278] GM-CSF=granulocyte-macrophage colony stimulating factor
[0279] HATU=O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylammonium
hexafluorophosphate
[0280] HCT=hematocrit
[0281] HGB=hemoglobin
[0282] His(Dnp)=L-histidine(side chain
N.sup.lmdinitrophenyl-protected)
[0283] HOBT=N-hydroxybenzotriazole
[0284] HPLC=high pressure liquid chromatography
[0285] IMDM=Iscove's modified Dulbecco's medium
[0286] IPA=isopropanol
[0287] Lev=levulinic acid
[0288] Lys(CIZ)=L-lysine(side chain
2-chlorobenzyloxycarbonyl)-protected
[0289] MBHA=4-methylbenzhydrylamine
[0290] MRT=mean residence time
[0291] MTT=4-methylTrityl
[0292] MTT=thiazolyl blue
[0293] NHS=N-hydroxysuccinimide
[0294] --OCH.sub.2-Pam-resin=--O--CH.sub.2--Bz-CH.sub.2CONHCH.sub.2
(copolystyrene-divinylbenzene)-resin
[0295] Pbo=4-(CH.sub.3S(O)-)benzyl
[0296] PBS=phosphate buffered saline
[0297] PCV=packed cell volume
[0298] RBC=red blood cell
[0299] RET=reticulocyte
[0300] rhEPO=recombinant human EPO
[0301] RSA=rat serum albumin
[0302] SDS=sodium dodecyl sulfate
[0303] SDS-PAGE=SDS-polyacrylamide gel electrophoresis
[0304] SEP=synthetic erythropoiesis protein
[0305] Ser(Bzl)=L-serine(side chain benzyl-protected)
Example 1
[0306] Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-0
[0307] A synthetic erythropoiesis stimulating protein (SEP) was
synthesized. The sequence of the full-length synthesized protein
(designated "SEP-0 (1-166)" is:
5 (SEQ ID NO:1) APPRLICDSR VLERYLLEAK EAEKITTGCA EHCSLNEKIT
VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW.psi.P
LQLHVDKAVS GLRSLTTLLR ALGAQK.psi.AIS PPDAASAAPL RTITADTFRK
LFRVYSNFLR GKLKLYTGEA CRTGDR
[0308] where .PSI. denotes a non-native amino acid residue
consisting of a cysteine that is carboxymethylated at the
sulfhydryl group. The SEP-0 protein was synthesized in solution
from four polypeptide segments:
[0309] Segment SEP-0:1 (GRFN 1711; Composed of Residues 1-32 of SEQ
ID NO: 1):
[0310] APPRLICDSR VLERYLLEAK EAEKITTGCA EH-thioester
[0311] Segment SEP-0:2 (GRFN 1712, Composed of Residues 33-88 of
SEQ ID NO: 1):
6 CSLNEKITVP DTKVNFYAWK RMEVGQQAVE VWQGLALLSE AVLRGQALLV
KSSQPW-thioester
[0312] (where Cys.sup.33 is Acm protected)
[0313] Segment SEP-0:3 (GRFN 1713, Composed of Residues 89-116 of
SEQ ID NO: 1):
[0314] CPLQLHVDKA VSGLRSLTTL LRALGAQK-thioester (where Cys.sup.89
is Acm protected)
[0315] Segment SEP-0:4 (GRFN 1714, Composed of Residues 117-166 of
SEQ ID NO: 1):
7 CAISPPDAAS AAPLRTITAD TFRKLFRVYS NFLRGKLKLY
TGEACRTGDR-carboxylate
[0316] (where the C-terminal cysteine (Cys.sup.161) carries a
picolyl (pico) protecting group)
[0317] The peptides SEP-0:1 and SEP-0:2 and SEP-0:3 were
synthesized on a thioester-generating resin by the in situ
neutralization protocol for Boc (tert-butoxycarbonyl) chemistry and
stepwise solid phase peptide synthesis (SPPS) using established
SPPS, side-chain protection and thioester-resin strategies
(Hackeng, et al., PNAS (1999) 96: 10068-10073; and Schnolzer, et
al., Int. J. Pept. Prot. Res., (1992) 40: 180-193)) on an ABI433A
automated peptide synthesizer or by manual chain assembly, or
ordered and acquired from commercial vendors. For instance, a
standard set of Boc SPPS protecting groups was used, namely:
Arg(Tos); Asp(cHex); Cys(4MeBzl) & Cys(Acm); Glu(cHex);
His(DNP); Lys(CIZ); Ser(Bzl); Thr(Bzl); Trp(formyl); Tyr(BrZ); Met,
Asn, Gin were side-chain unprotected. Segment SEP-0:4 was
synthesized analogously on a --OCH.sub.2-Pam-resin. The peptides
were deprotected and simultaneously cleaved from the resin support
using HF/p-cresol according to standard Boc chemistry procedure;
however, for those peptides containing protecting groups not
removed in HF/p-cresol, the protecting groups were retained. The
peptides were purified by preparative C4 reverse-phase-high
pressure liquid chromatography (HPLC). Fractions containing pure
peptide were identified using ES-MS (electrospray ionization mass
spectrometry), pooled and lyophilized for subsequent ligation.
[0318] Step 1: Ligation #1 Segment SEP-0:4 and segment SEP-0:3 were
dissolved in TFE at 15 mM concentration. Saturated phosphate buffer
(pH 7.9) containing 6 M guanidinium chloride and 1% thiophenol was
added, resulting in a clear solution of the peptide segments. After
ligation, the ligation mix was added to a solution of 2 ml TFE
(trifluoroethanol), 6 ml 6M guanidinium chloride, 100 mM phosphate
containing 25% .beta.-mercaptoethanol and incubated for 20 minutes.
The solution was acidified with a solution of 15 mg/ml TCEP
(tris(2-carboxyethyl)phosphine- .HCl) in glacial acetic acid and
loaded onto a preparative C4 reverse-phase HPLC column (1 inch
diameter). The peptides were then purified by preparative gradient
reverse-phase HPLC. Fractions containing the desired ligated
product SEP-0:Acm+SEP-0:3+SEP-0:4 were identified by ES-MS and
pooled.
[0319] Step 2: Acm-removal #1 For Acm removal, the aqueous
acetonitrile solution containing the pooled fractions of
SEP-0:Acm+SEP-0:3+SEP-0:4 was diluted 1.times. with HPLC grade
water, and solid urea was added for a final concentration of 2
molar. A threefold molar excess (relative to the total expected
cysteine concentration) of a 30 mg/ml Hg(acetate).sub.2 solution in
3% aqueous acetic acid was added and the solution is stirred for
one hour. The solution was then made 20% in .beta.-mercaptoethanol,
loaded onto a semi-preparative reverse-phase HPLC column and
purified with a step gradient. Fractions containing the desired
product SEP-0:3+SEP-0:4 were identified by ES-MS and lyophilized
overnight.
[0320] Step 3: Ligation #2 Equal amounts of SEP-0:3+SEP-0:4 and
SEP-0:2 were jointly dissolved in neat TFE at 15 mM concentration.
250 mM Phosphate buffer (pH 7.5) containing 6 M guanidinium
chloride and 1% thiophenol was added, resulting in a clear solution
of the peptide segments. After one day of ligation, the ligation
mix was added to a solution of 10 ml TFE, 10 ml
.beta.-mercaptoethanol, 10 ml piperidine and 20 ml 6M guanidinium
chloride, pH 4, and incubated for 20 minutes to remove any
remaining protecting groups. The solution was acidified with a
solution of 15 mg/ml TCEP in 20% aqueous acetic acid, loaded onto a
preparative reverse-phase HPLC column and purified with a linear
gradient. Fractions containing the desired ligated product
SEP-0:Acm+SEP-0:2+SEP-0:3+SEP-0:4 were identified by ES-MS and
lyophilized overnight.
[0321] Step 4: Carboxymethylation SEP-0:Acm+SEP-0:2+SEP-0:3+SEP-0:4
was dissolved in TFE at 15 mM concentration. A two-fold excess
(v/v) of 200 mM Phosphate buffer (pH 7.9) containing 6 M
guanidinium chloride was added, resulting in a clear solution of
the peptide segment. A 25-fold excess of bromo-acetic acid
dissolved in a minimum amount of methanol was added, and the
solution was allowed to react for two hours. The solution was
acidified with a solution of 15 mg/ml TCEP in 20% aqueous acetic
acid, loaded onto a preparative reverse-phase HPLC column and
purified with a step gradient. Fractions containing the desired
carboymethylated product SEP-0:Acm+SEP-0:2+SEP-0:3+SEP-0:4+Et were
identified by ES-MS and pooled.
[0322] Step 5: Picolyl Removal Zinc dust was activated in 2M HCl
for 30 minutes. Peptide SEP-0:Acm+SEP-0:2+SEP-0:3+SEP-0:4+Et was
dissolved in neat TFE at about 10 mg/ml concentration. The solution
was diluted with 4.times. (v/v relative to TFE) 6M guanidinium
chloride, 100 mM acetate, pH 4, containing (freshly added) 35 mg/ml
L-methionine and 35 mg/ml dodecylsarcosine (i.e. sodium
N-dodecanoylsarcosine). The solution was added to the activated Zn
powder. The reaction was monitored at .about.1 hr intervals and is
complete after five hours. After completion, the supernatant was
removed and the remaining Zn powder washed twice for five minutes
with 6M guanidinium chloride, pH 4, 100 mM acetate containing 35
mg/ml L-methionine and 35 mg/ml dodecylsarcosine containing 20% TFE
as well as once with the same solution containing 20%
.beta.-mercaptoethanol. The combined product was acidified with a
solution of 15 mg/ml TCEP in 20% aqueous acetic acid, loaded onto a
preparative reverse-phase HPLC column and purified with a step
gradient. Fractions containing the desired modified product
SEP-0:Acm+SEP-0:2+SEP-0- :3+SEP-0:4+Et-Pico were identified by
ES-MS and pooled.
[0323] Step 6: Acm-removal #2 The pooled solution of
SEP-0:Acm+SEP-0:2+SEP-0:3+SEP-0:4+Et-Pico was diluted 3.times. with
HPLC grade water, and solid urea was added for a final
concentration of 2 molar. A threefold molar excess (relative to the
total expected cysteine concentration) of a 30 mg/ml
Hg(acetate).sub.2 solution in 3% aqueous acetic acid was added and
the solution stirred for one hour. The solution was then made 20%
in .beta.-mercaptoethanol, loaded onto a semi-preparative
reverse-phase HPLC column and purified with a step gradient.
Fractions containing the desired product
SEP-0:2+SEP-0:3+SEP-0:4+Et-Pico were identified by ES-MS, diluted
2.times. (v/v) with water containing 2.times. (w/w relative to
peptide mass) DPC (dodecylphosphocholine) and lyophilized
overnight.
[0324] Step 7: Ligation #3 Equal amounts of
SEP-0:2+SEP-0:3+SEP-0:4+Et-Pic- o and SEP-0:1 were jointly
dissolved in neat TFE at 15 mM concentration and 250 mM Phosphate
buffer (pH 7.5) containing 6 M guanidinium chloride is added. To
the solution 1% thiophenol was added. After one day of ligation,
the ligation mix was added to a solution of 10 ml TFE, 10 ml
.beta.-mercaptoethanol, 10 ml piperidine and 20 ml 6M guanidinium
chloride, pH 4, and incubated for 20 minutes to remove any
remaining protecting groups. The solution was acidified with a
solution of 15 mg/ml TCEP in 20% aqueous acetic acid, loaded onto a
preparative reverse-phase HPLC column and purified with a linear
gradient. Fractions containing the desired ligated product SEP-0
(1-166): (SEQ ID NO: 1) were identified by electrospray mass
spectrometry, diluted 2.times. (v/v) with water containing 2.times.
(w/w relative to peptide mass) dodecylsarcosine and
lyophilized.
[0325] Step 8 Folding: Full-length ligated peptide SEP-0 (1-166)
was dissolved in 200 mM Tris buffer (pH 8.7) containing 6 M
guanidinium chloride and 20% TFE and a ten-fold molar excess
(relative to Cys residues in protein) of cysteine. This solution
was dialyzed overnight against a solution of 200 mM Tris buffer (pH
8.7).containing 3 M guanidinium chloride at room temperature. The
solution was then dialyzed against a solution of 200 mM Tris buffer
(pH 8.7) containing 1 M guanidinium chloride at room temperature
for 4 hours at 4.degree. C. and finally against 10 mM phosphate
buffer (pH 7.0) for 4 hours at 4.degree. C. to yield the final
folded product. Folding was verified by electrospray ES-MS and CD
(circular dichroism) spectrometry.
[0326] Step 9 Purification: The folded polypeptide was concentrated
5.times. in centricon concentrator vials and loaded on to Resource
S cation exchange column equilibrated at 10 mM phosphate, pH 7.0.
The folded protein was eluted in a linear salt gradient to 500 mM
NaCl in 10 minutes. Fractions containing the desired folded product
SEP-0 (1-166) were identified by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), and frozen and stored at -80.degree. C.
An analytical reverse-phase HPLC chromatogram and an ES-MS spectrum
of the folded protein product as well as a CD spectrum demonstrated
the presence of folded protein.
Example 2
[0327] Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-L30
[0328] A second synthetic erythropoiesis stimulating protein
(designated SEP-1-L30) was synthesized to contain oxime-forming
groups at positions 24 and 126 of SEP-0. These groups were then
used to form SEP-1-L30, in which linear (EDA-Succ-).sub.18
carboxylate (EDA=(4,7,10)-trioxatridecane- -1,13diamine, also
called TTD; Succ=--CO--CH.sub.2CH.sub.2CO--) polymers have been
joined to the protein backbone. The sequence of the full-length
SEP-1 (1-166) is:
8 (SEQ ID NO:2) APPRLICDSR VLERYLLEAK EAEK.sup.oxITTGCA EHCSLNEKIT
VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW.psi.P
LQLHVDKAVS GLRSLTTLLR ALGAQK.psi.AIS PPDAAK.sup.oxAAPL RTITADTFRK
LFRVYSNFLR GKLKLYTGEA CRTGDR
[0329] where .PSI. denotes an non-native amino acid residue
consisting of a cysteine that is carboxymethylated at the
sulfhydryl group, and where K.sup.ox denotes a non-native lysine
that is chemically modified at the .epsilon.-amino group with an
oxime linker group coupled to a designated water-soluble polymer
through an oxime bond. The structure of SEP-1-L30 also is shown in
FIG. 15.
[0330] A schematic illustrating the chemical synthesis of SEP1-L30
described below is shown in FIG. 14. Briefly, in FIGS. 14(A) and
14(B), water-soluble polymer GRFN32 (GP32) is attached to peptides
SEP-1:1 and SEP-1:4 through an oxime bond-forming chemical ligation
reaction. As shown, peptide SEP-1:1 bears a C-terminal Yaa group at
position 32 comprising a histidine alpha-carboxyl thioester for
subsequent native chemical ligation, and a non-native lysine
residue at position 24 (a glycosylation site of natural human EPO)
the side chain of which has been chemically modified to bear a
U.sub.n=1 group comprising an aminooxy acyl moiety. The N-terminal
alanine corresponding to position 1 of the final full-length
product is shown for reference. Peptide SEP-1:4 has an unprotected
N-terminal Xaa group at position 117 comprising a cysteine, a
U.sub.n=1 group comprising an aminooxy acyl modified Lysine at
position 126 (a glycosylation site of natural human EPO), and
cysteine at position 161 (a disulfide forming cysteine) that has
its side-chain thiol protected with a picolyl group. Cys.sup.161 is
protected to prevent its thioalkylation in a subsequent conversion
of cysteines introduced for native chemical ligation sites (See
FIG. 14(D)); also, a picolyl protecting group is used since its
removal is orthogonal to the conditions required for removing an
Acm (acetamidomethyl) protected cysteine in a first native chemical
ligation reaction (See FIG. 14(C)). Site-specific, and exclusive
attachment of the GP32 polymer constructs at positions 24 and 126
is achieved through oxime-forming chemical ligation to produce the
precision polymer-modified peptides SEP-1:1+GP32 and SEP-1:4+GP32.
FIG. 14(C) shows native chemical ligation of SEP-1:4+GP32 to a
middle peptide segment SEP-1:3 and generation of
SEP-1:3-SEP+1:4+GP32, the ligation site of which is at Lys.sup.116
and Cys.sup.117. As shown peptide SEP-1:3 comprises an N-terminal
Xaa group at position 89 comprising an Acm protected cysteine, and
a C-terminal Yaa group at position 116 comprising a lysine
alpha-carboxyl thioester. Following native chemical ligation, the
Acm protecting group is removed to prepare this ligation product
for the next ligation reaction. FIG. 14(D) shows native chemical
ligation of SEP-1:3+SEP-1:4+GP32 to a middle peptide segment
SEP-1:2 and generation of SEP-1:2+SEP-1:3+SEP-1:4+GP32, the
ligation site of which is at Trp.sup.88 and Cys.sup.89. As shown
peptide SEP-1:2 comprises an N-terminal Xaa group at position 33
comprising an Acm cysteine, and a C-terminal Yaa group at position
89 comprising a tryptophan alpha-carboxyl thioester. Following
native chemical ligation, the ligation product is exposed to
bromoacetic acid for carboxymethylation of the side chain thiols of
ligation site cysteines 89 and 117, and thus their conversion to
pseudo glutamic acids. Following carboxymethylation, the Acm and
Picolyl protecting groups are removed to prepare this unprotected,
polymer-modified ligation product for the next ligation reaction.
FIG. 14(E) shows native chemical ligation of SEP-1:3-SEP1:4+GP32 to
peptide segment SEP-1:1+GP32 (corresponding the N-terminal segment
of the full length product) and generation of the full length
SEP-1-L30 product SEP-1:1+GP32+SEP-1:2+SEP-1:3+SEP-1:4+GP32, the
final ligation site of which is at His.sup.32 and Cys.sup.33. As
shown the full-length SEP-1-L30 product comprises two water-soluble
polymers (G32) attached exclusively at user-defined sites, i.e.,
glycosylation sites corresponding to position 24 and position 126
of natural-human EPO. The details of the synthesis are described
below.
[0331] A. Oximation of GRFN1776 and GRFN1711 with GRFNP32
[0332] Oxime formation was performed to attach water-soluble
polymers bearing an aminooxyacetyl group to peptides carrying a
ketone carbonyl group. To accomplish this, the following peptide
segments were synthesized:
[0333] Segment SEP-1:4 (GRFN 1776; Composed of Residues 117-166 of
SEQ ID NO: 2):
9 CAISPPDAAK AAPLRTITAD TFRKLFRVYS NFLRGKLKLY
TGEACRTGDR-carboxylate
[0334] (where Lys.sup.126 is modified with a levulinic acid residue
at the .epsilon.-amino group, and where Cys.sup.117 is Acm
protected)
[0335] Segment SEP-1:1 (GRFN 1711, Composed of Residues 1-32 of SEQ
ID NO: 2):
[0336] APPRLICDSR VLERYLLEAK EAEKITTGCA EH-thioester (where Lys24
is modified with a levulinic acid residue)
[0337] Segment SEP-1:1 (GRFN 1711) was synthesized on a
thioester-generating resin, and Segment SEP-1:4 (GRFN 1776) on a
--OCH.sub.2-Pam-resin as in Example 1. Lysines 24 and 126 of these
two peptide segments were initially protected with an Fmoc group at
the .epsilon.-amino group. After completion of the chain assembly,
the Fmoc-bearing amino groups were deprotected following standard
Fmoc deprotection procedures and modified by attachment of
levulinic acid to each peptide resin, respectively. The peptides
were then deprotected and simultaneously cleaved from the resin
support as described in Example 1. The peptides were purified by
preparative C4 reverse phase HPLC. Fractions containing pure
peptide were identified using ES-MS, pooled and lyophilized for
subsequent ligation. GRFNP32 [--(EDA-Succ).sub.18carboxyl- ate] was
assembled on a Sasrin carboxyl-generating resin following standard
protocols (Rose, K. et al., U.S. patent application Ser. No.
09/379,297; Rose, et al., J Am Chem Soc. (1999) 121: 7034). An
aminooxyacetyl (AoA) moiety was attached to the N-terminal amino
group of the polymer by coupling a fivefold excess of activated
Boc-aminooxyacetic acid. The polymer chain was cleaved from the
resin support using classic Fmoc-chemistry procedures. The polymer
chain was purified by preparative C4 reverse-phase HPLC. Fractions
containing pure polymer were identified using ES-MS, pooled and
lyophilized for subsequent ligation.
[0338] Segment SEP-1:4 and GRFNP32 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was then lyophilized. The dried powder was dissolved
and the polymer-modified peptide separated from unmodified peptide
and unreacted polymer by preparative gradient C4 reverse-phase
HPLC. Fractions containing the desired oximated product
SEP-1:4+GP32 were identified by ES-MS and pooled and
lyophilized.
[0339] Segment SEP-1:1 and GRFNP32 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was then lyophilized. The dried powder was dissolved
and the polymer-modified peptide separated from unreacted polymer
by preparative gradient C4 reverse-phase HPLC. Fractions containing
the desired oximated product SEP-1:1+GP32 were identified by ES-MS
and pooled and lyophilized.
[0340] B. Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-L30
[0341] SEP-1-L30 was synthesized in solution from four polypeptide
segments:
[0342] Segment SEP-1:1+GP32 (GRFN 1711+GRFNP32, Corresponding to
Residues 1-32 of SEQ ID NO: 2):
[0343] APPRLICDSR VLERYLLEAK EAEK.sup.oxITTGCA EH-thioester (where
Lys.sup.24 is modified at the .epsilon.-amino group with a
levulinic oxime linker group that is coupled to GRFNP32 through a
levulinic-aminooxyacetyl (Lev-AoA) oxime bond denoted K.sup.ox)
[0344] Segment SEP-1:2 (GRFN 1712, Corresponding to Residues 33-88
of SEQ ID NO: 2):
10 CSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL
LVKSSQPW-thioester
[0345] Segment SEP-1:3 (GRFN 1713, Corresponding to Residues 89-116
of SEQ ID NO: 2):
[0346] CP LQLHVDKAVS GLRSLTTLLR ALGAQK-thioester (where Cys.sup.89
is Acm protected)
[0347] Segment SEP:1:4+GP32 (GRFN 1776+GRFNP32, Corresponding to
Residues 117-166 of SEQ ID NO: 2):
11 CAIS PPDAAK.sup.oxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA
CRTGDR-carboxylate
[0348] (where Lys.sup.126 is modified at the .epsilon.-amino group
with a levulinic oxime linker group that is coupled to GRFNP32
through a levulinic-aminooxycetyl (Lev-AoA) oxime bond denoted
K.sup.ox, and where the C-terminal cysteine carries a picolyl
(pico) protecting group)
[0349] Synthesis of additional peptides, ligation reactions,
carboxymethylation, protecting group removal reactions, folding and
purification were performed as described in Example 1 to yield
full-length, folded SEP-1-L30. An analytical reverse-phase HPLC
chromatogram and an ES-MS spectrum of the folded protein product as
well as a CD spectrum demonstrated the presence of folded
protein
Example 3
[0350] Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-L26
[0351] A third synthetic erythropoiesis stimulating protein
(designated SEP-1-L26) was synthesized to contain oxime-forming
groups at positions 24 and 126 of SEP-0. These groups were then
used to form SEP-1-L26, in which the linear polymers
(EDA-Succ).sub.18 carboxylate and (EDA-Succ).sub.6-amide have been
joined to the protein backbone through oxime linkages at positions
24 and 126, respectively. The sequence of the full-length SEP-1
(1-166) is:
12 (SEQ ID NO:2) APPRLICDSR VLERYLLEAK EAEK.sup.oxITTGCA EHCSLNEKIT
VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW.psi.P
LQLHVDKAVS GLRSLTTLLR ALGAQK.psi.AIS PPDAAK.sup.oxAAPL RTITADTFRK
LFRVYSNFLR GKLKLYTGEA CRTGDR
[0352] where .PSI. denotes an non-native amino acid residue
consisting of a cysteine that is carboxymethylated at the
sulfhydryl group, and where K.sup.ox denotes a non-native lysine
that is chemically modified at the .epsilon.-amino group with an
oxime linker group coupled to a designated water-soluble polymer
through an oxime bond.
[0353] In contrast to SEP-1-L30, the SEP-1-L26 construct was
designed to bear a smaller and uncharged water-soluble polymer
attached at position 126. The polymer attached at position 24 was
the same as in SEP-1-L30. Assembly of the full-length product was
as described in Example 2. The structure of SEP-1-L26 is shown in
FIG. 16.
[0354] A. Oximation of GRFN1776 with GRFNP6 and Oximation of
GRFN1711 with GRFNP32
[0355] Oxime formation was performed to attach water-soluble
polymers bearing an aminooxyacetyl group to peptides carrying a
ketone carbonyl group. To accomplish this, the following peptide
segments were synthesized:
[0356] Segment SEP-1:4 (GRFN 1776; Composed of Residues 117-166 of
SEQ ID NO: 2):
13 CAISPPDAAK AAPLRTITAD TFRKLFRVYS NFLRGKLKLY
TGEACRTGDR-carboxylate
[0357] (where Lys.sup.126 is modified with a levulinic acid residue
at the .epsilon.-amino group, and where Cys.sup.117 is Acm
protected)
[0358] Segment SEP-1:1 (GRFN 1711, Composed of Residues 1-32 of SEQ
ID NO: 2):
[0359] APPRLICDSR VLERYLLEAK EAEKITTGCA EH-thioester (where
Lys.sup.24 is modified with a levulinic acid residue)
[0360] Segment SEP-1:1 (GRFN 1711) was synthesized on a
thioester-generating resin, and Segment SEP-1:4 (GRFN 1776) on a
--OCH.sub.2-Pam-resin as in Example 1. Lysines 24 and 126 of these
two peptide segments were initially protected with an Fmoc group at
the .epsilon.-amino group. After completion of the chain assembly,
the Fmoc-bearing amino groups were deprotected following standard
Fmoc deprotection procedures and modified by attachment of
levulinic acid to each peptide resin, respectively, following
standard coupling protocols. The peptides were then deprotected and
simultaneously cleaved from the resin support according to standard
Boc-chemistry procedures as in Example 1. The peptides were
separately purified by preparative C4 reverse-phase HPLC. For each
peptide, fractions containing pure peptide were identified using
ES-MS, pooled and lyophilized for subsequent ligation.
[0361] The water soluble polymer (EDA-Succ).sub.18 carboxylate
(GRFNP32) was assembled on a Sasrin carboxy-generating resin
following standard protocols (Rose, K. et at., U.S. patent
application Ser. No. 09/379,297; Rose, et al., J Am Chem Soc.
(1999) 121: 7034). The water soluble polymer (EDA-Succ).sub.6-amide
(GRFNP6) was assembled on a Sieber amide-generating resin following
standard protocols (Rose, K. et al., U.S. patent application Ser.
No. 09/379,297; Rose, et al., J Am Chem Soc. (1999) 121: 7034). An
aminooxyacetyl (AoA) moiety was attached to the N-terminal amino
group of each resin-bound polymer by coupling a fivefold excess of
activated Boc-aminooxyacetic acid. The two polymer chains were
separately cleaved from the respective resin supports using classic
Fmoc--chemistry procedures. Each polymer chain was purified by
preparative reverse phase HPLC. For each polymer, fractions
containing pure polymer were identified using ES-MS, pooled and
lyophilized for subsequent ligation.
[0362] Segment SEP-1:4 and GRFNP6 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was then lyophilized. The dried powder was dissolved
and the polymer-modified peptide separated from unmodified peptide
and unreacted polymer by preparative gradient C4 reverse-phase
HPLC. Fractions containing the desired oximated product SEP-1:4+GP6
were identified by ES-MS and pooled and lyophilized.
[0363] Segment SEP-1:1 and GRFNP32 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was then lyophilized. The dried powder was dissolved
and the polymer-modified peptide separated from unreacted polymer
by preparative gradient C4 reverse-phase HPLC. Fractions containing
the desired oximated product SEP-1:1+GP32 were identified by ES-MS
and pooled and lyophilized.
[0364] B. Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-L26
[0365] SEP-1-L26 was synthesized in solution from four polypeptide
segments:
[0366] Segment SEP:1:1+GP32 (GRFN 1711+GRFNP32, Corresponding to
Residues 1-32 of SEQ ID NO: 2):
[0367] APPRLICDSR VLERYLLEAK EAEK.sup.oxITTGCA EH-thioester (where
Lys.sup.24 is modified at the .epsilon.-amino group with a
levulinic oxime linker group that is coupled to GRFNP32 through a
levulinic-aminooxyacetyl (Lev-AoA) oxime bond denoted K.sup.ox)
[0368] Segment SEP-1:2 (GRFN 1712, Corresponding to Residues 33-88
of SEQ ID NO: 2):
14 CSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL
LVKSSQPW-thioester
[0369] (where Cys.sup.33 is Acm protected)
[0370] Segment SEP-1:3 (GRFN 1713, Corresponding to Residues 89-116
of SEQ ID NO: 2):
[0371] CP LQLHVDKAVS GLRSLTTLLR ALGAQK-thioester (where Cys.sup.89
is Acm protected)
[0372] Segment SEP:1:4+GP6 (GRFN 1776+GRFNP6, Corresponding to
Residues 117-166 of SEQ ID NO: 2):
15 CAISPPDAAK.sup.oxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTG
DR-carboxylate
[0373] (where Lys.sup.126 is modified at the .epsilon.-amino group
with a levulinic oxime linker group that is coupled to GRFNP6
through a levulinic-aminooxyacetyl (Lev-AoA) oxime bond denoted
K.sup.ox, and where the C-terminal cysteine [i.e. Cys.sup.161]
carries a picolyl (pico) protecting group)
[0374] Synthesis of additional peptides, ligation reactions,
carboxymethylation, protecting group removal reactions, folding and
purification were performed as described in Examples 1 and 2 to
yield full-length, folded SEP-1-L26. An analytical C4 reverse-phase
HPLC chromatogram and an ES-MS spectrum of the folded protein
product as well as a CD spectrum demonstrated the presence of
folded protein.
Example 4
[0375] Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-B50
[0376] A fourth synthetic erythropoiesis stimulating protein
(designated SEP-1-B50) was synthesized. The amino acid sequence of
the full-length SEP-1-B50 is the same as that of SEP-1-L30:
16 (SEQ ID NO:2) APPRLICDSR VLERYLLEAK EAE.sup.oxITTGCA EHCSLNEKIT
VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW.psi.P
LQLHVDKAVS GLRSLTTLLR ALGAQK.psi.AIS PPDAAK.sup.oxAAPL RTITADTFRK
LFRVYSNFLR GKLKLYTGEA CRTGDR
[0377] where .PSI. denotes an non-native amino acid residue
consisting of a cysteine that is carboxymethylated at the
sulfhydryl group, and where K.sup.ox denotes a non-native lysine
that is chemically modified at the C-amino group with an oxime
linker group coupled to a designated water-soluble polymer through
an oxime bond.
[0378] However, the protein was derivatized with a branched polymer
construct having four linear (Succ-EDA).sub.12 moieties rather than
the linear (Succ-EDA).sub.18 polymer of SEP-1-L30. Derivatization
was accomplished via oxime linkages.
[0379] FIG. 17 is a schematic illustrating the chemical synthesis
of the branched water-soluble polymer GRFNP29 (GP29) that was
joined to the SEP-1-B50 through oxime forming ligation as described
in detail below. As shown in FIG. 17, an orthogonally protected
lysine U--B precursor was used to generate U--B component GRFN17
(GP17) bearing an aminooxy acyl as the U group, and having a
3.times. lysine branching core B bearing four pendant thiol groups
for subsequent coupling through thioether linkages to a linear
water-soluble polyamide ethylene oxide construct designated GP29,
which bears a pendant carboxylate donated by a succinic acid
residue. Assembly of the full-length product was as described in
Example 2. The structure of SEP-1-B50 is shown in FIG. 18.
[0380] A. Synthesis of Template GRFNP17 Carrying Multiple Thiol
Groups
[0381] The template GRFNP17 was synthesized manually on an amide
generating (4-methyl)benzhydrylamine(MBHA)-resin on a 0.4 mmol
scale. Fmoc-Lys(Boc)-OH was coupled using standard coupling
protocols (Schnolzer, M., Int J Pept Protein Res. (1992)
40:180-93). 2.1 mmol amino acid, 10% DIEA in 3.8 ml 0.5M HBTU was
used; i.e. 5-fold excess of amino acid. After removal of the Fmoc
protecting group, Fmoc-Lys(Fmoc)-OH was coupled using standard
amino acid coupling protocols (2.1 mmol amino acid, 10% DIEA in 3.8
ml 0.5M HBTU; i.e. 5-fold excess amino acid). After a second Fmoc
removal step, Fmoc-Lys(Fmoc)-OH was coupled using standard amino
acid coupling protocols (4.2 mmol amino acid, 10% DIEA in 7.6 ml
0.5M HBTU; i.e. 5-fold excess amino acid relative to free amine).
After a final Fmoc deprotection step, a five-fold excess (relative
to free amines) of S-acetyl thioglycolic acid pentafluorophenyl
ester (SAMA-oPfp) in DMF was coupled for 30 minutes. The Boc
protecting group of the side chain of the C-terminal lysyl residue
was removed by two times one minute batch washes with neat TFA,
followed by neutralization of the resin by washing with 10% DIEA in
DMF. 2 mmol Boc-aminooxyacetic acid and 2 mmol N-hydroxysuccinimide
(NHS) were dissolved in 3 ml DMF. After addition of 2 mmol DIC
(diisopropylcarbodiimide), the acid was activated for 30-60
minutes. The solution was added to the neutralized resin and
coupled 1 hr. Finally, the S-linked acetyl groups were removed with
20% piperidine in DMF for 30 minutes. The template was deprotected
and simultaneously cleaved from the resin support using HF/p-cresol
according to standard Boc-chemistry procedures in the presence of
cysteine as a scavenger for free aldehyde (Schnolzer, M., Int J
Pept Protein Res. (1992) 40:180-93). The recovered polyamide in 50%
B [i.e. 50% aqueous acetonitrile containing 0.1% TFA] (aldehyde
free) was frozen and lyophilized. For purification, the template
crude product was dissolved in 2 ml 50% B, and 100 ml 100% A [i.e.
0.1% TFA in water] was added to dilute the sample (Avoid
guanidinium chloride or acetate addition, since the addition of
aldehyde is guaranteed). The template was loaded onto a C4
preparative reverse-phase HPLC column equilibrated at T=40.degree.
C. at 3% B. Salts were eluted isocratically and the desired
template, GRFNP17 purified in a linear gradient. Fractions
containing the desired product were identified by ES-MS, pooled and
lyophilized.
[0382] B. Synthesis of Branched Water-Soluble Polymer GRNP29
[0383] GRFNP29, a branched (EDA-Succ).sub.12 polymer of 15 kDa
molecular weight was synthesized by thioether-generating ligation
of purified thiol-containing template GRFNP17 and a linear polymer
GRFNP31, Br-acetyl-(EDA-Succ).sub.12 carboxylate, where GRFNP31 was
synthesized on a Sasrin carboxy-generating resin following standard
protocols (Rose, K. et al., U.S. patent application Ser. No.
09/379,297; Rose, et al., J Am Chem Soc. (1999) 121: 7034).
[0384] A 1.3.times. molar excess (over total thiols) of the
purified GRFNP31, Br-acetylated (EDA-Succ).sub.12, and purified
thiol-containing template GRFNP17 were jointly dissolved 0.1 M
Tris-HCl/6 M guanidinium chloride, pH 8.7 at .about.10 mM
concentration. After dissolution, the solution was diluted
threefold (v/v) with 0.1 M Tris-HCl, pH 8.7 buffer. The ligation
mixture was stirred at room temperature and the reaction monitored
by reversed-phase HPLC and ES/MS. Additional GRFNP31 reactant was
added on an as-needed basis until the desired reaction product was
the major product. For workup, 3.times. (v/v to ligation mix) 0.1M
acetate/6 M guanidinium chloride, pH 4 was added, and the solution
was loaded onto a preparative C4 reverse-phase HPLC column, and
purified with a linear gradient. Fractions containing pure GRFNP29
construct were identified using ES-MS, pooled and lyophilized.
[0385] C. Oximation of GRFN1776 and GRFN1711 with GRFNP29
[0386] Segments SEP-1:4, and Segment SEP-1:1 were synthesized as
described in Example 2. Segment SEP-1:4 and GRFNP29 were jointly
dissolved at an equimolar ratio in 50% aqueous acetonitrile
containing 0.1% TFA . The solution was lyophilized. The dried
powder was loaded onto a preparative reverse-phase HPLC column (1
inch diameter). The polymer-modified peptide was separated from
unmodified peptide and unreacted polymer by preparative gradient C4
reverse-phase HPLC. Fractions containing the desired oximated
product SEP:1:4+GP29 were identified by ES-MS and pooled.
[0387] Segment SEP-1:1 and GRFNP29 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was frozen and lyophilized. The dried powder was
dissolved in 50% aqueous acetonitrile containing 0.1% TFA and
loaded onto a preparative GPC (gel permeation chromatography)
column (1 inch diameter). The polymer-modified peptide was
separated from unmodified peptide and unreacted polymer by
isocratic elution. Fractions containing the desired oximated
product SEP-1:1+GP29 were identified by ES-MS and pooled.
[0388] D. Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-B50
[0389] SEP-1-B50 (SEQ ID NO: 2) was synthesized in solution from
four polypeptide segments:
[0390] Segment SEP-1:1+GP29 (GRFN 1711+GRFNP29, Corresponding to
Residues 1-32 of SEQ ID NO: 2):
[0391] APPRLICDSR VLERYLLEAK EAEK.sup.oxITTGCA EH-thioester (where
Lys.sup.24 is modified at the .epsilon.-amino group with a
levulinic oxime linker group that is coupled to GRFNP29 through a
levulinic-aminooxyacetyl (Lev-AoA) oxime bond denoted K.sup.ox)
[0392] Segment SEP-1:2 (GRFN 1712, Corresponding to Residues 33-88
of SEQ ID NO: 2):
17 CSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL
LVKSSQPW-thioester
[0393] (where Cys.sup.33 is Acm protected)
[0394] Segment SEP-1:3 (GRFN 1713, Corresponding to Residues 89-116
of SEQ ID NO: 2):
[0395] CP LQLHVDKAVS GLRSLTTLLR ALGAQK-thioester (where Cys.sup.89
is Acm protected)
[0396] Segment SEP11:4+GP29 (GRFN 1776+GRFNP29, Corresponding to
Residues 117-166 of SEQ ID NO: 2):
18 CAIS PPDAA.sup.oxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA
CRTGDR-carboxylate
[0397] (where Lys.sup.126 is modified at the .epsilon.-amino group
with a levulinic oxime linker group that is coupled to GRFNP29
through a levulinic-aminooxyacetyl (Lev-AoA) oxime bond denoted
K.sup.ox and where the C-terminal cysteine carries a picolyl (pico)
protecting group)
[0398] Synthesis of additional peptides, ligation reactions,
carboxymethylation, protecting group removal reactions, folding and
purification were performed as described in Examples 1 and 2,
except that purification was on a Resource Q column, to yield
full-length, folded SEP-1-B50 (SEQ ID NO: 2). An analytical C4
reverse-phase HPLC chromatogram and an ES-MS spectrum of the folded
protein product SEP-1-B50 as well as a CD spectrum demonstrated the
presence of folded protein.
Example 5
[0399] Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-3-L42
[0400] A fifth synthetic erythropoiesis stimulating protein
(designated SEP-3-L42) was synthesized. The amino acid sequence of
the full-length SEP-3 protein is:
19 (SEQ ID NO:3) APPRLICDSR VLERYLLEAK EAECITTGCA EHCSLNECIT
VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LACSSQPWEP LQLHVDKAVS
GLRSLTTLLR ALGAQKEAIS PPDAACAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA
CRTGDR
[0401] The cysteine residues in positions: 24, 38, 83, and 126 were
modified with maleimide-functionalized (EDA-Succ).sub.18 (GRFNP32)
polymer units (via a Michael addition reaction) to form SEP-3-L42.
The structure of SEP-3-L42 is shown in FIG. 20.
[0402] A schematic illustrating the chemical synthesis of SEP-3-L42
described below is shown in FIG. 19. Briefly, FIGS. 19(A) and 19(B)
show the native chemical ligation of the four SEP-3-L42 peptide
segments SEP-3:1 through SEP-3:4, which generates cysteines at the
ligation sites corresponding to all four native glycosylation sites
in human EPO. In particular, FIG. 19(B) shows full-length
polypeptide having all disulfide-forming cysteines protected,
permitting the site-specific attachment of four charged linear
water soluble polymers (designated GRFN32-maleimide
(GP32-maleimide)) at the cysteine ligation sites through a Michael
addition reaction. FIG. 19(B) also shows the final deprotection
step of the disulfide-forming cysteines, and generation of the
full-length, polymer-modified product.
[0403] In detail, SEP-3 was synthesized in solution from four
polypeptide segments:
[0404] Segment SEP-3:1 (GRFN 1747, Corresponding to Residues 1-37
of SEQ ID NO: 3):
[0405] APPRLICDSR VLERYLLEAK EAECITTGCA EHCSLNE-thioester (where
Cys.sup.7, Cys.sup.29, and Cys.sup.33 are Acm protected)
[0406] Segment SEP-3:2 (GRFN 1774, Corresponding to Residues 38-82
of SEQ ID NO: 3):
20 CIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LA-thioester
[0407] (where Cys.sup.38 is side chain protected with the Acm
group)
[0408] Segment SEP-3:3 (GRFN 1749, Corresponding to Residues 83-125
of SEQ ID NO: 3):
21 CSSQPWEP LQLHVDKAVS GLRSLTTLLR ALGAQKEAIS PPDAA-thioester
[0409] (where Cys.sup.83 is side chain protected with the Acm
group)
[0410] Segment SEP-3:4 (GRFN 1750, Corresponding to Residues
126-166 of SEQ ID NO: 3):
22 CAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDR-carboxylate
[0411] (where Cys161 is Pbo [i.e., 4-(CH.sub.3S(O)-)benzyl-]
protected)
[0412] Peptide synthesis. The peptides SEP-3:1 and SEP-3:2 and
SEP-3:3 were synthesized on a thioester-generating resin by the in
situ neutralization protocol for Boc chemistry SPPS, using
established side-chain protection strategies as described in
Example 1, with changes in protecting group strategy as noted in
the specific peptides above. Segment SEP-3:4 was synthesized
analogously on a --OCH.sub.2-Pam-resin. The peptides were
deprotected and simultaneously cleaved from the resin support as
described in Example 1. The resulting peptides described above were
purified by preparative RP-HPLC. Fractions containing pure peptide
were identified using ES-MS, pooled and lyophilized for subsequent
ligation.
[0413] Step 1: Ligation #1. Segment SEP-3:4 and segment SEP-3:3
were dissolved in TFE at 15 mM concentration. Saturated phosphate
buffer (pH 7.5) containing 6 M guanidinium chloride and 1%
thiophenol was added, resulting in a clear solution of the peptide
segments. After ligation, the ligation mix (defined as 1 volume)
was added to 2 volumes of a solution of {2 ml TFE, 6 ml 6 M
guanidinium chloride, 100 mM phosphate containing 25%
.beta.-mercaptoethanol} and incubated for 20 minutes. The solution
was acidified with a solution of 15 mg/ml TCEP in glacial acetic
acid, loaded onto a preparative C4 reverse-phase HPLC column and
purified with a linear gradient. Fractions containing the desired
ligated product SEP-3:Acm+SEP-3:3+SEP-3:4 were identified by ES-MS
and pooled.
[0414] Step 2: Acm-removal #1 For Acm removal, the aqueous
acetonitrile solution containing the pooled fractions of
SEP-3:Acm+SEP-3:3+SEP-3:4 was diluted 1.times. with HPLC grade
water, and solid urea was added for a final concentration of 2
molar. A threefold molar excess (relative to the total expected
cysteine concentration) of a 30 mg/ml Hg(acetate).sub.2 solution in
3% aqueous acetic acid was added and the solution was stirred for
one hour. The solution was then made 20% in .beta.-mercaptoethanol,
loaded onto a semi-preparative C4 reverse-phase HPLC column and
purified with a step gradient. Fractions containing the desired
ligated product SEP-3:3+SEP-3:4 were identified by ES-MS and
lyophilized overnight.
[0415] Step 3: Ligation #2 Equal amounts of SEP-3:3+SEP-3:4 and
SEP-3.2 were jointly dissolved in neat TFE trifluoroethanol at 15
mM concentration. 250 mM Phosphate buffer (pH 7.5) containing 6 M
guanidinium and 1% thiophenol was added, resulting in a clear
solution of the peptide segments. After one day of ligation, the
ligation mix (defined as 1 volume) was added to 2 volumes of a
solution of 10 ml TFE, 10 ml .beta.-mercaptoethanol, 10 ml
piperidine and 20 ml 6M guanidinium, pH 4, and incubated for 20
minutes to remove any remaining protecting groups. The solution was
acidified with a solution of 15 mg/ml TCEP in 20% aqueous acetic
acid, loaded onto a preparative C4 reverse-phase HPLC column and
purified with a linear gradient. Fractions containing the desired
ligated product SEP-3:Acm+SEP-3:2+SEP-3:3+SEP-3:4 were identified
by ES-MS and lyophilized overnight.
[0416] Step 4 Acm removal For Acm removal, the aqueous acetonitrile
solution containing the pooled fractions of
SEP-3:Acm+SEP-3:2+SEP-3:3+SEP- -3:4 was diluted 1.times. with HPLC
grade water, and solid urea added for a final concentration of 2
molar. A threefold molar excess (relative to the total expected
cysteine concentration) of a 30 mg/ml Hg(acetate).sub.2 solution in
3% aqueous acetic acid was added and the solution stirred for one
hour. The solution was then made 20% in .beta.-mercaptoethanol,
loaded onto a C4 semi-preparative reverse-phase HPLC column and
purified with a step gradient. Fractions containing the desired
ligated product SEP-3:2+SEP-3:3+SEP-3:4 were identified by ES-MS
and lyophilized overnight.
[0417] Step 5: Ligation #3 Equal amounts of SEP-3:2+SEP-3:3+SEP-3:4
and SEP:3:1 were jointly dissolved in neat TFE at 15 mM
concentration. 250 mM Phosphate buffer (pH 7.5) containing 6 M
guanidinium and 1% thiophenol was added, resulting in a clear
solution of the peptide segments. After one day of ligation, the
ligation mix (defined as 1 volume) was added to 2 volumes of a
solution of 10 ml TFE, 10 ml .beta.-mercaptoethanol, 10 ml
piperidine and 20 ml 6M guanidinium, pH 4, and incubated for 20
minutes to remove any remaining protecting groups. The solution was
acidified with a solution of 15 mg/ml TCEP in 20% aqueous acetic
acid, loaded onto a preparative C4 reverse-phase HPLC column and
purified with a linear gradient. Fractions containing the desired
ligated product SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4 were identified by
ES-MS and lyophilized overnight.
[0418] Step 6: Attachment of the Polymer GRFNP32. A
maleimide-functionalized linear (EDA-Succ).sub.18 polymer called
GRFNP32-maleimide was prepared by functionalizing GRFNP32 with BMPS
(3-maleimido propionic acid NHS ester, Pierce, USA) following the
manufacturers protocols to form a maleimide-functionalized
(EDA-Succ).sub.18 polymer [i.e., maleimide-(EDA-Succ).sub.18].
SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4 was dissolved in the minimum amount
of TFE required. A threefold excess of GRFNP32-maleimide was
dissolved in 6M guanidinium chloride, 100 mM phosphate, pH 7.5 and
added to the TFE solution. The progress of the Michael addition
reaction was followed by analytical reverse-phase HPLC. After the
reaction was complete, the solution was loaded onto a preparative
C4 reverse-phase HPLC column and purified with a linear gradient.
Fractions containing the desired polymer-modified product
SEP3:Acm+SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4+pPEG [i.e. the ligated
full-length 166 residue polypeptide chain with four copies of
GRFNP32 attached to the side chain thiols of Cys.sup.24,
Cys.sup.38, Cys.sup.83 and Cys.sup.126, and thus also called
SEP3:Acm+SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4+GP32], were identified by
ES-MS and lyophilized overnight.
[0419] Step 7: Pbo removal For Pbo reduction, the lyophilized
powder of SEP3:Acm+SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4+GP32 was
dissolved in neat TFA containing 5% ethanedithiol. The Pbo group
was then cleaved by addition of 10% thioanisole and 15%
bromotrimethylsilane for 30 minutes. The solution was dried in a
rotator-evaporator and taken up in aqueous acetonitrile containing
0.1% TFA. The resulting solution was loaded onto a semi-preparative
reverse-phase HPLC column and purified with a step gradient.
Fractions containing the desired Cys.sup.161-deprotected product
SEP3:Acm+SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4+GP32-Pbo were identified
by ES-MS and lyophilized overnight.
[0420] Step 8: Acm removal For final Acm removal from the side
chains of Cys.sup.7, Cys.sup.29, and Cys.sup.33, the aqueous
acetonitrile solution containing the pooled fractions of
SEP3:Acm+SEP-3:1+SEP-3:2+SEP-3:3+SEP-3- :4+GP32-Pbo was diluted
1.times. with HPLC grade water, and solid urea was added for a
final concentration of 2 molar. A threefold molar excess (relative
to the total expected cysteine concentration) of a 30 mg/ml
Hg(acetate).sub.2 solution in 3% aqueous acetic acid was added and
the solution was stirred for one hour. The solution was then made
20% in .beta.-mercaptoethanol, loaded onto a semi-preparative C4
reverse-phase HPLC column and purified with a step gradient.
Fractions containing the desired ligated, polymer-modified product
SEP-3 (1-166) were identified by ES-MS and lyophilized
overnight.
[0421] Step 9 Folding: Full-length ligated, polymer-modified
peptide SEP-3 (1-166) was dissolved in 200 mM Tris buffer (pH 8.7)
containing 6 M guanidinium chloride and 20% TFE and a ten-fold
molar excess (relative to Cys residues in SEP-3) of cysteine. This
solution was dialyzed overnight against a solution of 200 mM Tris
buffer (pH 8.7) containing 3 M guanidinium chloride at room
temperature. The solution was then dialyzed against a solution of
200 mM Tris buffer (pH 8.7) containing 1 M guanidinium chloride for
4 hours at 4.degree. C. and finally against 10 mM phosphate buffer
(pH 7.0) for 4 hours at 4.degree. C. to yield the final folded
product. Folding was verified by electrospray ES-MS and CD
spectrometry.
[0422] Step 10 Purification: The folded polypeptide was
concentrated 5.times. in centricon concentrator vials and loaded on
to Resource S cation exchange column equilibrated at 10 mM
phosphate, pH 7.0. The folded protein was eluted in a linear salt
gradient to 500 mM NaCl in 10 minutes. Fractions containing the
desired folded product SEP-3-L42 were identified by SDS-PAGE, and
frozen and stored at -80 .degree. C.
Example 6
[0423] Bioactivity Assay of Synthetic Erythropoiesis Stimulating
Proteins
[0424] The bioactivity of folded synthetic erythropoiesis
stimulating proteins, SEP-0, SEP-1-L26, SEP-1-L30, SEP-1-B50, and
SEP-3-L42 was determined using UT/7 and 32D 103197 cell lines, in
factor-dependent cell-line proliferation assays using commercial
recombinant erythropoietin as a control standard. UT-7 is a human
megakaryoblastic leukemia cell line with absolute dependence on one
of interleukin-3, granulocyte-macrophage colony-stimulating factor
(GM-CSF), or erythropoietin (EPO) for growth and survival (Miura Y,
et al., Acta Haematol (1998) 99:180-184); Komatsu N, et al., Cancer
Res. (1991) 51:341-8). 32D 103197 is a murine hemopoietic cell line
(Metcalf, D. Int J Cell Cloning (1992) 10:116-25).
[0425] Stock-solutions of the SEP constructs were made in Iscove's
modified Dulbecco's medium (IMDM), 10% FBS (Fetal bovine serum),
glutamine and Penstrep, and serial 2.times. dilutions of these
stock solutions were added to multi-well plates to which human UT/7
EPO cells at a concentration of 5000 cells/50 .mu.l were added. The
plates were incubated at 37.degree. C. in the presence of 5%
CO.sub.2 and monitored daily for growth. After four days, 20 .mu.l
2.5 mg/ml MTT (methylthiazol tetrazolium) in PBS (phosphate
buffered saline) was added and the plates were incubated for four
hours. 150 .mu.l IPA was added and the absorbance of each well was
read at 562 nm. The ED50 (effective dose to reach 50% of maximum
effect) values for the SEP compounds was determined and compared to
that of CHO (Chinese hamster ovary)-cell produced rhEPO
(recombinant human erythropoietin). The results from these
experiments demonstrated that all of the synthetic erythropoiesis
stimulating proteins exhibited bioactivity. ED50 results for SEP-0,
SEP-1-L26, SEP-1-L30, and SEP-1-B50 are shown in Table VI.
23 TABLE VI In Vitro ED50 Values (pM) Erythropoiesis UT-7 32D
103197 Stimulating Protein (Human) Cells (Mouse) Cells SEP-0 1,570
863 SEP-1-L26 46.5 100.8 SEP-1-L30 71.5 182.5 SEP-1-B50 182 6200 rh
EPO 32.5 136.3
[0426] Additional results from these experiments when normalized
for peptide content only using an extinction coefficient for
calculating protein concentration by absorbance for
polymer-modified SEP-1-L30 are shown in Table VII and FIG. 26 for
SEP-0, SEP-1-L26, SEP-1-L30, SEP-1-B50, SEP-3-L42, and SEP-1-B51
(synthesis of the SEP-1-B51 compound is described in Example 7
below). Collectively, these in vitro assays demonstrate the
differential effect of varying the polymer structures and sites of
attachment on in vitro bioactivity, where these compounds have the
following relative order of potency when tested for (1) human EPO
receptor activity (from most potent to least potent):
SEP-1-L26.apprxeq.SEP-1-L30>SEP-1-B50.apprxeq.SEP-1-B51>SEP-3-L42&g-
t;SEP-0; and (2) mouse EPO receptor activity (from most potent to
least potent):
SEP-1-L26>SEP-1-L30>SEP-0>SEP-3-L42>SEP-1-B50.apprxe-
q.SEP-1-B51.
24 TABLE VII In Vitro ED50 Values (ng/ml) Erythropoiesis UT-7 Cells
32D 103197 Cells Stimulating Protein (Human EPO R) (Mouse EPO R)
SEP-0 11 20 SEP-1-L26 0.8 1 SEP-1-L30 0.8 4 SEP-3-L42 7 70
SEP-1-B50 2.5 125 SEP-1-B51 2 125
[0427] The results in Table VII and FIG. 26 also show a significant
difference in receptor preference for the polymer-modified SEP
constructs compared to the non-polymer modified construct SEP-0. In
particular, SEP-1-L26 is modified with different linear polymers
attached at corresponding human EPO glycosylation site position 24
(a 5.5 kDa linear polymer with a pendant negative charge attached
at this site) and position 126 (a 1.8 kDa linear polymer with a
pendant neutral charge attached at this site); this construct had a
similar activity against both human and mouse receptors. SEP-1-L30
is modified with a 5.5 kDa linear polymer having a pendant negative
charge at corresponding human EPO glycosylation site positions 24
and 126; this construct was about 5.times. more active against the
human receptor compared to the mouse receptor. SEP-3-L42, which is
modified with a 5.5 kDa linear polymer having a pendant negative
charge at corresponding human EPO glycosylation site positions 24,
38, 83 and 126 was about 10.times. more active against the human
receptor compared to the mouse receptor. The largest differential
was observed for SEP-1-B50 and SEP-1-B51, which are modified with
branched, negatively charged 15 kDa polymers attached at
corresponding human EPO glycosylation site positions 24 and 126,
and have a pl of approximately 5 (similar to human EPO); these two
constructs were .about.60.times. more active against the human
receptor compared to the mouse receptor.
[0428] As mouse EPO is missing a glycosylation site at a position
corresponding to human EPO position 126 (mouse EPO has
non-glycosylated proline instead of the O-glycosylated serine found
in human EPO), the altered receptor activity may be due in part to
this difference, and thus preference of the mouse receptor for an
EPO that is missing O-linked glycosylation at corresponding
position 126. This is in line with the comparison of SEP-1-L26 and
SEP-1-L30 to SEP-1-B50 and SEP-1-B52. For example, SEP-1-L26 has a
1.8 kDa uncharged polymer at position 126 whereas SEP-1-L30 has a
5.5 kDa negatively charged polymer at position 126. Although
equivalent activity was observed against the human EPO receptor, a
4-fold difference was found against the mouse EPO receptor. The
largest difference was observed for the SEP-1-B50 and SEP-1-B51
constructs, with negatively charged 15 kDa polymers attached at
corresponding human EPO glycosylation site position 126. Addition
of the 5.5 kDa negatively charged polymers at sites corresponding
to all four native glycosylation positions in human EPO, i.e., 24,
38, 83 and 126 as with the SEP-3-L42 construct reduced preference
for mouse even further, but less than the SEP-1-B50 and SEP-1-B51
constructs. These results demonstrate that receptor-specificity can
be modulated by precision modification of sites corresponding to
glycosylation sites in a biologically produced, glycosylated
protein, and by adjusting the size, the nature of branching and
charge of the polymer.
Example 7
[0429] Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-B51
[0430] A. Composition of SEP-1-B51. A sixth synthetic
erythropoiesis stimulating protein (designated SEP-1-B51) was
synthesized. The amino acid sequence of the full-length SEP-1-B51
is the same as that of SEP-1-B50:
25 (SEQ ID NO:2) APPRLICDSR VLERYLLEAK EAEK.sup.oxITTGCA EHCSLNEKIT
VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW.psi.P
LQLHVDKAVS GLRSLTTLLR ALGAQK.psi.AIS PPDAAK.sup.oxAAPL RTITADTFRK
LFRVYSNFLR GKLKLYTGEA CRTGDR
[0431] where .PSI. denotes an non-native amino acid residue
consisting of a cysteine that is carboxymethylated at the
sulfhydryl group, and where K.sup.ox denotes a non-native lysine
that is chemically modified at the &-amino group with an oxime
linker group coupled to a designated water-soluble polymer through
an oxime bond.
[0432] However, in contrast to SEP-1-B50, the protein was
derivatized with a branched polymer construct having four linear
(Succ-TTD).sub.12-Succ-Al- anine-OH [i.e.,
(Succ-TTD).sub.12-Succ-Ala-OH, or (Succ-TTD).sub.12Succ-Al- a]
polymers coupled through amide bonds to the lysine branching core.
Coupling of the linear polymers to the branching core through amide
bonds was designed to improve stability. The four linear polymers
also are designed to carry an alanine moiety and pendant negative
charges, with the negative charges mimicking sialic acids and the
alanine a hydrophobic character similar to pendant sugar moieties
of carbohydrate chains. The branched polymers also were designed to
have a water-soluble spacer between the branching core and the
protein backbone attachment site to improve synthesis and handling
properties of the branching core template, and to mimic the spacing
found in natural carbohydrate chains between the site of sugar
attachment to the protein backbone and the first branch point of
the carbohydrate.
[0433] FIG. 21 is a schematic illustrating the chemical synthesis
of the branched water-soluble polymer GRFNP41 (GP41) that was
joined to the SEP-1-B51 through oxime-forming ligation as described
below. Assembly of the full-length product was as described in
Example 2. The structure of SEP-1-B51 is shown in FIG. 22.
[0434] B. Synthesis of (Succ-TTD).sub.12Succ-AlaOtBu (GRFNP39) for
Coupling to the Branching Template (GRFNP40).
[0435] Linear polymer (Succ-TTD).sub.12Succ-AlaOtBu (GRFNP39) was
synthesized on a 0.5 mmol scale on Sasrin acid labile, carboxylic
acid-generating resin. 0.5 mmole (.about.0.5 grams) Sasrin acid
labile, carboxylic acid-generating polystyrene resin (hydroxyl
substitution 1.02 mmole/g;) was swelled in DMF for 15 minutes, and
then drained. To this hydroxyl-functionalized resin was added 450
mg (4.5 mmole) succinic anhydride and 488 mg (4 mmole)
4-(dimethylamino)pyridine dissolved in 8 ml of DMF containing 500
microliter (3.9 mmole) DIEA (diisopropylethylamine) and allowed to
react for 30 minutes with vortex agitation, and drained. The
coupling was repeated and excess reactants and soluble coproducts
were removed by a 1 minute vortexing flow wash with DMF (.about.50
ml) and then drained. The HOOC--CH.sub.2CH.sub.2CO--O- -resin (0.5
mmole) was activated by addition of 8 ml of fresh 1.0 M (8 mmole)
CDI (carbonyldiimidazole) solution in DMF and allowed to react for
40 minutes, and then drained. Excess reactants and soluble
coproducts were removed by a 1 minute vortexing flow wash with DMF
(.about.50 ml), then drained. 4 ml (4 grams, 18.2 mmole)
(4,7,10)-trioxatridecane-1,13dia- mine (TTD) dissolved in 4 ml 0.5M
(2 mmole) HOBT solution in DMF was added and allowed to react with
vortex agitation for 30 minutes, then drained. Excess reactants and
soluble coproducts were removed by a 1 minute vortexing flow wash
with DMF (.about.50 ml), then drained. Succinic anhydride (450 mg,
4.5 mmole) dissolved in 8 ml of 0.5M (4 mmole) HOBT
(N-hydroxybenzotriazole) solution containing 500 microliter (3.9
mmole) DIEA was added to the resin and allowed to react with vortex
agitation for 15 minutes, then drained. The three steps (CDI
activation; TTD coupling; succinic anhydride reaction) were
repeated eleven times [i.e. a total of twelve times]. Excess
reactants and soluble coproducts were removed by a 1 minute
vortexing flow wash with DMF (.about.50 ml), and drained. The
HOOC--CH.sub.2CH.sub.2CO(TTD-succinyl).sub.12-O-resin (0.5 mmole)
was activated with 8 ml of fresh 1.0 M (8 mmole) CDI solution in
DMF and allowed to react for 40 minutes, and then drained. Excess
reactants and soluble coproducts were removed by a 1-minute
vortexing flow wash with DMF (.about.50 ml), then drained. 2.5
mmole H-AlaOtBu.HCl was dissolved in 4.75 ml 0.5 M (2.375 mmole)
HOBT in DMF containing 150 microliter (111 mg, 0.825 mmole) DIEA,
and allowed to react with the CDI-activated
HOOC--CH.sub.2CH.sub.2CO(TTD-succinyl).sub.12-O-resin (0.5 mmole)
for 1 hour with vortex agitation, then drained. Excess reactants
and soluble coproducts were removed by a 1-minute vortexing flow
wash with DMF (.about.50 ml), and then drained. The product
tertBuOOC--CH(CH3)-NH--OC--CH.sub.2CH.sub.2CO(TTD-succinyl).sub.12-O-resi-
n was washed extensively with DCM (dichlormethane), drained and
then the resin was dried under vacuum to constant weight. Typical
weight of product-resin was around 2 grams.
[0436] The linear (Succ-TTD).sub.12Succ-AlaOtBu polymer was cleaved
from the resin support according to standard Fmoc-chemistry
procedures using 4% TFA in DCM. The precipitated crude product was
dissolved in 50% aqueous acetonitrile containing 0.1% TFA and
lyophilized. The lyophilized polymer was dissolved in a small
amount of 50% aqueous acetonitrile containing 0.1% TFA and diluted
to reduce the concentration of organic below 1%. The crude product
was loaded onto a C4 preparative reverse-phase column equilibrated
at T=40.degree. C. at 3% B. Salts were eluted isocratically and the
desired template was purified with a linear gradient of 20-35%
Buffer B (acetonitrile containing 0.1% TFA) versus 0.1% aqueous TFA
over 60 minutes. Fractions containing the desired
(Succ-TTD).sub.12-Succ-AlaOtBu material (GRFNP39) were identified
by ES-MS, frozen and lyophilized.
[0437] C. Synthesis of a Template Carrying Multiple Amine Groups
for Branching and a Protected Aminooxy Group for (Later) Attachment
to the Protein (GRFNP40)
[0438] The template GRFNP40 was synthesized manually on a Sieber
amide-generating resin on a 0.4 mmol scale. There was a one-minute
flow-washing step with DMF between every coupling, deprotection and
activation step. 2 mmol
N.sup..alpha.-Fmoc-N.sup..beta.(Boc-aminooxyacety-
l)-L-diaminopropionic acid was coupled to the resin, using 30
minute NHS-ester pre-activation with 2 mmol DIC and 2 mmol NHS in
DCM/DMF (dimethylformamide), for 1 hour. After removal of the Fmoc
protecting group (2.times.3 minutes 20% v/v piperidine in DMF) and
washing, 4 mmol succinic anhydride dissolved in 8 ml of 0.5M HOBT
(N-hydroxybenzotriazole- ) solution containing 2.2 mmol DIEA was
coupled to the resin for 10 minutes. After this step, the carboxyl
group was activated with 8 ml of fresh 0.5 M (4 mmole) CDI solution
in DMF. 4 ml (18.2 mmole) TTD was added in 4 ml 0.5M HOBT solution
in DMF and coupled for 30 minutes.
[0439] Fmoc-Lys(Fmoc)-OH (2 mmol) was coupled to the resulting
amino-TTD-Succ-Dpr(BocAoA)-resin (where Dpr=diaminopropionic acid)
using 30 minutes pre-activation with 2 mmol DIC and 2 mmol NHS in
DMF and coupling for 1 hour. This coupling was repeated once. After
an Fmoc removal step (2.times.3 minutes 20% v/v piperidine in DMF),
4 mmol Fmoc-Lys(Fmoc)-OH was coupled as described above, including
recoupling. After a final Fmoc protection step (2.times.3 minutes
20% v/v piperidine in DMF), the template was cleaved from the resin
support according to standard Fmoc-chemistry procedures using 4%
TFA in DCM. The precipitated product was dissolved in 50% aqueous
acetonitrile containing 0.1% TFA and lyophilized. The lyophilized
crude product was dissolved in a small amount of 50% aqueous
acetonitrile containing 0.1% TFA and diluted to reduce the
concentration of organic below 1%. The template was loaded onto a
C4 preparative reverse-phase HPLC column equilibrated at
T=40.degree. C. at 3% Buffer B (0.1% TFA in acetonitrile). Salts
and other non-amide material were eluted isocratically, and the
desired template was purified with a linear gradient of 5-12%
Buffer B (acetonitrile containing 0.1% TFA) versus 0.1% aqueous TFA
over 60 minutes. Fractions containing the desired
Lys-Lys(Lys)-TTD-Succ-Dpr(BocAO- A).amide material (GRFNP40) were
identified by ES-MS, frozen and lyophilized.
[0440] D. Assembly and Deprotection of the Amide-Coupled Branched
Polymer (GRFNP41)
[0441] GRFNP41, a branched (TTD-Succ).sub.49--polymer of 16 kDa
molecular weight was synthesized by coupling of GRFNP39
[(Succ-TTD).sub.12-Succ-Ala- OtBu] to the purified template
GRFNP40. Purified (Succ-TTD).sub.12-Succ-Al- aOtBu (GRFNP39) (1.0
mmole) dissolved in DMSO (dimethylsulfoxide) at 60.degree. C. to a
concentration of 20 mg/ml was activated with 0.95 mole of HATU
{O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate} in DMSO at a concentation of 10 mg/ml in the
presence of a twenty-fold (molar) excess of DIEA. Purified template
GRFNP40 (0.24 mole) dissolved in DMSO at a concentration of 3.9
mg/ml was added immediately. Progress of the reaction was monitored
by C4 analytical reverse-phase HPLC and ES-MS. Typically, the
coupling was complete within minutes. For work-up, 4 volumes
(relative to ligation mix) 0.1M sodium acetate/6 M guanidinium
chloride, pH 4 was added, and the solution was loaded onto a
preparative (C4) reverse-phase HPLC column. Salts and other
non-amide containing material were eluted isocratically and the
desired branched polymer product was purified with a linear
gradient of 20-35% Buffer B (acetonitrile containing 0.1% TFA)
versus 0.1% aqueous TFA in 80 minutes. Fractions containing the
desired material were identified by ES-MS, frozen and
lyophilized.
[0442] The resulting purified polymer was dissolved in neat TFA at
a concentration of 1 mg/ml for 1 hour to remove the Boc group from
the aminooxyacetyl moiety and to remove the tertButyl ester groups
from the--Ala-OtBu moieties. The solution was rotated to dryness in
a rotator evaporator and the dried polymer was dissolved in 50%
Buffer B (acetonitrile containing 0.1% TFA). Aminooxyacetic acid
(a.k.a. `Carboxymethoxylamine`) was added to a final concentration
of 0.5M to scavenge adduct-forming impurities. After 20 minutes,
the solution was diluted out with 3.3 volumes of Buffer A (0.1% TFA
in water), and loaded onto a preparative C4 reverse phase HPLC
column and pumped at 10% Buffer B until all the aminooxyacetic acid
was removed, then the polymer was purified with a step gradient
from 15% to 45% Buffer B versus 0.1% aqueous TFA over 80 minutes.
Pooled fractions containing the desired branched polymer material
(GRFNP41) were frozen and lyophilized.
[0443] E. Oxime-Forming Ligation (Oximation) of the Peptide
Segments GRFN1776 and GRFN1711 with the Branched Polymer
GRFNP41
[0444] Segments SEP-1:4 (GRFN1776, composed of residues 117-166 of
SEQ ID NO: 2 and Segment SEP-1:1 (GRFN1711, composed of residues
1-32 of SEQ ID NO: 2 were synthesized as described above in Example
1 using standard in situ neutralization Boc chemistry SPPS. Peptide
GRFN1776 was synthesized on an --OCH.sub.2-Pam-Resin following
standard protocols also as described above. Peptide GRFN1711 was
synthesized on a thioester-generating resin following standard
protocols as described above. Segment GRFN1776 containing a
levulinic acyl moiety on Lys.sup.126 and the AoA-containing GRFNP41
were jointly dissolved at an equimolar ratio in 50% aqueous
acetonitrile containing 0.1% TFA. The solution was lyophilized. The
dried powder crude product was redissolved in 50% aqueous
acetonitrile containing 0.1% TFA and loaded onto a preparative
reverse-phase (C4) HPLC column. The polymer-modified peptide was
separated from unmodified peptide and unreacted polymer by
preparative gradient reverse phase HPLC. Fractions containing the
desired oxime-linked (oximated) product SEP-1:4+GP41 were
identified by ES-MS and pooled and lyophilized.
[0445] Segment GRFN1711 containing a levulinic acyl moiety on
Lys.sup.24 and the AoA-containing GRFNP41 were jointly dissolved at
an equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was frozen and lyophilized. The dried powder was
dissolved in 50% aqueous acetonitrile containing 0.1% TFA and
loaded onto a C4 preparative reverse-phase HPLC column. The
polymer-modified peptide was separated from unmodified peptide and
unreacted polymer by preparative gradient elution C4 reverse-phase
HPLC. Fractions containing the desired oxime-linked (oximated)
product SEP-1:1+GP41 were identified by ES-MS, and pooled and
lyophilized.
[0446] F. Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-B51
[0447] SEP-1-B51, having (SEQ ID NO: 2), was synthesized in
solution from four polypeptide segments:
[0448] Segment SEP-1:1+GP41 (GRFN 1711+GRFNP41; Corresponding to
Residues 1-32 of SEQ ID NO: 2):
[0449] APPRLICDSR VLERYLLEAK EAEK.sup.oxITTGCA EH-thioester (where
Lys.sup.24 has a levulinic acyl pendant moiety oxime-linked to the
branched polymer GRFNP41, as denoted by K.sup.ox; and where
His.sup.32 is Dnp protected)
[0450] Segment SEP-1:2 (GRFN 1712; Corresponding to Residues 33-88
of SEQ ID NO: 2):
26 CSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL
LVKSSQPW-thioester
[0451] (where Cys.sup.33 is Acm protected; and where the three Trp
residues are formyl protected)
[0452] Segment SEP-1:3 (GRFN 1713, Corresponding to Residues 89-116
of SEQ ID NO: 2):
[0453] CP LQLHVDKAVS GLRSLTTLLR ALGAQK-thioester (where Cys.sup.89
is Acm protected; and where His.sup.94 is Dnp protected)
[0454] Segment SEP-1:4+GP41 (GRFN 1776+GRFNP41, Corresponding to
Residues 117-166 of SEQ ID NO: 2):
27 CAIS PPDAAK.sup.oxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA
CRTGDR-carboxylate
[0455] (where the C-terminal cysteine (i.e. Cys.sup.161) carries a
picolyl (pico) protecting group, and where Lys.sup.126 has a
levulinic acyl pendant moiety oxime-linked to the branched polymer
GRFNP41, as denoted by K.sup.ox)
[0456] Synthesis of additional peptides, ligation reactions,
carboxymethylation, protecting group removal reactions, folding and
purification to yield full-length, folded SEP-1-B51 (SEQ ID NO: 2)
were performed as described in Examples 1-4, with the following
modifications:
[0457] Step 1: Ligation #1 Segment SEP-1:4+GP41 was dissolved in
TFE (1 volume) at 3 mM concentration, Segment SEP-1:3 was dissolved
in 2 volumes of 300 mM Na phosphate buffer (pH 7.9) containing 6 M
guanidinium chloride to a concentration of 2.25 mM. The two
solutions were mixed, giving final segment concentrations of 1 mM
SEP-1:4+GP41 and 1.5 mM SEP-1:3, and 1% thiophenol was added,
resulting in a solution (`3 volumes`) of the peptide segments at a
pH of 6.8-7.2. Reaction was allowed to proceed overnight at room
temperature. After ligation, .beta.-mercaptoethanol (3 volumes) was
added to the ligation mix, followed by 3 volumes of 6 M guanidinium
chloride 300 mM Na phosphate pH7.9 buffer, and TCEP was added (0.25
by weight of the total weight of peptide segments), and the
solution stirred for 20 minutes. The solution was acidified to
pH4.0.+-.0.1 with 0.6 volumes of glacial acetic acid to give a
clear solution to which was added 30 volumes of pH4.0, 100 mM Na
acetate dilution buffer, 6M in guanidinium chloride. The resulting
solution was pumped onto a preparative reverse-phase (C4) HPLC
column. Buffer was pumped at 5% B [thus the remainder is 95% Buffer
A (0.1% TFA in water)] until all non-peptide materials had eluted
from the column, then the ligation product was purified by a
gradient of 25-45% Buffer B over 80 minutes. Fractions containing
the desired ligated product (Cys.sup.89(Acm)){SEP-1:3+SEP-1:4+GP41}
were identified by electrospray mass spectrometry and pooled.
[0458] Step 2: Acm-removal #1 For Acm removal, the aqueous
acetonitrile solution containing the pooled fractions of
(Cys.sup.89(Acm)){SEP-1:3+SEP- -1:4+GP41} was diluted 1.times. with
HPLC grade water, and solid urea was added for a final
concentration of 2 M. A threefold molar excess (relative to the
total cysteine concentration) of a 30 mg/ml Hg(acetate).sub.2
solution in 3% aqueous acetic acid was added and the solution was
stirred for one hour. The solution was then made 20% in
.beta.-mercaptoethanol, loaded onto a semi-preparative
reverse-phase HPLC column and pumped at 25% Buffer B until all
non-peptide material had eluted, and the product was then purified
with a step gradient to 50% Buffer B. Fractions containing the
desired ligated product {SEP-1:3+SEP-1:4+GP41} were identified by
electrospray mass spectrometry, pooled and lyophilized.
[0459] Step 3: Ligation #2 The {SEP-1:3+SEP-1:4+GP41} product [i.e.
1713-1776-GP41] from Step 2 was dissolved in TFE (1 volume) at 3 mM
concentration. Segment SEP-1:2 (segment GRFN1712) was dissolved in
2 volumes of 300 mM Na phosphate buffer (pH 7.9) containing 6 M
guanidinium chloride to a concentration of 2.25 mM. The two
solutions were mixed, giving final segment concentrations of 1 mM
{SEP-1:3+SEP-1:4+GP41} and 1.5 mM SEP-1:2, and 1% thiophenol was
added, resulting in a solution (`3 volumes`) of the peptide
segments at a pH of 6.8-7.2. Reaction was allowed to proceed
overnight at room temperature. Then the ligation mix was diluted
with 3 volumes (i.e. relative to the volume of TFE used above) of
dilution buffer: 100 mM sodium acetate pH4.0 containing 6M
guanidinium chloride, and was then added to a solution, cooled to
4.degree. C., consisting of 3 volumes TFE, 3 volumes
.beta.-mercaptoethanol, 3 volumes piperidine and 6 volumes of 6M
guanidinium chloride, 100 mM Na acetate pH 4.0 and was stirred for
20 minutes at room temperature to remove any remaining protecting
groups. The solution was acidified with 1.8 volumes of frozen
glacial acetic acid to give a clear solution to which was added
TCEP (0.25 by weight of the total weight of peptide segments), and
the solution was incubated for 20 minutes. Then 30 volumes of
pH4.0, 100 mM Na acetate dilution buffer, 6 m in guanidinium
chloride was added and the solution was mixed. The resulting
solution was pumped onto a preparative reverse-phase (C4) HPLC
column. Buffer was pumped at 30% C (Buffer C: 0.1% TFA in 60%
isopropanol/30% acetonitrile/10% water) until all non-peptide
materials had eluted from the column, then the ligation product was
purified by a gradient of 37-57%% Buffer C over 80 minutes.
Fractions containing the desired ligated product
(Cys.sup.33(Acm)){SEP-1:2+SEP-1:3+SEP-1:4+GP41} were, identified by
ES-MS and pooled and lyophilized.
[0460] Step 4: Carboxymethylation of residues Cys.sup.89 and
Cys.sup.117 The (Cys.sup.33(Acm)){SEP-1:2+SEP-1:3+SEP-1:4+GP41}
[i.e. (Cys.sup.33(Acm))-1712-1713-1776-GP41] was dissolved in TFE
at 1 mM concentration. 10 volumes of 300 mM Na phosphate buffer (pH
7.9) containing 6 M guanidinium chloride was added. A 25-fold
excess (over sulfhydryl groups) of bromoacetic acid dissolved in
methanol at 75 mg/ml was added, and the solution was allowed to
react with stirring for two hours at room temperature The reaction
mixture was then diluted with 11 volumes of 100 mM Na acetate
pH4.0, 6M guanidinium chloride, and loaded onto a preparative
reverse-phase (C4) HPLC column and pumped at 20% Buffer C until all
non-peptide components had eluted, then the ligation product was
purified with a step gradient to 55% Buffer C. Fractions containing
the desired modified product (Cys.sup.33(Acm);
.PSI..sup.89,117){SEP-1:2+SEP-1:3+SEP-1:4+GP41} were identified by
ES-MS and pooled and lyophilized.
[0461] Step 5: Picolyl Removal Zinc dust (71 milligrams per
milligram of peptide) was activated in 2M HCl for 5 minutes, the
activation was repeated once, then the zinc was washed for 10
minutes with 6M guanidinium chloride, 50 mM glycine pH2.2
containing (freshly added) 35 mg/ml L-methionine and 35 mg/ml Na
dodecanoylsarcosine and 10% v/v TFE. to remove excess acid. The
wash was repeated once. The Cys.sup.161(Pico)-containing peptide
(Cys.sup.33(Acm); .PSI..sup.89,117){SEP-1:2+SEP-1:3+SEP-1:4+GP41}
was dissolved in neat TFE at about 30 mg/ml concentration. The
solution was diluted with 4 volumes (relative to TFE) 6M
guanidinium chloride, 50 mM glycine pH2.2 containing (freshly
added) 35 mg/ml L-methionine and 35 mg/ml Na dodecanoylsarcosine
and 10% v/v TFE. The solution was added to the activated zinc
powder and stirred for 50 minutes at room temperature. The reaction
was monitored by analytical C4 reverse-phase HPLC of aliquots
(treated with an equal volume of .beta.-mercaptoethanol and a few
grains of TCEP, then diluted with 2 volumes of 6M guanidinium
chloride, 100 mM Na acetate pH4.0 for analysis) at .about.1 hr
intervals and was complete after 2 to 5 hours. After picolyl group
removal was complete, as shown by ES-MS analysis of the analytical
HPLC peaks (i.e. loss of 91 Da mass), the supernatant was removed
by filtration and the remaining Zn powder was washed 3 times with
6M guanidinium chloride, pH 4, 100 mM acetate containing 35 mg/ml
L-methionine and 35 mg/ml dodecylsarcosine containing 20% TFE.
BioRad SM-2 beads (approximately one-third the volume of the
solution) were added to the combined supernatant and washes and
stirred at room temperature for 30 minutes, then filtered.
.beta.-mercaptoethanol was added to 10% v/v, then 0.25.times.
(combined weight of peptides) of TCEP was added and the solution
stirred for 5 minutes at room temperature. The solution was diluted
1:1 with an equal volume of 100 mM Na acetate pH4.0, 6M guanidinium
chloride and loaded onto a preparative (C4) reverse-phase HPLC
column and 35% Buffer C pumped until all the non-peptide material
was eluted, and the desired product was purified with a step
gradient to 55% Buffer C. Fractions containing the desired
Cys.sup.161-deprotected product (Cys.sup.33(Acm);
.PSI..sup.89,117){SEP-1:2+SEP-1:3+SEP-1:4+GP41-- Pico} were
identified by electrospray mass spectrometry and pooled.
[0462] Step 6: Acm-removal #2 The pooled solution of
(Cys.sup.33(Acm);
.PSI..sup.89,117){SEP-1:2+SEP-1:3+SEP-1:4+GP41-Pico} [i.e.
(Cys.sup.33(Acm); .PSI..sup.89,117)-1712-1713-1776-GP41] was
diluted 3.times. with HPLC grade water, and solid urea was added
for a final concentration of 2M. A threefold molar ratio (relative
to the total cysteine concentration) of a 30 mg/ml
Hg(acetate).sub.2 solution in 3% aqueous acetic acid was added and
the solution was stirred for one hour at room temperature. The
solution was then made 20% in .beta.-mercaptoethanol, and was
loaded onto a semi-preparative (C4) reverse-phase HPLC column.
Buffer C (20%) was pumped until all non-peptide material had been
eluted, and then the desired product was purified with a step
gradient to 55% Buffer C. Fractions containing the desired product
(Cys.sup.33; .PSI..sup.89,117){SEP-1:2+SEP-1:3+SEP-1:4+GP- 41-Pico}
were identified by ES-MS, diluted 2.times. (v/v) with water
containing 2.times. (w/w relative to peptide mass) DPC
(dodecylphosphocholine) and were lyophilized overnight.
[0463] Step 7: Ligation #3 (Cys.sup.33;
.PSI..sup.89,117){SEP-1:2+SEP-1:3+- SEP-1:4+GP41-Pico} [i.e.
(Cys.sup.33; .PSI..sup.89,117)-1712-1713-1776-GP4- 1] was dissolved
in neat TFE (1 volume) at 3 mM concentration. SEP-1:1 [i.e.
1711-GP41] was dissolved in 2 volumes 300 mM Na phosphate buffer
(pH 7.9) containing 6 M guanidinium chloride. The solutions were
combined, and 10% v/v thiophenol was added. The ligation mix was
stirred overnight at room temperature. To the solution was added 3
volumes (relative to TFE volume above) of .beta.-mercaptoethanol,
and 9 volumes of ligation buffer (300 mM Na phosphate buffer pH
7.9, containing 6 M guanidinium chloride). To the solution was
added 0.25.times. (combined weight of peptides) TCEP and the
solution was stirred at room temperature for 20 minutes. Glacial
acetic acid (0.6 volumes) was added to acidify the solution to
pH4.0, and the solution was then diluted with 30 volumes of 100 mM
Na acetate pH4.0 containing 6M guanidinium chloride, and loaded
onto a preparative (C4) reverse-phase HPLC column. Buffer C (25%)
was pumped until all non-peptide material was eluted, then the
ligation product was purified with a linear gradient of Buffer C
from 35-55% over 80 minutes. Fractions containing the desired
ligated product SEP-1 (1-166) (.PSI..sup.89,
117)(SEP-1:1(+GP41)+SEP-1:2+SEP-1:3+SEP-1:4+GP41}: (SEQ ID NO: 2)
were identified by electrospray mass spectrometry, and pooled.
[0464] Step 8 Folding: To the combined fractions containing
full-length ligated peptide SEP-1 (1-166)
(.PSI..sup.89,117){SEP-1:1(+GP41)+SEP-1:2+S- EP-1:3+SEP-1:4+GP41}
[i.e. 1711 (GP41)-1712-1713-1776-GP41] was added solid guanidinium
chloride, 1M Tris buffer (pH 8.7) and distilled water to make the
final solution 0.1 milligrams per ml ligated peptide SEP-1 (1-166),
6M guanidinium chloride, 100 mM Tris, This ligated peptide
(`protein`) solution was loaded into 15 ml dialysis cassettes and
dialyzed overnight at 4.degree. C. against a solution of 100 mM
Tris buffer (pH 8.5) containing 3 M guanidinium chloride, 5
micromolar cysteine and 2 micromolar cystine. The protein solution
was then dialyzed against a solution of 100 mM Tris buffer (pH 8.5)
containing 1 M guanidinium chloride for 8 hours at 4.degree. C. and
finally dialyzed against 10 mM Tris buffer (pH 7.0) for 14 hours at
4.degree. C. to yield the final folded product. The folded
protein-containing solutions from the dialysis cassettes were
combined. Folding was verified by ES-MS, analytical RP-HPLC, and by
CD spectrometry.
[0465] Step 9 Purification: The folded protein-containing solution
was loaded on to Q-Sepharaose ion exchange column equilibrated at
10 mM Tris, pH 7.0. The folded protein was eluted using a linear
salt gradient to 125 mM NaCl. Fractions containing the desired
folded product SEP-1-B51 were identified by non-reducing SDS-PAGE,
and pooled. The combined fractions were concentrated by
ultrafiltration to a concentration of approximately 2 milligrams
per milliliter, then the protein solution was loaded onto a
2.6.times.100 cm S-300 gel filtration column. The purified
SEP-1-B51 was eluted with 10 mM Tris pH7.0, 137 mM sodium chloride.
Fractions containing high purity SEP-1-B51 were identified by
non-reducing SDS-PAGE, and were pooled, frozen and stored at
-80.degree. C. The final, purified folded protein SEP-1-B51 was
characterized by analytical (C4) reverse-phase HPLC, ES-MS, by
non-reducing SDS-PAGE, and CD spectrometry. FIG. 25 shows a
representative Isoelectric Focusing Gel (IEF) and non-reducing
SDS-PAGE gel showing the relative molecular weight of the folded,
purified SEP1-B51. A molecular weight standard run on the same gels
are shown for comparison. As shown, the relative molecular weight
of SEP1-B51 by determined by non-reducing SDS-PAGE is approximately
73 kDa. The relative pl is approximately 5.0. Also shown is a
representative RP-HPLC chromatogram of the folded, purified
SEP-1-B51 product. This illustrates the purity and increased
relative molecular weight of the precision polymer-modified
proteins of the present invention.
Example 8
[0466] Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-B52
[0467] A. Composition of SEP-1-B52. A seventh synthetic
erythropoiesis stimulating protein (designated SEP-1-B52) was
synthesized. The amino acid sequence of the full-length SEP-1-B52
is the same as that of SEP-1-B51:
28 (SEQ ID NO:2) APPRLICDSR VLERYLLEAK EAEK.sup.oxITTGCA EHCSLNEKIT
VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW.psi.P
LQLHVDKAVS GLRSLTTLLR ALGAQK.psi.AIS PPDAAK.sup.oxAAPL RTITADTFRK
LFRVYSNFLR GKLKLYTGEA CRTGDR
[0468] where .PSI. denotes an non-native amino acid residue
consisting of a cysteine that is carboxymethylated at the
sulfhydryl group, and where K.sup.ox denotes a non-native lysine
that is chemically modified at the .epsilon.-amino group with an
oxime linker group coupled to a designated water-soluble polymer
through an oxime bond.
[0469] The SEP-1-B52 protein was derivatized at residues 24 and 126
with a branched (Succ-TTD).sub.12-Succ-Ala polymer construct
similar to SEP-1-B51, but for this analog the polymer was attached
via an oxime bond between pyruvic acid and aminooxyacetic acid.
Moreover, the .epsilon.-amino group of lysine residues 24 and 126
were modified to bear an aminooxyacetyl functional group instead of
a levulinic acyl moiety, and the branched
(Succ-TTD).sub.12-Succ-Ala polymer construct was made to bear a
pedant pyruvic acyl moiety. These changes were designed to improve
synthesis and handling, and to further increase stability of the
oxime linkages.
[0470] FIG. 23 is a schematic illustrating the chemical, synthesis
of the branched water-soluble polymer GRFNP43 (GP43) that was
joined to the SEP-1-B52 through oxime-forming ligation as described
below. Assembly of the full-length product was as described in
Example 2. The structure of SEP-1-B52 is shown in FIG. 24.
[0471] B. Synthesis of (Succ-TTD).sub.12-Succ-AlaOtBu (GRFNP39) for
Coupling to the Branching Template (GRFNP42).
[0472] (Succ-TTD).sub.12-Succ-AlaOtBu (GRFNP39) was synthesized on
a 0.5 mmol scale. 0.5 mmole (.about.0.5 grams) Sasrin acid labile,
carboxylic acid-generating polystyrene resin (hydroxyl substitution
1.02 mmole/g;) was swelled in DMF for 15 minutes and then drained.
To this hydroxyl-functionalized resin was added 450 mg (4.5 mmole)
succinic anhydride and 488 mg (4 mmole) 4-(dimethylamino)pyridine
dissolved in 8 ml of DMF containing 500 microliter (3.9 mmole) DIEA
(diisopropylethylamine) and allowed to react for 30 minutes with
vortex agitation, then drained. The coupling was repeated and
excess reactants and soluble coproducts were removed by a 1 minute
vortexing flow wash with DMF (.about.50 ml), then drained. The
HOOC--CH.sub.2CH.sub.2CO--O-re- sin (0.5 mmole) was activated by
addition of 8 ml of fresh 1.0 M (8 mmole) CDI solution in DMF and
allowed to react for 40 minutes, then drained. Excess reactants and
soluble coproducts were removed by a 1 minute vortexing flow wash
with DMF (.about.50 ml), and drained. 4 ml (4 grams, 18.2 mmole)
TTD dissolved in 4 ml 0.5M (2 mmole) HOBT solution in DMF was added
and allowed to react with vortex agitation for 30 minutes and
drained. Excess reactants and soluble coproducts were removed by a
1 minute vortexing flow wash with DMF (.about.50 ml) and drained.
Succinic anhydride (450 mg, 4.5 mmole) dissolved in 8 ml of 0.5M (4
mmole) HOBT (N-hydroxybenzotriazole) solution containing 500
microliter (3.9 mmole) DIEA was added to the resin and allowed to
react with vortex agitation for 15 minutes, then drained. The three
steps (CDI activation; TTD coupling; succinic anhydride reaction)
were repeated eleven times [i.e. a total of twelve times]. Excess
reactants and soluble coproducts were removed by a 1 minute
vortexing flow wash with DMF (.about.50 ml), and drained. The
HOOC--CH.sub.2CH.sub.2CO(TTD-succinyl).sub.12-O-resin (0.5 mmole)
was activated with 8 ml of fresh 1.0 M (8 mmole) CDI solution in
DMF, allowed to react for 40 minute, and drained. Excess reactants
and soluble coproducts were removed by a 1-minute vortexing flow
wash with DMF (.about.50 ml), and drained. 2.5 mmole H-AlaOtBu.HCl
was dissolved in 4.75 ml 0.5 M (2.375 mmole) HOBT in DMF containing
150 microliter (111 mg, 0.825 mmole) DIEA, and allowed to react
with the CDI-activated
HOOC--CH.sub.2CH.sub.2CO(TTD-succinyl).sub.12-O-resin (0.5 mmole)
for 1 hour with vortex agitation, then drained. Excess reactants
and soluble coproducts were removed by a 1-minute vortexing flow
wash with DMF (.about.50 ml), and then drained. The product
tertBuOOC--CH(CH3)-NH--OC---
CH.sub.2CH.sub.2CO(TTD-succinyl).sub.12-O-resin was washed
extensively with DCM, drained and then the resin was dried under
vacuum to constant weight. Typical weight of product-resin was
around 2 grams.
[0473] The linear GRFNP39 was cleaved from the resin support
according to standard Fmoc-chemistry procedures using 4% TFA in
DCM. The precipitated crude product was dissolved in 50% aqueous
acetonitrile containing 0.1% TFA and lyophilized. The lyophilized
polymer was dissolved in a small amount of 50% aqueous acetonitrile
containing 0.1% TFA and diluted to reduce the concentration of
organic below 1%. The crude product was loaded onto a C4
preparative reverse-phase HPLC column equilibrated at T=40.degree.
C. at 3% B. Salts were eluted isocratically and the desired
template was purified with a linear gradient of 20-35% Buffer B
(acetonitrile containing 0.1% TFA) versus 0.1% aqueous TFA over 60
minutes. Fractions containing the desired
(Succ-TTD).sub.12-Succ-AlaOtBu material (GRFNP39) were identified
by ES-MS, frozen and lyophilized.
[0474] C. Synthesis of a Template Carrying Multiple Amine Groups
for Branching and a Pendant Pyruvic Acyl Moiety for (Later)
Attachment to the Protein (GRFNP42)
[0475] The template was synthesized manually on a
Boc-Leu-OCH.sub.2-Pam-re- sin on a 0.4 mmol scale. A one-minute
flow-washing step with DMF was used between every coupling,
deprotection and activation step. The Boc group was removed by
treatment with neat (i.e. 100%) TFA. After DMF washing, 2 mmol
Fmoc-(Rink-linker)-OH was coupled to the resin after activation
with 1.8 mmol HBTU in 3.8 ml DMF containing 1 ml DIEA. After
removal of the Fmoc protecting group (2.times.3 minutes 20%
piperidine in DMF), 2 mmol Fmoc-Lys(MTT)-OH [MTT=4-methyltrityl]
was coupled to the resin using NHS-ester activation with 2 mmol DIC
and 2 mmol NHS in DMF. After removal of the Fmoc protecting group
(2.times.1 minute 0.5% DBU in DMF), 4 mmol succinic anhydride
dissolved in 8 ml of 0.5M HOBT solution containing 2.2 mmol DIEA
was coupled to the resin for 10 minutes. After this step, the
resin-bound carboxyl group was activated with 8 ml of fresh 0.5 M
CDI solution in DMF. 4 ml TTD was added in 4 ml 0.5M HOBT solution
and coupled for 30 minutes.
[0476] 2 mmol Fmoc-Lys(Fmoc)-OH was coupled to the resin using
NHS-ester activation with 2 mmol DIC and 2 mmol NHS in DMF. After
an Fmoc removal step (2.times.1 minute 0.5% DBU in DMF), 4 mmol
Boc-Lys(Boc)-NHS is coupled in 3 ml DMF.
[0477] The MTT protecting group was removed by multiple washes with
2% TFA in DCM. Deprotection was complete when the supernatant lost
its yellow color. The resin was neutralized with 10% DIEA in DMF
for one minute. 2 mmol pyruvic acid was coupled to the resin using
NHS-ester activation with 2 mmol DIC and 2 mmol NHS in DMF for 45
minutes. The template was deprotected and cleaved from the resin
support using neat TFA containing 5% water. The cleavage solution
was evaporated to dryness in a rotator evaporator. The residue was
dissolved in 50% aqueous acetonitrile containing 0.1% TFA and
lyophilized. The lyophilized template was dissolved in a small
amount of 50% aqueous acetonitrile containing 0.1% TFA and diluted
to reduce the concentration of organic below 1%. The
pyruvate-containing template was loaded onto a C4 Prep column
equilibrated at T=40.degree. C. at 0% Buffer B [i.e. 100% Buffer
A=0.1% TFA in water]. Salts were eluted isocratically and the
desired template was purified with a linear gradient of 5-12%
Buffer B versus 0.1% aqueous TFA in 60 minutes. Fractions
containing the desired material (GRFNP42) were identified by
ESI-MS, frozen and lyophilized.
[0478] D. Assembly and Deprotection of the Amide-Coupled Branched
Polymer (GRFNP43)
[0479] GRFNP43, a branched (TTD-Succ).sub.49--polymer of 16 kDa
molecular weight was synthesized by coupling GRFNP39 to the
purified template GRFNP42. Purified (Succ-TTD).sub.12-Succ-AlaOtBu
(GRFNP39) (1.0 mmole) dissolved in DMSO at 60.degree. C. to a
concentration of 20 mg/ml was activated with 0.95 mole of HATU in
DMSO at a concentation of 10 mg/ml in the presence of a twenty-fold
(molar) excess of DIEA. Purified template (0.24 mole) GRFNP42
dissolved in DMSO at a concentration of 3.9 mg/ml was added
immediately. Progress of the reaction was monitored by analytical
C4 reversed-phase HPLC and ES-MS. Typically, the coupling was
complete within minutes. For work-up, 4 volumes (relative to
ligation mix) 0.1M acetate/6 M guanidinium chloride, pH 4 was
added, and the solution was loaded onto a preparative (C4)
reverse-phase HPLC column. Salts and other non-amide containing
material were eluted isocratically and the desired product branched
polymer was purified with a linear gradient of 20-35% Buffer B
(acetonitrile containing 0.1% TFA) versus 0.1% aqueous TFA over 80
minutes. Fractions containing the desired material were identified
by ES-MS, frozen and lyophilized.
[0480] The resulting purified branched polymer construct GRFNP43
was dissolved in neat TFA at a concentration of 1 mg/ml for 1 hour
to remove the Ala-OtBu tertButyl ester protection. The solution was
evaporated to dryness in a rotary evaporator and the dried polymer
was dissolved in 50% Buffer B (acetonitrile containing 0.1% TFA).
The polymer was desalted on a preparative reverse phase HPLC with a
step gradient from 15% to 45% Buffer B versus 0.1% aqueous TFA in
80 minutes. Pooled fractions containing the desired material
(GRFNP43) were frozen and lyophilized, and the dried powder used
for the oximation-forming ligation step.
[0481] E. Oxime-Forming Ligation (Oximation) of GRFN1776 and
GRFN1711 With the Branched Polymer GRFNP43
[0482] Segments SEP-1:4, and Segment SEP-1:1 were synthesized as
described above in Examples 2, 3, 4, and 7, only that instead of
levulinic acid an aminooxyacetyl (AoA) moiety was appended to
Lys.sup.24 and Lys.sup.126 in the respective peptide segments
following standard coupling protocols. Thus, after assembly of the
peptide-resins, the side chain Fmoc group was removed from each
peptide-resin and 2 mmol Boc-aminooxyacetic acid was added to the
resin using NHS-ester activation with 2 mmol DIC and 2 mmol NHS in
DMF. The two peptides were deprotected and cleaved from the resin
with HF and purified by C4 reverse-phase HPLC. Suitable precautions
were taken to avoid exposure to carbonyl compounds. Segment SEP-1:4
and GRFNP43 were jointly dissolved at an equimolar ratio in 50%
aqueous acetonitrile containing 0.1% TFA (trifluoroacetic acid).
The solution was lyophilized. The dried powder was re-dissolved in
50% aqueous acetonitrile containing 0.1% TFA and loaded onto a
preparative C4 reverse-phase HPLC column. The polymer-modified
peptide was separated from unmodified peptide and unreacted polymer
by preparative gradient reverse-phase HPLC. Fractions containing
the desired oximated product SEP-1:4+GP43 were identified by ES-MS
and pooled.
[0483] Segment SEP-1:1 and GRFNP43 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was frozen and lyophilized. The dried powder was
dissolved in 50% aqueous acetonitrile containing 0.1% TFA and
loaded onto a preparative gradient C4 reverse-phase HPLC column.
The polymer-modified peptide was separated from unmodified peptide
and unreacted polymer by preparative reverse-phase gradient
elution. Fractions containing the desired oximated product
SEP-1:1+GP43 were identified by ES-MS and pooled.
[0484] F. Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-B52
[0485] SEP-1-B52 (SEQ ID NO: 2) was synthesized in solution from
four polypeptide segments:
[0486] Segment SEP:1:1+GP43 (GRFN 1711+GRFNP43; Corresponding to
Residues 1-32 of SEQ ID NO: 2):
[0487] APPRLICDSR VLERYLLEAK EAEK.sup.oxITTGCA EH-thioester (where
Lys.sup.24 has an AoA pendant moiety oxime-linked to the branched
polymer GRFNP43, as denoted by K.sup.ox; and where His.sup.32 is
Dnp protected)
[0488] Segment SEP-1:2 (GRFN 1712; Corresponding to Residues 33-88
of SEQ ID NO: 2):
29 CSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL
LVKSSQPW-thioester
[0489] (where Cys.sup.33 is Acm protected; and where the three Trp
residues are formyl protected)
[0490] Segment SEP-1:3 (GRFN 1713, Corresponding to Residues 89-116
of SEQ ID NO: 2):
[0491] CP LQLHVDKAVS GLRSLTTLLR ALGAQK-thioester (where Cys.sup.89
is Acm protected; and where His.sup.94 is Dnp protected)
[0492] Segment SEP-1:4+GP43 (GRFN 1776+GRFNP43, Corresponding to
Residues 117-166 of SEQ ID NO: 2):
30 CAIS PPDAAK.sup.oxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA
CRTGDR-carboxylate
[0493] (where the C-terminal cysteine (i.e. Cys.sup.161) carries a
picolyl (pico) protecting group, and where Lys.sup.q126 has an AoA
pendant moiety oxime-linked to the branched polymer GRFNP43, as
denoted by K.sup.ox).
[0494] Synthesis of additional peptides, ligation reactions,
carboxymethylation, protecting group removal reactions, folding and
purification are performed as described above in Examples 1, 2, 3,
4, and 7, to yield full-length, folded SEP-1-B52 (SEQ ID NO: 2),
which was characterized by analytical (C4) reverse-phase HPLC,
ES-MS, and non-reducing SDS-PAGE. Bioassays are performed as
described for the other SEP constructs.
Example 9
[0495] Efficacy Studies for SEP-3-L42
[0496] SEP-3-L42 was reformulated in Citrate Buffer (20 mM sodium
citrate+100 mM sodium chloride) plus 0.25% rat serum albumin (RSA)
and was administered intravenously to normal male rats (5 rats per
group) at doses of 0, 1, 5, or 10 .mu.g/kg, tiw, on Days 1, 3, and
6. Blood samples were collected 4 days after the last injection
(Day 9) and analyzed for hematologic parameters. There were no
statistically significant differences in red blood cell (RBC),
hemoglobin (HGB), hematocrit (HCT), and reticulocyte count (RET)
values at 4 days after the last injection with SEP-3-L42 at these
doses when compared with those of the control group.
Example 10
[0497] Pharmacokinetic Studies for SEP-3-L42
[0498] SEP-3-L42 was reformulated in Citrate Buffer (20 mM sodium
citrate+100 mM sodium chloride) plus 0.25% RSA, pH6.9, and was
administered intravenously as a single dose to normal male rats at
a dose level of 5 ug/kg. Blood samples were collected at 0, 1, 2,
4, 6, 12, 24, 48, 72, 96, 120, 144, 168 hours after the dosing. The
plasma SEP-3-L42 concentration was determined by anti-EPO ELISA kit
(R & D Systems, Human Erythropoietin Quantikine IVD Immunoassay
Kit #DEP00) according to the manufacturers instructions. The
results are shown in FIG. 27.
[0499] As shown in FIG. 27, SEP-1-B50 exhibited slightly slower
clearance compared to SEP3-L42. Despite its circulating half-life,
SEP3-L42 failed to exhibit statistically significant differences in
promoting red blood cell production at the doses tested. In
contrast, the SEP-1-B50 stimulated red blood cell production at the
same doses. This illustrates that polymer structure can be
exploited for fine-tuning in vivo biological properties, including
pharmacokinetic behavior and potency.
Example 11
[0500] Efficacy Studies for SEP-1-L30
[0501] SEP-1-L30 was reformulated in Citrate Buffer (20 mM sodium
citrate+100 mM sodium chloride) plus 0.25% RSA and was administered
intravenously to normal male rats (5 rats per group) at doses of 0,
1, 5, 25, or 50 .mu.g/kg, tiw, on Days 1, 3, and 5. Blood samples
were collected 4 and 8 days after the last injection (Days 9 and
13) and analyzed for hematologic parameters. There were no
statistically significant differences in RBC, HGB, and HCT values
at any interval after treatment with SEP-1-L30, when compared with
those of the control group. Reticulocyte counts were higher at 4
days after the last injection (Day 9) for rats treated with 25 and
50 .mu.g/kg (statistically significant difference when compared
with those of the control group). No other differences in
hematologic parameters were observed at either Day 9 or Day 13 for
any of the animals treated with SEP-1-L30. [Data not shown]
Example 12
[0502] Pharmacokinetic Studies for SEP-1-L30
[0503] SEP-1-L30 was reformulated in Citrate Buffer (20 mM sodium
citrate+100 mM sodium chloride) plus 0.25% RSA, pH6.9, and was
administered intravenously to normal male rats as a single dose at
a dose level of 5 or 25 ug/kg. Blood samples were collected at 0,
1, 2, 4, 6, 12, 24, 48, 72, 96, 120, 144, and 168 hours after the
dosing. Plasma concentration of SEP-1-L30 was determined by ELISA
kit (R & D Systems, Human Erythropoietin Quantikine IVD
Immunoassay Kit #DEP00) according to manufacturers instructions. No
SEP-1-L30 was detectable at any of the time points at the doses
given.
Example 13
[0504] Efficacy Studies for SEP-1-B50
[0505] SEP-1-B50 was reformulated in sterile PBS (phosphate
buffered saline) plus 0.25% RSA and was administered intravenously
to normal male rats (5 rats per group) at doses of 0, 1, 5, 25, or
125 .mu.g/kg, tiw, on Days 1, 3, and 6. Blood samples were
collected 4, 9, and 14 days after the last injection (Days 10, 15,
and 20) and analyzed for hematologic parameters. The data is shown
in Table VIII below. A dose-related response in increased RBC, HGB,
HCT, and RET was observed at 4 days after the last injection (Day
10) for animals receiving 5, 25, and 125 .mu.g/kg SEP-1-B50
(statistically significant difference when compared with those of
the control group). RBC, HGB, and HCT for these animals remained
higher than control values on Day 15 and continued to be
significantly higher than control values through Day 20 (14 days
after the last injection) for animals treated at 125 .mu.g/kg. RET
production was reduced after Day 10, with significantly lower
counts on Days 15 and 20 for the 25 and 125 .mu.g/kg animals.
31TABLE VIII Hematology Findings Following Multiple Doses of
SEP-1-B50 Day 10 Day 15 Day 20 RBC Control 7.01 .+-. 0.301 6.86
.+-. 0.391 6.96 .+-. 0.178 1 .mu.g/kg 7.07 .+-. 0.482 6.85 .+-.
0.446 7.00 .+-. 0.315 5 .mu.g/kg 7.16 .+-. 0.402 7.27 .+-. 0.229
6.92 .+-. 0.238 25 .mu.g/kg 7.98 .+-. 0.484** 7.73 .+-. 0.448 7.20
.+-. 0.331 125 .mu.g/kg 8.71 .+-. 0.512** 8.89 .+-. 0.598** 8.25
.+-. 0.641** HGB Control 14.2 .+-. 0.49 14.0 .+-. 0.61 13.9 .+-.
0.36 1 .mu.g/kg 14.4 .+-. 1.18 14.2 .+-. 0.44 14.1 .+-. 0.30 5
.mu.g/kg 15.6 .+-. 0.43* 15.3 .+-. 0.33* 14.4 .+-. 0.30 25 .mu.g/kg
16.1 .+-. 0.49** 15.1 .+-. 0.57 13.8 .+-. 0.22 125 .mu.g/kg 16.9
.+-. 0.82** 16.4 .+-. 0.32** 15.0 .+-. 1.07* HCT Control 41.1 .+-.
1.39 40.5 .+-. 0.93 40.9 .+-. 1.33 1 .mu.g/kg 42.6 .+-. 1.82 40.6
.+-. 1.83 41.2 .+-. 1.13 5 .mu.g/kg 44.7 .+-. 1.69* 44.1 .+-. 1.27
41.5 .+-. 0.90 25 .mu.g/kg 46.6 .+-. 1.50** 43.6 .+-. 1.61 40.3
.+-. 1.05 125 .mu.g/kg 49.8 .+-. 2.13** 48.4 .+-. 3.95** 44.3 .+-.
3.39* RET Control 2.4 .+-. 1.14 2.5 .+-. 1.10 0.9 .+-. 0.13 1
.mu.g/kg 3.8 .+-. 1.14 2.3 .+-. 0.46 1.3 .+-. 0.41 5 .mu.g/kg 6.6
.+-. 3.60* 1.8 .+-. 0.48 0.8 .+-. 0.26 25 .mu.g/kg 6.2 .+-. 1.12*
1.1 .+-. 0.24* 0.2 .+-. 0.21** 125 .mu.g/kg 8.4 .+-. 1.60** 0.9
.+-. 0.50** 0.3 .+-. 0.36** *Significant difference from control, p
< 0.05 **Significant difference from control, p < 0.01
Example 14
[0506] Pharmacokinetic Studies for SEP-1-B50
[0507] SEP-1-B50 was reformulated in Citrate Buffer (20 mM sodium
citrate+100 mM sodium chloride) plus 0.25% RSA, pH6.9, and was
administered intravenously to normal male rats as a single dose at
a dose level of 5 or 25 ug/kg. Blood samples were collected at 0,
1, 2, 4, 6, 12, 24, 48, 72, 96, 120, 144, 168 hours after dosing.
Plasma concentration of SEP-1-B50 was determined by ELISA kit (R
& D Systems, Human Erythropoietin Quantikine IVD Immunoassay
Kit #DEP00) according to manufacturers instructions. The
elimination half-lives were determined to be 9.7 and 9.9 hours for
5 and 25 ug/kg dose, respectively. The observed MRT (mean residence
time) was 13.9 and 14.4 hours for the 5 and 25 ug/kg dose,
respectively. A representative pharmacokinetic profile for
SEP-1-B50 is shown in FIG. 27.
Example 15
[0508] Efficacy Studies for SEP-1-B51
[0509] SEP-1-B51 was administered intravenously to normal male rats
in two experiments.
[0510] Experiment 1: SEP-1-B51 was reformulated in Citrate Buffer
(20 mM sodium citrate+100 mM sodium chloride) plus 0.25% RSA and
was administered intravenously to male rats (5 rats per group) at
doses of 0, 1, 5, or 25 .mu.g/kg, tiw, on Days 1, 3, and 6, and
blood samples collected at 4, 9, and 14 days after the last
injection (Days 10, 15, and 20) for analysis of hematologic
parameters. The data is shown in Table IX below. Four days after
the third and last intravenous injection with SEP-1-B51 (Day 10), a
statistically significant increase in RBC, HGB, HCT, and absolute
reticulocyte counts (ART) was observed for animals receiving 25
.mu.g/kg SEP-1-B51, when compared with the values for the control
group. The RBC value for these animals remained higher than the
control value on Day 15 and was comparable to the control value on
Day 20. Reticulocyte production was reduced after Day 10, with
significantly lower counts observed for the 5 and 25 .mu.g/kg
animals on Day 15.
32TABLE IX Hematology Findings Following Multiple Doses of
SEP-1-B51 Day 10 Day 15 Day 20 RBC Control 5.66 .+-. 0.497 5.62
.+-. 0.385 5.90 .+-. 0.286 1 ug/kg 5.32 .+-. 0.275 5.23 .+-. 0.470
5.77 .+-. 0.200 5 ug/kg 6.22 .+-. 0.377 6.18 .+-. 0.298 6.39 .+-.
0.369 25 ug/kg 6.70 .+-. 0.257** 6.25 .+-. 0.348* 5.94 .+-. 0.294
HGB Control 14.3 .+-. 1.10 14.2 .+-. 0.88 14.8 .+-. 0.72 1 ug/kg
13.3 .+-. 0.41 13.2 .+-. 0.93 14.3 .+-. 0.37 5 ug/kg 15.1 .+-. 0.53
14.8 .+-. 0.30 15.4 .+-. 0.25 25 ug/kg 16.5 .+-. 0.52** 15.0 .+-.
0.91 14.3 .+-. 0.64 HCT Control 36.8 .+-. 3.08 36.2 .+-. 2.59 37.9
.+-. 1.93 1 ug/kg 34.5 .+-. 1.21 33.4 .+-. 2.56 37.1 .+-. 0.82 5
ug/kg 39.9 .+-. 1.51 38.6 .+-. 0.91 40.2 .+-. 1.36 25 ug/kg 43.8
.+-. 1.83** 39.3 .+-. 2.77 37.2 .+-. 1.60 ART Control 0.29 .+-.
0.089 0.28 .+-. 0.086 0.21 .+-. 0.034 1 ug/kg 0.34 .+-. 0.50 0.23
.+-. 0.055 0.19 .+-. 0.075 5 ug/kg 0.28 .+-. 0.059 0.11 .+-.
0.021** 0.20 .+-. 0.081 25 ug/kg 0.48 .+-. 0.121* 0.06 .+-. 0.054**
1.45 .+-. 3.100 **significant difference from control p < 0.01
*significant difference from control p < 0.05
[0511] Experiment 2. SEP-1-B51 was reformulated in Citrate Buffer
(20 mM sodium citrate+100 mM sodium chloride) plus 0.25% human
serum albumin and was administered intravenously to male rats (5
rats per group) at doses of 0, 1, 5, or 25 .mu.g/kg, tiw, on Days
1, 3, and 5, and blood samples collected at 2, 4, and 6 days after
the last injection (Days 7, 9, and 11) for analysis of hematologic
parameters. In addition, blood samples for measurement of
hematocrit only [as packed cell volume (PCV)] were collected daily
on the remaining study days (Days 2, 3, 4, 5, 6, 8, 10, 12, and
13). The data is shown in Table X below. HCT (PCV or calculated)
values were significantly increased for animals receiving 5 and 25
.mu.g/kg beginning 2 days after the first injection (Day 3) and
continuing through 6 days after the third and last injection (Day
11). RBC and HGB were also significantly increased on the days that
they were measured (2, 4, and 6 days after the last dose; Days 7,
9, and 11) for animals receiving 5 and 25 .mu.g/kg. ART was
significantly increased only for the 25 .mu.g/kg group at 2 and 4
days after the last dose (Days 7 and 9).
33TABLE X Hematology Findings Following Multiple
Doses.sup..gradient.of SEP-1-B51 Day 7 Day 9 Day 11 RBC Control
5.61 .+-. 0.679 5.23 .+-. 0.467 5.34 .+-. 0.405 1 ug/kg 6.08 .+-.
0.141 5.70 .+-. 0.300 5.67 .+-. 0.547 5 ug/kg 6.29 .+-. 0.459 6.07
.+-. 0.308** 6.11 .+-. 0.248* 25 ug/kg 6.61 .+-. 0.295** 6.24 .+-.
0.268** 6.38 .+-. 0.173** HGB Control 13.7 .+-. 1.07 12.9 .+-. 0.79
13.3 .+-. 0.93 1 ug/kg 14.6 .+-. 0.27 13.9 .+-. 0.19 13.9 .+-. 0.90
5 ug/kg 15.8 .+-. 0.50** 14.9 .+-. 0.53** 14.8 .+-. 0.18* 25 ug/kg
16.7 .+-. 0.86** 15.1 .+-. 0.66** 14.6 .+-. 0.69* HCT Control 35.6
.+-. 3.61 33.2 .+-. 2.39 34.5 .+-. 3.19 1 ug/kg 38.9 .+-. 0.82 35.9
.+-. 0.94 35.9 .+-. 2.43 5 ug/kg 41.3 .+-. 1.92** 39.1 .+-. 1.01*
39.2 .+-. 0.78* 25 ug/kg 44.4 .+-. 2.08** 39.7 .+-. 2.23** 39.6
.+-. 2.32** ART Control 0.50 .+-. 0.158 0.36 .+-. 0.093 0.34 .+-.
0.079 1 ug/kg 0.30 .+-. 0.044* 0.30 .+-. 0.026 0.25 .+-. 0.054 5
ug/kg 0.45 .+-. 0.125 0.36 .+-. 0.056 0.25 .+-. 0.045 25 ug/kg 0.78
.+-. 0.117** 0.71 .+-. 0.152** 0.32 .+-. 0.099 *Significant
difference from control, p < 0.05 **Significant difference from
control, p < 0.01 .sup..gradient.Note: Day 7 (2 days post 3rd
dose); Day 9 (4 days post 3rd dose); Day 11 (6 days post 3rd
dose)
[0512] Additional groups of rats were treated with a single
intravenous dose of SEP-1-B51 at 25 .mu.g/kg, and blood samples
were collected at 48 hr after injection for analysis of hematologic
parameters. Blood samples for measurement of hematocrit only (as
packed cell volume) were collected at 8, 24, and 72 hr and at 7
days after injection (Days 1, 2, 4, and 8). HCT, HGB, and ART
values for the rats treated intravenously with SEP-1-B51 were
higher than those of the control animals (statistically
significantly) on Day 3 (2 days after injection), and HCT was also
significantly increased in these animals on Day 4.
Example 16
[0513] Efficacy Studies in Polycythemic and Hypoxic Model for
SEP-1-B51
[0514] Groups of 10 normal mice each (vehicle control, recombinant
glycosylated human erythropoietin produced in CHO-cells ("rhEPO")
at 4 dose levels, and SEP-1-B51 at 4 dose levels: 0.32, 1, 5, 25
ug/kg/dose assuming 100 mU/ng) were exposed 18 hr per day to
atmospheric air maintained at 506.5 mb in a simulated high altitude
chamber for 20 days. The rhEPO or SEP-1-B51, formulated in Citrate
Buffer plus 0.25% human serum albumin, was injected intravenously
(IV) in a 200 .mu.L-volume on the 4.sup.th post-hypoxic day. Two
days later, each mouse was injected intraperitoneally (i.p.) with
0.2 .mu.Ci of .sup.59Fe. RBC-radioiron uptake was estimated 3 days
later by measuring the amount of radioactivity present in 500 .mu.L
of blood taken by cardiac puncture.
[0515] The 72-hour RBC-.sup.59Fe uptake (% of dose) for SEP-1-B51
and rhEPO showed a clear dose related response in increased
.sup.59Fe uptake with increasing dose up to 5 .mu.g/kg, above which
the response plateaued (25 .mu.g/kg). The three lowest dose levels
were therefore used to calculate linear regression. The linear
regression analyses of SEP-1-B51 and rhEPO are presented in FIG.
28. The responses of SEP-1-B51 and rhEPO were essentially the same.
The "potency ratio" of SEP-1-B51 to rhEPO was 1.0035 (95%
confidence interval of 0.6947 to 1.4498) and the potency of
SEP-1-B51 determined in this assay was 100 mU/ng (95% confidence
interval of 69-145).
[0516] SEP-1-B51 exhibits in vivo activity similar to that of rhEPO
and acts in dose-dependent manner, including a response plateau at
higher doses due to induction of a negative feedback mechanism
typical for rhEPO in this model. This illustrates that the polymer
structures attached at precise user-defined glycosylation sites in
the SEP-1-B51 molecule mimic the in vivo activity contributed by
the sugar chains of rhEPO.
Example 17
[0517] Pharmacokinetic Studies for SEP-1-B51
[0518] Groups of normal male rats were dosed intravenously with a
singe dose of 5 ug/kg SEP-1-B51, formulated in Citrate Buffer plus
0.25% human serum albumin, or rhEPO (equivalent to 500 U/kg) and
bled at 5, 30, 60 minutes, 2, 4, 8, and 24 hours, and 2, 3, 4, 5,
6, and 7 days following the dose. The SEP-1-B51 or rhEPO
concentrations in the plasma samples were determined by ELISA
assays using a R&D Systems anti-EPO ELISA kit (R & D
Systems, Human Erythropoietin Quantikine IVD Immunoassay Kit
#DEP00) according to manufacturers instructions.
[0519] A least squares analysis of the logarithms of the
concentrations was performed, yielding the pharmacokinetic
parameters listed in Table XI below. The change in plasma
concentration of SEP-1-B51 with time in male rats receiving a
single intravenous dose of 5 .mu.g/kg could be described by a
mono-exponential pharmacokinetic disposition function with
half-life of 10.5.+-.0.5 hours. The volume of distribution of the
central compartment (Vc) for SEP-1-B51 was 32.5.+-.2.0 mL/kg. The
clearance (CL) was 2.15 mL/hr/kg, with mean residence time (MRT) of
15.1.+-.0.7 hours. By comparison, the change in plasma
concentration of rhEPO in male rats receiving an intravenous dose
of 5 .mu.g/kg was best described using a bi-exponential
pharmacokinetic disposition function, with .alpha. half-life of
1.24.+-.0.22 hours and .beta. half-life of 5.51.+-.0.40 hours. The
Vc for rhEPO was 57.0.+-.3.2 mL/kg. The CL was 16.0.+-.0.5
mL/hr/kg, about 8 fold greater than that of SEP-1-B51. The mean
residence time (MRT) of rhEPO was 5.73.+-.0.17 hours, about 3 fold
shorter than that of SEP-1-B51.
34TABLE XI Pharmacokinetic Parameters for SEP-1-B51 Compared to
rhEPO AUC Cmax Vc CL MRT T1/2.alpha. T1/2.beta. Curve (ng/mL-hr)
(ng/mL) (mL/kg) (mL/hr/kg) (Hours) (Hours) (Hours) SEP-1-B51 2326
.+-. 121 154 .+-. 9 32.5 .+-. 2.0 2.15 .+-. 0.11 15.1 .+-. 0.7 10.5
.+-. 0.5 N/A rhEPO 312 .+-. 9 87.9 .+-. 5.0 57.0 .+-. 3.2 16.0 .+-.
0.5 5.73 .+-. 0.17 1.24 .+-. 0.22 5.51 .+-. 0.40
[0520] The graphic presentation of SEP-1-B51 and rhEPO plasma
clearance is in FIG. 29. These data illustrate a significant
increase in circulating half-life for SEP-1-B51 over the
glycosylated recombinant human EPO, and that this increase is due
to the polymer structures attached at precise user-defined sites in
the molecule.
[0521] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
Sequence CWU 1
1
3 1 166 PRT Homo sapiens 1 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg
Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Lys
Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Lys
Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys
Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55 60 Gln Gly
Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80
Leu Val Lys Ser Ser Gln Pro Trp Cys Pro Leu Gln Leu His Val Asp 85
90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala
Leu 100 105 110 Gly Ala Gln Lys Cys Ala Ile Ser Pro Pro Asp Ala Ala
Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg
Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu
Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg
165 2 166 PRT Homo sapiens 2 Ala Pro Pro Arg Leu Ile Cys Asp Ser
Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu
Lys Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu
Lys Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp
Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55 60 Gln
Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70
75 80 Leu Val Lys Ser Ser Gln Pro Trp Cys Pro Leu Gln Leu His Val
Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu
Arg Ala Leu 100 105 110 Gly Ala Gln Lys Cys Ala Ile Ser Pro Pro Asp
Ala Ala Lys Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr
Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly
Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly
Asp Arg 165 3 166 PRT Homo sapiens 3 Ala Pro Pro Arg Leu Ile Cys
Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu
Ala Glu Cys Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu
Asn Glu Cys Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr
Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55
60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
65 70 75 80 Leu Ala Cys Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His
Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu
Leu Arg Ala Leu 100 105 110 Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro
Asp Ala Ala Cys Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp
Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg
Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr
Gly Asp Arg 165
* * * * *