U.S. patent application number 11/004461 was filed with the patent office on 2005-05-05 for g-csf conjugates.
This patent application is currently assigned to Maxygen Holdings Ltd.. Invention is credited to Andersen, Kim Vilbour, Hansen, Christian Karsten, Mikkelsen, Jan Moller, Nissen, Torben Lauesgaard, Schambye, Hans Thalsgaard.
Application Number | 20050095684 11/004461 |
Document ID | / |
Family ID | 34557952 |
Filed Date | 2005-05-05 |
United States Patent
Application |
20050095684 |
Kind Code |
A1 |
Nissen, Torben Lauesgaard ;
et al. |
May 5, 2005 |
G-CSF conjugates
Abstract
Polypeptide conjugates with G-CSF activity comprising a
polypeptide having at least one introduced lysine residue and at
least one removed lysine residue compared to the sequence of human
G-CSF, and which are conjugated to 2-6 polyethylene glycol
moieties. The conjugates have a low in vitro bioactivity, a long in
vivo half-life, a reduced receptor-mediated clearance, and provide
a more rapid stimulation of production of white blood cells and
neutrophils than non-conjugated recombinant human G-CSF.
Inventors: |
Nissen, Torben Lauesgaard;
(San Francisco, CA) ; Andersen, Kim Vilbour;
(Broenshoej, DK) ; Hansen, Christian Karsten;
(Vedbaek, DK) ; Mikkelsen, Jan Moller; (Gentofte,
DK) ; Schambye, Hans Thalsgaard; (Frederiksberg,
DK) |
Correspondence
Address: |
MAXYGEN, INC.
INTELLECTUAL PROPERTY DEPARTMENT
515 GALVESTON DRIVE
RED WOOD CITY
CA
94063
US
|
Assignee: |
Maxygen Holdings Ltd.
|
Family ID: |
34557952 |
Appl. No.: |
11/004461 |
Filed: |
December 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11004461 |
Dec 3, 2004 |
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10192294 |
Jul 10, 2002 |
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6831158 |
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10192294 |
Jul 10, 2002 |
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09904196 |
Jul 11, 2001 |
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6555660 |
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09904196 |
Jul 11, 2001 |
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09760008 |
Jan 10, 2001 |
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6646110 |
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60176376 |
Jan 14, 2000 |
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60189506 |
Mar 15, 2000 |
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60215644 |
Jun 30, 2000 |
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Current U.S.
Class: |
435/69.5 ;
435/320.1; 435/325; 530/351; 536/23.5 |
Current CPC
Class: |
C07K 14/535 20130101;
A61K 9/0019 20130101; A61K 38/00 20130101; A61K 47/42 20130101;
C07K 14/53 20130101 |
Class at
Publication: |
435/069.5 ;
530/351; 536/023.5; 435/320.1; 435/325 |
International
Class: |
C12P 021/02; C07H
021/04; C07K 014/535 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 22, 2002 |
DK |
PA 2002 00447 |
May 8, 2002 |
DK |
PA 2002 00708 |
Jan 10, 2000 |
DK |
PA 2000 00024 |
Mar 2, 2000 |
DK |
PA 2000 00341 |
Jun 16, 2000 |
DK |
PA 2000 00943 |
Claims
We claim:
1. A polypeptide conjugate exhibiting G-CSF activity, comprising a
polypeptide comprising at least one substitution selected from the
group consisting of K16R/Q, K34R/Q and K40R/Q, and at least one
substitution selected from the group consisting of T105K and S159K,
relative to the amino acid sequence of hG-CSF shown in SEQ. ID NO:1
or in a corresponding position relative to an amino acid sequence
having at least 80% sequence identity with SEQ ID NO:1, the
conjugate having 2-6 polyethylene glycol moieties with a molecular
weight of about 1000-10,000 Da attached to attachment groups of the
polypeptide.
2. The polypeptide conjugate of claim 1, comprising three, four or
five of said substitutions.
3. The polypeptide conjugate of claim 1, further comprising the
substitution H170K, H1170Q or H170R.
4. The polypeptide conjugate of claim 1, comprising substitutions
selected from the group consisting of: Q70K+S 159K, Q70K+H170K,
Q90K+S 159K, Q90K+H170K, T105K+S159K, T105K+H170K, Q120K+S159K,
Q120K+H170K, T133K+S159K, T133K+H170K, S159K+H170K,
Q70K+Q90K+S159K, Q70K+Q90K+H170K, Q70K+T105K+S159K,
Q70K+T105K+H170K, Q70K+Q120K+S159K, Q70K+Q120K+H170K,
Q70K+T133K+S159K, Q70K+T133K+H170K, Q70K+S159K+H170K,
Q90K+T105K+S159K, Q90K+T105K+H170K, Q90K+Q120K+S159K,
Q90K+Q120K+H170K, Q90K+T133K+S159K, Q90K+T133K+H170K,
Q90+S159K+H170K, T 05K+Q120K+S159K, T105K+Q120K+H170K,
T105K+T133K+S159K, T105K+T133K+H170K, T105K+S159K+H170K,
Q120K+T133K+S159K, Q120K+T133K+H170K, Q120K+S159K+H170K,
T133K+S159K+H170K, Q70K+Q90K+T105K+S159K, Q70K+Q90K+T105K+H170K,
Q70K+Q90K+Q120K+S159K, Q70K+Q90K+Q120K+H170K,
Q70K+Q90K+T133K+S159K, Q70K+Q90K+T133K+H170K,
Q70K+Q90K+S159K+H170K, Q70K+T105K+Q120K+S159K,
Q70K+T105K+Q120K+H170K, Q70K+T105K+T133K+S159K,
Q70K+T105K+T133K+H170K, Q70K+T105K+S159K+H170K,
Q70K+Q120K+T133K+S159K, Q70K+Q120K+T133K+H170K,
Q70K+T133K+S159K+H170K, Q90K+T105K+Q120K+S159K,
Q90K+T105K+Q120K+H170K, Q90K+T105+T133K+S159K,
Q90K+T105+T133K+H170K, Q90K+T105+S159K+H170K,
Q90K+Q120K+T133K+S159K, Q90K+Q120K+T133K+H170K,
Q90K+Q120K+S159K+H170K, Q90K+T133K+S159K+H170K,
T105K+Q120K+T133K+S159K, T105K+Q120K+T133K+H170K,
T105K+Q120K+S159K+H170K, T105K+T133K+S159K+H170K and
Q120K+T133K+S159K+H170K.
5. The polypeptide conjugate of claim 1, comprising substitutions
selected from the group consisting of K16R+K23R, K16R+K34R,
K16R+K40R, K23R+K34R, K23R+K40R, K34R+K40R, K16R+K23R+K34R,
K16R+K23R+K40R, K23R+K34R+K40R, K16R+K34R+K40R and
K16R+K23R+K34R+K40R.
6. The polypeptide conjugate of claim 5, comprising the
substitutions K16R+K34R+K40R, and comprising a lysine residue in
position 23.
7. The polypeptide conjugate of claim 1, wherein said substitutions
are in a corresponding position relative to an amino acid sequence
having at least about 90% sequence identity with SEQ ID NO:1.
8. The polypeptide conjugate of claim 7, wherein said substitutions
are in a corresponding position relative to an amino acid sequence
having at least about 95% sequence identity with SEQ ID NO:1
9. The polypeptide conjugate of claim 1, wherein the polyethylene
glycol moieties are attached to at least one lysine residue and
optionally also to the N-terminal amino group.
10. The polypeptide conjugate of claim 1, comprising a polypeptide
comprising the substitutions K16R, K34R, K40R, T105K and S159K
relative to the amino acid sequence of hG-CSF shown in SEQ ID NO:1
and having 2-6 polyethylene glycol moieties with a molecular weight
of about 1000-10,000 Da attached to attachment groups of the
polypeptide.
11. The polypeptide conjugate of claim 1, which is glycosylated at
T133.
12. The polypeptide conjugate of claim 1, having 3-5, 4-6, 3-4, 4-5
or 5-6 attached PEG moieties.
13. The polypeptide conjugate of claim 12, having 3-6 polyethylene
glycol moieties with a molecular weight of about 5000-6000 Da
attached.
14. The polypeptide conjugate of claim 13, having 4 PEG moieties of
about 5 kDa attached.
15. The polypeptide conjugate of claim 13, having 5 PEG moieties of
about 5 kDa attached.
16. The polypeptide conjugate of claim 1, having an in vitro
bioactivity in the range of about 2-30% of the bioactivity of
non-conjugated hG-CSF as determined by the luciferase assay
described herein.
17. A method for preparing a G-CSF conjugate which provides a
reduced duration of neutropenia compared to hG-CSF, the method
comprising preparing a polypeptide comprising an amino acid
sequence that differs in at least one amino acid residue from the
amino acid sequence of hG-CSF shown in SEQ ID NO:1, and attaching a
non-polypeptide moiety to each of at least two attachment groups of
said polypeptide to result in a conjugate having an in vitro
bioactivity in the range of about 2-30% of the bioactivity of
non-conjugated hG-CSF as determined by the luciferase assay
described herein.
18. The method of claim 17, wherein a desired in vitro bioactivity
is obtained by alteration of at least one amino acid residue
selected from amino acid position 11-41 (helix A), 71-95 (helix B),
102-125 (helix C), and 145-170 (helix D), relative to the amino
acid sequence of SEQ ID NO:1, and by conjugation of at least one
non-polypeptide moiety to the at least one altered amino acid
residue.
19. A composition comprising the polypeptide conjugate of claim 1
and at least one pharmaceutically acceptable carrier or
excipient.
20. A method for treating a mammal suffering from an insufficient
neutrophil level, comprising administering to a mammal in need
thereof a therapeutically effective amount of the polypeptide
conjugate of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of co-pending
U.S. application Ser. No. 09/904,196 filed Jul. 11, 2001. Pursuant
to 35 U.S.C. .sctn.119(a)-(d), this application also claims
priority from and benefit of Danish Patent Application No. PA 2002
00447 filed Mar. 22, 2002, and Danish Patent Application No. PA
2002 00708 filed May 8, 2002. U.S. Ser. No. 09/904,196 is a
continuation-in-part of U.S. application Ser. No. 09/760,008 filed
Jan. 10, 2001. U.S. Ser. No. 09/760,008 claims priority to and
benefit of U.S. Provisional Application Ser. No. 60/176,376 filed
Jan. 14, 2000, U.S. Provisional Application Ser. No. 60/189,506
filed Mar. 15, 2000, U.S. Provisional Application Ser. No.
60/215,644 filed Jun. 30, 2000, Danish Patent Application No. PA
2000 00024 filed Jan. 10, 2000, Danish Patent Application No. PA
2000 00341 filed Mar. 2, 2000, and Danish Patent Application No. PA
2000 00943 filed Jun. 16, 2000. The disclosure of each application
listed above is incorporated herein in its entirety for all
purposes.
COPYRIGHT NOTIFICATION
[0002] Pursuant to 37 C.F.R. .sctn. 1.71 (e), Applicants note that
a portion of this disclosure contains material which is subject to
copyright protection. The copyright owner has no objection to the
facsimile reproduction by anyone of the patent document or the
patent disclosure, as it appears in the Patent and Trademark Office
patent file or records, but otherwise reserves all copyright rights
whatsoever.
FIELD OF THE INVENTION
[0003] The present invention relates to new polypeptides exhibiting
granulocyte colony-stimulating factor (G-CSF) activity, to
conjugates between a polypeptide exhibiting G-CSF activity and a
non-polypeptide moiety, to methods for preparing such polypeptides
or conjugates and the use of such polypeptides or conjugates in
therapy, in particular for the treatment of neutropenia or
leukopenia.
BACKGROUND OF THE INVENTION
[0004] The process by which white blood cells grow, divide and
differentiate in the bone marrow is called hematopoiesis (Dexter
and Spooncer, Ann. Rev. Cell. Biol., 3:423, 1987). Each of the
blood cell types arises from pluripotent stem cells. There are
generally three classes of blood cells produced in vivo: red blood
cells (erythrocytes), platelets and white blood cells (leukocytes),
the majority of the latter being involved in host immune defense.
Proliferation and differentiation of hematopoietic precursor cells
are regulated by a family of cytokines, including
colony-stimulating factors (CSF's) such as G-CSF and interleukins
(Arai et al., Ann. Rev. Biochem., 59:783-836, 1990). The principal
biological effect of G-CSF in vivo is to stimulate the growth and
development of certain white blood cells known as neutrophilic
granulocytes or neutrophils (Welte et al., PNAS-USA 82:1526-1530,
1985, Souza et al., Science, 232:61-65, 1986). When released into
the blood stream, neutrophilic granulocytes function to fight
bacterial and other infection.
[0005] The amino acid sequence of human G-CSF (hG-CSF) was reported
by Nagata et al. Nature 319:415-418, 1986. hG-CSF is a monomeric
protein that dimerizes the G-CSF receptor by formation of a 2:2
complex of 2 G-CSF molecules and 2 receptors (Horan et al. (1996),
Biochemistry 35(15): 4886-96). Aritomi et al. Nature 401:713-717,
1999 have described the X-ray structure of a complex between hG-CSF
and the BN-BC domains of the G-CSF receptor. They identify the
following hG-CSF residues as being part of the receptor binding
interfaces: G4, P5, A6, S7, S8, L9, P10, Q11, S12, L15, K16, E19,
Q20, L108, D109, D112, T115, T116, Q119, E122, E123, and L124.
Expression of rhG-CSF in Escherichia coli, Saccharomyces cerevisiae
and mammalian cells has been reported (Souza et al., Science
232:61-65, 1986, Nagata et al., Nature 319: 415-418, 1986, Robinson
and Wittrup, Biotechnol. Prog. 11:171-177, 1985).
[0006] Leukopenia (a reduced level of white blood cells) and
neutropenia (a reduced level of neutrophils) are disorders that
result in an increased susceptibility to various types of
infections. Neutropenia can be chronic, e.g. in patients infected
with HIV, or acute, e.g. in cancer patients undergoing chemotherapy
or radiation therapy. For patients with severe neutropenia, e.g. as
a result of chemotherapy, even relatively minor infections can be
serious and even life-threatening. Recombinant human G-CSF
(rhG-CSF) is generally used for treating various forms of
leukopenia/neutropenia. Thus, commercial preparations of rhG-CSF
are available under the names filgrastim (Gran.RTM. and
Neupogen.RTM.), lenograstim (Neutrogin.RTM. and Granocyte.RTM.) and
nartograstim (Neu-up.RTM.). Gran.RTM. and Neupogen.RTM. are
non-glycosylated and produced in recombinant E. coli cells.
Neutrogin.RTM. and Granocyte.RTM. are glycosylated and produced in
recombinant CHO cells and Neu-up.RTM. is non-glycosylated with five
amino acids substituted at the N-terminal region of intact rhG-CSF
produced in recombinant E. coli cells.
[0007] Various protein-engineered variants of hG-CSF have been
reported (e.g. U.S. Pat. No. 5,581,476, U.S. Pat. No. 5,214,132,
U.S. Pat. No. 5,362,853, U.S. Pat. No. 4,904,584 and Riedhaar-Olson
et al. Biochemistry 35: 9034-9041, 1996). Modification of hG-CSF
and other polypeptides so as to introduce at least one additional
carbohydrate chain as compared to the native polypeptide has been
suggested (U.S. Pat. No. 5,218,092). It is stated that the amino
acid sequence of the polypeptide may be modified by amino acid
substitution, amino acid deletion or amino acid insertion so as to
effect addition of an additional carbohydrate chain. In addition,
polymer modifications of native hG-CSF, including attachment of PEG
groups, have been reported (Satake-Ishikawa et al., Cell Structure
and Function 17:157-160, 1992, U.S. Pat. No. 5,824,778, U.S. Pat.
No. 5,824,784, WO 96/11953, WO 95/21629, WO 94/20069).
[0008] Bowen et al., Experimental Hematology 27 (1999), 425-432
disclose a study of the relationship between molecule mass and
duration of activity of PEG-conjugated G-CSF mutein. An apparent
inverse correlation was suggested between molecular weight of the
PEG moieties conjugated to the protein and in vitro activity,
whereas in vivo activities increased with increasing molecular
weight. It is speculated that a lower affinity of the conjugates
act to increase the half-life, because receptor-mediated
endocytosis is an important mechanism regulating levels of
hematopoietic growth factors.
[0009] The commercially available rhG-CSF has a short-term
pharmacological effect and must therefore be administered once a
day for the duration of the leukopenic state. A molecule with a
longer circulation half-life would decrease the number of
administrations necessary to alleviate the leukopenia and prevent
consequent infections. Another, more significant problem with
currently available rG-CSF products is that patients become
neutropenic after chemotherapy even after administration of G-CSF.
For these patients, it is important to be able to reduce the
duration and degree of the neutropenic state as much as possible in
order to minimize the risk of serious infections. A further problem
is the occurrence of dose-dependent bone pain. Since bone pain is
experienced by patients as a significant side effect of treatment
with rG-CSF, it would be desirable to provide a rG-CSF product that
does not cause bone pain, either by means of a product that
inherently does not have this effect or that is effective in a
sufficiently small dose that no bone pain is caused. Thus, there is
clearly a need for improved recombinant G-CSF-like molecules.
[0010] With respect to the half-life, one way to increase the
circulation half-life of a protein is to ensure that clearance of
the protein, in particular via renal clearance and
receptor-mediated clearance, is reduced. This may be achieved by
conjugating the protein to a chemical moiety which is capable of
increasing the apparent size, thereby reducing renal clearance and
increasing the in vivo half-life. Furthermore, attachment of a
chemical moiety to the protein may effectively block proteolytic
enzymes from physical contact with the protein, thus preventing
degradation by non-specific proteolysis. Polyethylene glycol (PEG)
is one such chemical moiety that has been used in the preparation
of therapeutic protein products. Recently, G-CSF molecule modified
with a single, N-terminally linked 20 kDa PEG group (Neulasta.TM.)
was approved for sale in the United States. This PEGylated G-CSF
molecule has been shown to have an increased half-life compared to
non-PEGylated G-CSF and thus may be administered less frequently
than current G-CSF products, but it does not reduce the duration of
neutropenia significantly compared to non-PEGylated G-CSF. Thus,
there is still substantial room for improvement of the known G-CSF
molecules.
[0011] A need therefore still exists for providing novel molecules
exhibiting G-CSF activity that are useful in the treatment of
leukopenia/neutropenia, and which have are improved in terms of
e.g. an increased half-life and in particular a reduction in the
duration of neutropenia. The present invention relates to such
molecules.
BRIEF DISCLOSURE OF THE INVENTION
[0012] The present invention relates to specific conjugates
comprising a polypeptide exhibiting G-CSF activity and a
non-polypeptide moiety, methods for their preparation and their use
in medical treatment and in the preparation of pharmaceuticals.
Accordingly, in a first aspect the invention relates to various
specific conjugates comprising a polypeptide exhibiting G-CSF
activity and having an amino acid sequence that differs from the
known amino acid sequence of human G-CSF as shown in SEQ ID NO:1 in
at least one specified altered amino acid residue comprising an
attachment group for a non-polypeptide moiety, and having at least
one non-polypeptide moiety attached to an attachment group of the
polypeptide. These conjugates have a substantially reduced in vitro
bioactivity compared to that of non-conjugated hG-CSF, which
surprisingly has been shown to result in a more rapid neutrophil
recovery. The conjugate of the present invention thus has one or
more improved properties as compared to commercially available
rhG-CSF, including increased stimulation of neutrophils, increased
functional in vivo half-life, increased serum half-life, reduced
side effects, reduced immunogenicity and/or increased
bioavailability. Consequently, medical treatment with a conjugate
of the invention offers a number of advantages over the currently
available G-CSF compounds.
[0013] In a further aspect the invention relates to polypeptides
exhibiting G-CSF activity and which form part of a conjugate of the
invention. The polypeptides of the invention are contemplated to be
useful as such for therapeutic, diagnostic or other purposes, but
find particular interest as intermediate products for the
preparation of a conjugate of the invention.
[0014] In a further aspect the invention relates to a polypeptide
conjugate comprising a polypeptide exhibiting G-CSF activity, which
comprises an amino acid sequence that differs from the amino acid
sequence of hG-CSF (with the amino acid sequence shown in SEQ ID
NO:1) in at least one amino acid residue selected from an
introduced or removed amino acid residue comprising an attachment
group for a non-polypeptide moiety, and a sufficient number or type
of non-polypeptide moieties to provide the conjugate with an
increased half-life and/or a more rapid neutrophil recovery
compared to known recombinant G-CSF products.
[0015] In a particular aspect the invention relates to a
polypeptide conjugate exhibiting G-CSF activity, comprising a
polypeptide having the substitutions K16R, K34R, K40R, T105K, and
S159K, and optionally a substitution in position H170, e.g. to R, K
or Q, relative to the amino acid sequence of hG-CSF shown in SEQ ID
NO:1, or in a corresponding position relative to an amino acid
sequence having at least 80% sequence identity with SEQ ID NO:1,
and having 2-6, typically 3-6 polyethylene glycol moieties with a
molecular weight of about 1000-10,000 Da attached to one or more
attachment groups of the polypeptide. Where these substitutions are
relative to a sequence with at least about 80% sequence identity
with SEQ ID NO:1, the degree of sequence identity is typically at
least about 90% or 95%, such as at least about 96%, 97%, 98% or
99%.
[0016] In still further aspects the invention relates to methods
for preparing a conjugate of the invention, including nucleotide
sequences encoding a polypeptide of the invention, expression
vectors comprising such a nucleotide sequence, and host cells
comprising such a nucleotide sequence or expression vector.
[0017] In final aspects the invention relates to a composition
comprising a conjugate or polypeptide of the invention, a method
for preparing a pharmaceutical composition, use of a conjugate or
composition of the invention as a pharmaceutical, and a method of
treating a mammal with such composition. In particular, the
polypeptide, conjugate or composition of the invention may be used
to prevent infection in cancer patients undergoing certain types of
radiation therapy, chemotherapy, and bone marrow transplantations,
to mobilize progenitor cells for collection in peripheral blood
progenitor cell transplantations, for treatment of severe chronic
or relative leukopenia, irrespective of cause, and to support
treatment of patients with acute myeloid leukemia. Additionally,
the polypeptide, conjugate or composition of the invention may be
used for treatment of AIDS or other immunodeficiency diseases as
well as bacterial infections.
DETAILED DISCLOSURE OF THE INVENTION
[0018] Definitions
[0019] In the context of the present application and invention the
following definitions apply:
[0020] The term "conjugate" is intended to indicate a heterogeneous
molecule formed by the covalent attachment of one or more
polypeptides, typically a single polypeptide, to one or more
non-polypeptide moieties such as polymer molecules, lipophilic
compounds, carbohydrate moieties or organic derivatizing agents.
The term covalent attachment means that the polypeptide and the
non-polypeptide moiety are either directly covalently joined to one
another, or else are indirectly covalently joined to one another
through an intervening moiety or moieties, such as a bridge,
spacer, or linkage moiety or moieties. Preferably, the conjugate is
soluble at relevant concentrations and conditions, i.e. soluble in
physiological fluids such as blood. The term "non-conjugated
polypeptide" may be used about the polypeptide part of the
conjugate.
[0021] The term "polypeptide" may be used interchangeably herein
with the term "protein".
[0022] The "polymer molecule" is a molecule formed by covalent
linkage of two or more monomers, wherein none of the monomers is an
amino acid residue, except where the polymer is human albumin or
another abundant plasma protein. The term "polymer" may be used
interchangeably with the term "polymer molecule". The term is
intended to cover carbohydrate molecules, although, normally, the
term is not intended to cover the type of carbohydrate molecule
which is attached to the polypeptide by in vivo N- or
O-glycosylation (as further described below), since such molecule
is referred to herein as "an oligosaccharide moiety". Except where
the number of polymer molecule(s) is expressly indicated every
reference to "a polymer", "a polymer molecule", "the polymer" or
"the polymer molecule" contained in a polypeptide of the invention
or otherwise used in the present invention shall be a reference to
one or more polymer molecule(s).
[0023] The term "attachment group" is intended to indicate an amino
acid residue group of the polypeptide capable of coupling to the
relevant non-polypeptide moiety. For instance, for polymer
conjugation, in particular to PEG, a frequently used attachment
group is the .epsilon.-amino group of lysine or the N-terminal
amino group. Other polymer attachment groups include a free
carboxylic acid group (e.g. that of the C-terminal amino acid
residue or of an aspartic acid or glutamic acid residue), suitably
activated carbonyl groups, oxidized carbohydrate moieties and
mercapto groups. Useful attachment groups and their matching
non-peptide moieties are apparent from the table below.
1 Conjugation Attachment Examples of non- method/- group Amino acid
peptide moiety Activated PEG Reference --NH.sub.2 N-terminal,
Polymer, e.g. PEG, mPEG-SPA Shearwater Corp. Lys, His, Arg with
amide or imine Tresylated Delgado et al., critical group mPEG
reviews in Therapeutic Drug Carrier Systems 9(3, 4): 249-304 (1992)
--COOH C-term, Asp, Polymer, e.g. PEG, mPEG-Hz Shearwater Corp. Glu
with ester or amide group Oligosaccharide In vitro coupling moiety
--SH Cys Polymer, e.g. PEG, PEG- Shearwater Corp. with disulfide,
vinylsulphone Delgado et al., critical maleimide or vinyl
PEG-maleimide reviews in Therapeutic sulfone group Drug Carrier
Systems 9(3, 4): 249-304 (1992) Oligosaccharide In vitro coupling
moiety --OH Ser, Thr, --OH, Oligosaccharide In vivo O-linked Lys
moiety glycosylation PEG with ester, ether, carbamate, carbonate
--CONH.sub.2 Asn as part of Oligosaccharide In vivo N- an N- moiety
glycosylation glycosylation Polymer, e.g. PEG site Aromatic Phe,
Tyr, Trp Oligosaccharide In vitro coupling residue moiety
--CONH.sub.2 Gln Oligosaccharide In vitro coupling Yan and Wold,
moiety Biochemistry, 1984, Jul 31; 23(16): 3759-65 Aldehyde
Oxidized Polymer, e.g. PEG, PEGylation Andresz et al., 1978, Ketone
oligosaccharide PEG-hydrazide Makromol. Chem. 179: 301, WO
92/16555, WO 00/23114 Guanidino Arg Oligosaccharide In vitro
coupling Lundblad and Noyes, moiety Chemical Reagents for Protein
Modification, CRC Press Inc., Florida, USA Imidazole His
Oligosaccharide In vitro coupling As for guanidine ring moiety
[0024] For in vivo N-glycosylation, the term "attachment group" is
used in an unconventional way to indicate the amino acid residues
constituting an N-glycosylation site (with the sequence
N-X'-S/T/C-X", wherein X' is any amino acid residue except proline,
X" any amino acid residue which may or may not be identical to X'
and which preferably is different from proline, N is asparagine,
and S/T/C is either serine, threonine or cysteine, preferably
serine or threonine, and most preferably threonine). Although the
asparagine residue of the N-glycosylation site is where the
oligosaccharide moiety is attached during glycosylation, such
attachment cannot be achieved unless the other amino acid residues
of the N-glycosylation site are present. Accordingly, when the
non-peptide moiety is an oligosaccharide moiety and the conjugation
is to be achieved by N-glycosylation, the term "amino acid residue
comprising an attachment group for the non-peptide moiety" as used
in connection with alterations of the amino acid sequence of the
polypeptide of interest is to be understood as meaning that one or
more amino acid residues constituting an N-glycosylation site are
to be altered in such a manner that either a functional
N-glycosylation site is introduced into the amino acid sequence or
removed from said sequence.
[0025] In the present application, amino acid names and atom names
(e.g. CA, CB, NZ, N, O, C, etc.) are used as defined by the Protein
DataBank (PDB) (www.pdb.org), which is based on the IUPAC
nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and
Peptides (residue names, atom names etc.), Eur. J. Biochem., 138,
9-37 (1984) together with their corrections in Eur. J. Biochem.,
152, 1 (1985). The term "amino acid residue" is intended to
indicate any naturally or non-naturally occurring amino acid
residue, in particular an amino acid residue contained in the group
consisting of the 20 naturally occurring amino acids, i.e. alanine
(Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic
acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G),
histidine (His or H), isoleucine (Ile or I), lysine (Lys or K),
leucine (Leu or L), methionine (Met or M), asparagine (Asn or N),
proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R),
serine (Ser or S), threonine (Thr or T), valine (Val or V),
tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.
[0026] The terminology used for identifying amino acid
positions/substitutions is illustrated as follows: F13 indicates
position number 13 occupied by a phenylalanine residue in the
reference amino acid sequence. F13K indicates that the
phenylalanine residue of position 13 has been substituted with a
lysine residue. Unless otherwise indicated, the numbering of amino
acid residues made herein is made relative to the amino acid
sequence of hG-CSF shown in SEQ ID NO:1. Alternative substitutions
are indicated with a "/", e.g. Q67D/E means an amino acid sequence
in which glutamine in position 67 is substituted with either
aspartic acid or glutamic acid. Multiple substitutions are
indicated with a "+", e.g. S53N+G55S/T means an amino acid sequence
which comprises a substitution of the serine residue in position 53
with an asparagine residue and a substitution of the glycine
residue in position 55 with a serine or a threonine residue.
[0027] The term "nucleotide sequence" is intended to indicate a
consecutive stretch of two or more nucleotide molecules. The
nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic or
synthetic origin, or any combination thereof.
[0028] The term "polymerase chain reaction" or "PCR" refers to the
well-known method for amplification of a desired nucleotide
sequence in vitro using a thermostable DNA polymerase.
[0029] "Cell", "host cell", "cell line" and "cell culture" are used
interchangeably herein and all such terms should be understood to
include progeny resulting from growth or culturing of a cell.
"Transformation" and "transfection" are used interchangeably to
refer to the process of introducing DNA into a cell.
[0030] "Operably linked" refers to the covalent joining of two or
more nucleotide sequences, by means of enzymatic ligation or
otherwise, in a configuration relative to one another such that the
normal function of the sequences can be performed. For example, the
nucleotide sequence encoding a presequence or secretory leader is
operably linked to a nucleotide sequence for a polypeptide if it is
expressed as a preprotein that participates in the secretion of the
polypeptide: a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence; a
ribosome binding site is operably linked to a coding sequence if it
is positioned so as to facilitate translation. Generally, "operably
linked" means that the nucleotide sequences being linked are
contiguous and, in the case of a secretory leader, contiguous and
in reading phase. Linking is accomplished by ligation at convenient
restriction sites. If such sites do not exist, then synthetic
oligonucleotide adaptors or linkers are used, in conjunction with
standard recombinant DNA methods.
[0031] The term "introduce" refers to introduction of an amino acid
residue comprising an attachment group for a non-polypeptide
moiety, in particular by substitution of an existing amino acid
residue, or alternatively by insertion of an additional amino acid
residue. The term "remove" refers to removal of an amino acid
residue comprising an attachment group for a non-polypeptide
moiety, in particular by substitution of the amino acid residue to
be removed by another amino acid residue, or alternatively by
deletion (without substitution) of the amino acid residue to be
removed.
[0032] When substitutions are performed in relation to a parent
polypeptide, they are preferably "conservative substitutions", in
other words substitutions performed within groups of amino acids
with similar characteristics, e.g. small amino acids, acidic amino
acids, polar amino acids, basic amino acids, hydrophobic amino
acids and aromatic amino acids.
[0033] Preferred substitutions in the present invention may in
particular be chosen from among the conservative substitution
groups listed in the table below.
[0034] Conservative Substitution Groups:
2 1 Alanine (A) Glycine (G) Serine (S) Threonine (T) 2 Aspartic
acid (D) Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4
Arginine (R) Histidine (H) Lysine (K) 5 Isoleucine (I) Leucine (L)
Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y)
Tryptophan (W)
[0035] The term "immunogenicity" as used in connection with a given
substance is intended to indicate the ability of the substance to
induce a response from the immune system. The immune response may
be a cell or antibody mediated response (see, e.g., Roitt:
Essential Immunology (8.sup.th Edition, Blackwell) for further
definition of immunogenicity). Normally, reduced antibody
reactivity will be an indication of reduced immunogenicity. The
reduced immunogenicity may be determined by use of any suitable
method known in the art, e.g. in vivo or in vitro.
[0036] The term "functional in vivo half-life" is used in its
normal meaning, i.e. the time at which 50% of the biological
activity of the polypeptide or conjugate is still present in the
body/target organ, or the time at which the activity of the
polypeptide or conjugate is 50% of the initial value. As an
alternative to determining functional in vivo half-life, "serum
half-life" may be determined, i.e. the time in which 50% of the
polypeptide or conjugate molecules circulate in the plasma or
bloodstream prior to being cleared. Alternative terms to serum
half-life include "plasma half-life", "circulating half-life",
"serum clearance", "plasma clearance" and "clearance half-life".
The polypeptide or conjugate is cleared by the action of one or
more of the reticuloendothelial systems (RES), kidney, spleen or
liver, by receptor-mediated degradation, or by specific or
non-specific proteolysis, in particular by the action of
receptor-mediated clearance and renal clearance. Normally,
clearance depends on size (relative to the cutoff for glomerular
filtration), charge, attached carbohydrate chains, and the presence
of cellular receptors for the protein. The functionality to be
retained is normally selected from proliferative or
receptor-binding activity. The functional in vivo half-life and the
serum half-life may be determined by any suitable method known in
the art as further discussed in the Materials and Methods section
below.
[0037] The term "increased" as used about the functional in vivo
half-life or serum half-life is used to indicate that the relevant
half-life of the conjugate or polypeptide is statistically
significantly increased relative to that of a reference molecule,
such as a non-conjugated hG-CSF (e.g. Neupogen.RTM.) as determined
under comparable conditions. For instance, the relevant half-life
may increased by at least about 25%, such as by at least about 50%,
e.g. by at least about 100%, 200%, 500% or 1000%.
[0038] The term "renal clearance" is used in its normal meaning to
indicate any clearance taking place by the kidneys, e.g. by
glomerular filtration, tubular excretion or tubular elimination.
Renal clearance depends on physical characteristics of the
conjugate, including size (diameter), symmetry, shape/rigidity and
charge. Reduced renal clearance may be established by any suitable
assay, e.g. an established in vivo assay. Typically, renal
clearance is determined by administering a labeled (e.g.
radioactive or fluorescent labeled) polypeptide conjugate to a
patient and measuring the label activity in urine collected from
the patient. Reduced renal clearance is determined relative to a
corresponding reference polypeptide, e.g. the corresponding
non-conjugated polypeptide, a non-conjugated corresponding
wild-type polypeptide or another conjugated polypeptide (such as a
conjugated polypeptide not according to the invention), under
comparable conditions. Preferably, the renal clearance rate of the
conjugate is reduced by at least 50%, preferably by at least 75%,
and most preferably by at least 90% compared to a relevant
reference polypeptide.
[0039] Generally, activation of the receptor is coupled to
receptor-mediated clearance (RMC) such that binding of a
polypeptide to its receptor without activation does not lead to
RMC, while activation of the receptor leads to RMC. The clearance
is due to internalization of the receptor-bound polypeptide with
subsequent lysosomal degradation. Reduced RMC may be achieved by
designing the conjugate so as to be able to bind and activate a
sufficient number of receptors to obtain optimal in vivo biological
response and avoid activation of more receptors than required for
obtaining such response. This may be reflected in reduced in vitro
bioactivity and/or increased off-rate. In a preferred embodiment,
the conjugates of the invention have a substantially reduced in
vitro bioactivity compared to that of non-conjugated hG-CSF.
[0040] Typically, reduced in vitro bioactivity reflects reduced
efficacy/efficiency and/or reduced potency and may be determined by
any suitable method for determining any of these properties. For
instance, in vitro bioactivity may be determined in a luciferase
based assay ("Primary assay 2"; see Materials and Methods). Another
method for determining the in vitro bioactivity is to determine the
binding affinity of a conjugate of the invention using the
cell-based assay described in the Materials and Methods section
("Secondary assay").
[0041] It has been found that a relatively low in vitro
bioactivity, compared to the activity of hG-CSF (SEQ ID NO:1), is
advantageous in terms of both a long plasma half-life and a high
degree of stimulation of neutrophils. Surprisingly, it has been
found that administration of G-CSF conjugates of the invention
having a low in vitro bioactivity results in a faster neutrophil
recovery, i.e. a faster recovery of the neutrophil count to a
normal level, than administration of hG-CSF. Since it is critical
to be able to reduce the duration of neutropenia as much as
possible in patients having a reduced neutrophil level due to e.g.
chemotherapy or radiation therapy, this is an important finding.
Thus, in a preferred embodiment, the in vitro bioactivity of a
conjugate of the invention is in the range of about 2-30%,
preferably about 3-25%, of the bioactivity of hG-CSF (where the
hG-CSF used as the reference polypeptide has SEQ ID NO:1,
optionally with an N-terminal methionine residue; the reference
hG-CSF may in particular be Neupogen.RTM., i.e. non-glycosylated
Met-hG-CSF) as determined by the luciferase assay described herein,
or, alternatively, using the cell-based receptor binding affinity
assay ("Secondary assay"). The in vitro bioactivity of the
conjugate is thus preferably reduced by at least 70%, such as by at
least 75%, e.g. by at least 80% or 85%, as compared to the in vitro
bioactivity of hG-CSF, determined under comparable conditions.
Expressed differently, the conjugate may have an in vitro
bioactivity that is as small as about 2%, typically at least about
3%, such as at least about 4% or 5%, of that of the wild-type
polypeptide. For instance, the in vitro bioactivity may be in the
range of about 4-20% of that of hG-CSF, determined under comparable
conditions. In cases where reduced in vitro bioactivity is desired
in order to reduce receptor-mediated clearance, it will be clear
that sufficient bioactivity to obtain the desired receptor
activation must nevertheless be maintained, which is why the
bioactivity should be at least about 2% of that of hG-CSF and
preferably slightly higher as explained above.
[0042] It has been found that amino acid alterations, in particular
substitutions, in the helix regions of G-CSF, i.e. in an amino acid
residue selected from amino acid position 11-41 (helix A), 71-95
(helix B), 102-125 (helix C), and 145-170 (helix D) (compared to
SEQ ID NO:1), result in a reduced receptor-mediated clearance and
thus an increased in vivo half-life when the resulting polypeptides
are conjugated to polyethylene glycol. In addition to a longer
half-life, it has surprisingly been found that administration of
such polypeptide conjugates is able to stimulate production of
white blood cells and neutrophils to the same degree as, or even
better than, administration of the commercially available G-CSF
products Neupogen.RTM. and Neulasta.TM.. G-CSF conjugates having a
reduced in vitro bioactivity may thus be prepared by altering,
typically by substitution, one or more amino acid residues in a
helix region of G-CSF, and by conjugating the resulting polypeptide
to one or more non-polypeptide moieties such as polyethylene
glycol.
[0043] Preferably, the off-rate between the polypeptide conjugate
and its receptor is increased by a magnitude resulting in the
polypeptide conjugate being released from its receptor before any
substantial internalization of the receptor-ligand complex has
taken place. The receptor-polypeptide binding affinity may be
determined as described in the Materials and Methods section
herein. The off-rate may be determined using the Biacore.RTM.
technology as described in the Materials and Methods section. The
in vitro RMC may be determined by labeling (e.g. radioactive or
fluorescent labeling) the polypeptide conjugate, stimulating cells
comprising the receptor for the polypeptide, washing the cells, and
measuring label activity. Alternatively, the conjugate may be
exposed to cells expressing the relevant receptor. After an
appropriate incubation time the supernatant is removed and
transferred to a well containing similar cells. The biological
response of these cells to the supernatant is determined relative
to a non-conjugated polypeptide or another reference polypeptide,
and this is a measure of the extent of the reduced RMC.
[0044] Normally, reduced in vitro bioactivity of the conjugate is
obtained as a consequence of its modification by a non-polypeptide
moiety. However, in order to further reduce in vitro bioactivity or
for other reasons it may be of interest to modify the polypeptide
part of the conjugate further. For instance, in one embodiment at
least one amino acid residue located at or near a receptor binding
site of the polypeptide may be substituted with another amino acid
residue as compared to the corresponding wild-type polypeptide so
as to obtain reduced in vitro bioactivity. The amino acid residue
to be introduced by substitution may be any amino acid residue
capable of reducing in vitro bioactivity of the conjugate.
Conveniently, the introduced amino acid residue comprises an
attachment group for the non-polypeptide moiety as defined herein.
In particular, when the non-polypeptide moiety is a polymer
molecule such as PEG molecule, the amino acid residue to be
introduced may be a lysine residue.
[0045] The term "exhibiting G-CSF activity" is intended to indicate
that the polypeptide or conjugate has one or more of the functions
of native G-CSF, in particular hG-CSF with the amino acid sequence
shown in SEQ ID NO:1, including the capability to bind to a G-CSF
receptor (Fukunaga et al., J. Bio. Chem, 265:14008, 1990). The
G-CSF activity is conveniently assayed using the primary assay
described in the Materials and Methods section hereinafter. The
polypeptide "exhibiting" G-CSF activity is considered to have such
activity when it displays a measurable function, e.g. a measurable
proliferative activity or a receptor binding activity (e.g. as
determined by the primary assay described in the Materials and
Methods section). The polypeptide exhibiting G-CSF activity may
also be termed "G-CSF molecule" herein for the sake of simplicity,
even though such polypeptides are in fact variants of G-CSF.
[0046] The term "parent G-CSF" or "parent polypeptide" is intended
to indicate the molecule to be modified in accordance with the
present invention. The parent G-CSF is normally hG-CSF or a variant
thereof. A "variant" is a polypeptide which differs in one or more
amino acid residues from a parent polypeptide, normally in 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.
Examples of rhG-CSF include filgrastim (Gran.RTM. and
Neupogen.RTM.), lenograstim (Neutrogin.RTM. and Granocyte.RTM.) and
nartograstim (Neu-up.RTM.).
[0047] Conjugate of the Invention
[0048] As stated above, in a first aspect the invention relates to
a conjugate comprising a polypeptide exhibiting G-CSF activity,
which comprises an amino acid sequence that differs from the amino
acid sequence of SEQ ID NO:1 in at least one amino acid residue
selected from specified introduced or removed amino acid residues
comprising an attachment group for a non-polypeptide moiety, and at
least one non-polypeptide moiety attached to an attachment group of
the polypeptide. The amino acid residues to be introduced and/or
removed are described in further detail in the following sections.
It will be understood that the conjugate itself also exhibits G-CSF
activity.
[0049] By removing and/or introducing an amino acid residue
comprising an attachment group for the non-polypeptide moiety it is
possible to specifically adapt the polypeptide so as to make the
molecule more susceptible to conjugation to the non-polypeptide
moiety of choice, to optimize the conjugation pattern (e.g. to
ensure an optimal distribution of non-polypeptide moieties on the
surface of the G-CSF molecule and to ensure that only the
attachment groups intended to be conjugated are present in the
molecule) and thereby obtain a new conjugate molecule which has
G-CSF activity and in addition one or more improved properties as
compared to G-CSF molecules available today.
[0050] While the polypeptide may be of any origin, in particular
mammalian origin, it is presently preferred to be of human origin,
in particular a variant of a polypeptide having the amino acid
sequence of SEQ ID NO:1.
[0051] In preferred embodiments of the present invention more than
one amino acid residue of the polypeptide with G-CSF activity is
altered, e.g. the alteration embraces removal as well as
introduction of amino acid residues comprising an attachment group
for the non-polypeptide moiety of choice.
[0052] In addition to the amino acid alterations disclosed herein
aimed at removing and/or introducing attachment sites for the
non-polypeptide moiety, it will be understood that the amino acid
sequence of the polypeptide of the invention may if desired contain
other alterations that need not be related to introduction or
removal of attachment sites, i.e. other substitutions, insertions
or deletions. These may, for example, include truncation of the N-
and/or C-terminus by one or more amino acid residues, or addition
of one or more extra residues at the N- and/or C-terminus, e.g.
addition of a methionine residue at the N-terminus.
[0053] The conjugate of the invention has one or more of the
following improved properties as compared to hG-CSF, in particular
as compared to rhG-CSF (e.g. filgrastim, lenograstim or
nartograstim) or known hG-CSF variants: increased ability to reduce
the duration of neutropenia, increased functional in vivo
half-life, increased serum half-life, reduced renal clearance,
reduced receptor-mediated clearance, reduced side effects such as
bone pain, and reduced immunogenicity.
[0054] It will be understood that the amino acid residue comprising
an attachment group for a non-polypeptide moiety, whether it be
removed or introduced, will be selected on the basis of the nature
of the non-polypeptide moiety of choice and, in most instances, on
the basis of the method by which conjugation between the
polypeptide and the non-polypeptide moiety is to be achieved. For
instance, when the non-polypeptide moiety is a polymer molecule
such as a polyethylene glycol or polyalkylene oxide derived
molecule amino acid residues comprising an attachment group may be
selected from the group consisting of lysine, cysteine, aspartic
acid, glutamic acid, histidine and arginine. When conjugation to a
lysine residue is to be achieved, a suitable activated molecule is
e.g. mPEG-SPA from Shearwater Corp.,
oxycarbonyl-oxy-N-dicarboxyimide-PEG (U.S. Pat. No. 5,122,614), or
PEG available from PolyMASC Pharmaceuticals plc. The first of these
will be illustrated further below.
[0055] In order to avoid too much disruption of the structure and
function of the parent hG-CSF molecule, the total number of amino
acid residues to be altered in accordance with the present
invention, e.g. as described in the subsequent sections herein, (as
compared to the amino acid sequence shown in SEQ ID NO:1) will
typically not exceed 15. The exact number of amino acid residues
and the type of amino acid residues to be introduced or removed
depends in particular on the desired nature and degree of
conjugation (e.g. the identity of the non-polypeptide moiety, how
many non-polypeptide moieties it is desirable or possible to
conjugate to the polypeptide, where conjugation is desired or
should be avoided, etc.). Preferably, the polypeptide part of the
conjugate of the invention or the polypeptide of the invention
comprises an amino acid sequence which differs in 1-15 amino acid
residues from the amino acid sequence shown in SEQ ID NO:1,
typically in 2-10 amino acid residues, e.g. in 3-8 amino acid
residues, such as 4-6 amino acid residues, from the amino acid
sequence shown in SEQ ID NO:1. Thus, normally the polypeptide part
of the conjugate or the polypeptide of the invention comprises an
amino acid sequence which differs from the amino acid sequence
shown in SEQ ID NO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14 or 15 amino acid residues.
[0056] The polypeptide part of the conjugate will typically have an
amino acid sequence with at least about 80% identity with SEQ ID
NO:1, preferably at least about 90%, such as at least about 95%,
e.g. at least about 96%, 97%, 98% or 99% sequence identity with SEQ
ID NO:1. Amino acid sequence homology/identity is conveniently
determined from aligned sequences, using e.g. the ClustalW program,
version 1.8, June 1999, using default parameters (Thompson et al.,
1994, ClustalW: Improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, position-specific
gap penalties and weight matrix choice, Nucleic Acids Research, 22:
4673-4680) or from the PFAM families database version 4.0
(http://pfam.wustl.edu/) (Nucleic Acids Res. 1999 Jan. 1;
27(1):260-2) by use of GENEDOC version 2.5 (Nicholas, K. B.,
Nicholas H. B. Jr., and Deerfield, D. W. II. 1997 GeneDoc: Analysis
and Visualization of Genetic Variation, EMBNEW.NEWS 4:14; Nicholas,
K. B. and Nicholas H. B. Jr. 1997 GeneDoc: Analysis and
Visualization of Genetic Variation).
[0057] In a preferred embodiment one difference between the amino
acid sequence of the polypeptide and the amino acid sequence shown
in SEQ ID NO:1 is that at least one and often more, e.g. 1-15,
amino acid residues comprising an attachment group for the
non-polypeptide moiety has been introduced, preferably by
substitution, into the amino acid sequence. Thereby, the
polypeptide part is altered in the content of the specific amino
acid residues to which the non-polypeptide moiety of choice binds,
whereby a more efficient, specific and/or extensive conjugation is
achieved. For instance, when the total number of amino acid
residues comprising an attachment group for the non-polypeptide of
choice is altered to an optimized level, the clearance of the
conjugate is typically significantly reduced, due to the altered
shape, size and/or charge of the molecule achieved by the
conjugation. Furthermore, when the total number of amino acid
residues comprising an attachment group for the non-polypeptide of
choice is increased, a greater proportion of the polypeptide
molecule is shielded by the non-polypeptide moieties of choice,
leading to a lower immune response.
[0058] The term "one difference" as used in the present application
is intended to allow for additional differences being present.
Accordingly, in addition to the specified amino acid difference,
other amino acid residues than those specified may be mutated.
[0059] In a further preferred embodiment one difference between the
amino acid sequence of the polypeptide and the amino acid sequence
shown in SEQ ID NO:1 is that at least one and preferably more, e.g.
1-15, amino acid residues comprising an attachment group for the
non-polypeptide moiety has/have been removed, preferably by
substitution, from the amino acid sequence. By removing one or more
amino acid residues comprising an attachment group for the
non-polypeptide moiety of choice it is possible to avoid
conjugation to the non-polypeptide moiety in parts of the
polypeptide in which such conjugation is disadvantageous, e.g. in
amino acid residues located at or near a functional site of the
polypeptide (since conjugation at such a site may result in
inactivation or reduced G-CSF activity of the resulting conjugate
due to impaired receptor recognition). In the present context the
term "functional site" is intended to indicate one or more amino
acid residues which is/are essential for or otherwise involved in
the function or performance of hG-CSF. Such amino acid residues are
a part of the functional site. The functional site may be
determined by methods known in the art and is preferably identified
by analysis of a structure of the polypeptide complexed to a
relevant receptor, such as the hG-CSF receptor (See Aritomi et al.
Nature 401:713-717, 1999).
[0060] In a still further preferred embodiment, the amino acid
sequence of the polypeptide differs from the amino acid sequence
shown in SEQ ID NO:1 in that a) at least one specified amino acid
residue comprising an attachment group for the non-polypeptide
moiety and present in the amino acid sequence shown in SEQ ID NO:1
has been removed, preferably by substitution, and b) at least one
specified amino acid residue comprising an attachment group for the
non-polypeptide moiety has been introduced into the amino acid
sequence, preferably by substitution, the specified amino acid
residues being any of those described in the subsequent sections
herein. This embodiment is considered of particular interest in
that it is possible to specifically design the polypeptide so as to
obtain an optimal conjugation to the non-polypeptide moiety of
choice. For instance, by introducing and removing selected amino
acid residues as disclosed in the following sections it is possible
to ensure an optimal distribution of attachment groups for the
non-polypeptide moiety of choice, which gives rise to a conjugate
in which the non-polypeptide moieties are placed so as to a)
effectively shield epitopes and other surface parts of the
polypeptide and b) ensure an optimal Stokes radius of the
conjugate, without causing too much structural disruption and
thereby impair the function of the polypeptide.
[0061] The conjugate of the invention will in general comprise a
sufficient number and type of non-polypeptide moieties to provide
the conjugate with an increased functional in vivo half-life and/or
serum half-life as compared to hG-CSF, e.g. filgrastim, lenograstim
or nartograstim, and preferably as compared to rhG-CSF comprising a
single N-terminally attached 20 kDa PEG moiety. The increased
functional in vivo half-life is conveniently determined as
described in the Materials and Methods section herein.
[0062] The conjugate of the invention may comprise at least one
non-conjugated, conjugatable attachment group for the
non-polypeptide moiety. In the present context the term
"conjugatable attachment group" is intended to indicate an
attachment group that is located in a position of the polypeptide
where it is accessible for conjugation, and that but for special
precautions is conjugated to the relevant non-polypeptide moiety
when subjected to conjugation. For instance, such attachment group
may be part of an amino acid residue involved in or otherwise
essential for the polypeptide to exert its activity. A convenient
way to avoid conjugation of an otherwise conjugatable attachment
group is to shield the attachment group by means of a helper
molecule, e.g. as described in the section entitled "Blocking of
the functional site". It will be understood that the number of
non-conjugated, conjugatable attachment groups depends on the
specific G-SCF polypeptide and the location of the conjugatable
attachment groups. For instance, the polypeptide conjugate
comprises one or two non-conjugated, conjugatable attachment
groups, and at least one, and preferably two or more conjugated
attachment groups.
[0063] The four helices of G-CSF comprise amino acid residues 11-41
(helix A), 71-95 (helix B), 102-125 (helix C), and 145-170 (helix
D) (Zink et al. (1994) Biochemistry 33: 8453-8463). Surprisingly,
it has been found that advantageous results may be obtained when
non-polypeptide moieties are attached to amino acid residues
located in one or more of the helices of G-CSF, even though
modification of protein helices, e.g. the helix structures of
four-helix bundle proteins such as G-CSF, is generally considered
to be accompanied by a risk of disturbance of protein function. In
one embodiment, the polypeptide conjugate of the invention
therefore comprises at least one non-polypeptide moiety attached to
an attachment group of an amino acid residue located in one of the
four helices, in particular in one or more of the B, C or D
helices.
[0064] Conjugate of the Invention Wherein the Non-Polypeptide
Moiety is Attached to a Lysine or the N-Terminal Amino Acid
Residue
[0065] In one aspect the invention relates to a polypeptide
conjugate comprising
[0066] i) a polypeptide exhibiting G-CSF activity, comprising an
amino acid sequence that differs from the amino acid sequence shown
in SEQ ID NO:1 in at least one substitution selected from the group
consisting of T1K, P2K, L3K, G4K, P5K, A6K, S7K, S8K, L9K, P10K,
Q11K, S12K, F13K, L14K, L15K, E19K, Q20K, V21K, Q25K, G26K, D27K,
A29K, A30K, E33K, A37K, T38K, Y39K, L41K, H43K, P44K, E45K, E46K,
V48K, L49K, L50K, H52K, S53K, L54K, I56K, P57K, P60K, L61 K, S62K,
S63K, P65K, S66K, Q67K, A68K, L69K, Q70K, L71K, A72K, G73K, S76K,
Q77K, L78K, S80K, F83K, Q86K, G87K, Q90K, E93K, G94K, S96K, P97K,
E98K, L99K, G100K, P101K, T102K, D104K, T105K, Q107K, L108K, D109K,
A111K, D112K, F113K, T115K, T116K, W118K, Q119K, Q120K, M121K,
E122K, E123K, L124K, M126K, A127K, P128K, A129K, L130K, Q131K,
P132K, T133K, Q134K, G135K, A136K, M137K, P138K, A139K, A141K,
S142K, A143K, F144K, Q145K, S155K, H156K, Q158K, S159K, L161K,
E162K, V163K, S164K, Y165K, V167K, L168K, H170K, L171K, A172K,
Q173K and P174K, and
[0067] ii) at least one non-polypeptide moiety attached to a lysine
residue of the polypeptide.
[0068] hG-CSF contains four lysine residues, of which K16 is
located in the receptor-binding domain and the others are located
in positions 23, 34 and 40, respectively, all relatively close to
the receptor-binding domain. In order to avoid conjugation to one
or more of these lysine residues (since this may inactivate or
severely reduce the activity of the resulting conjugate) it may be
desirable to remove at least one lysine residue, e.g. two, three or
all of these residues. Accordingly, in another, more preferred
aspect the invention relates to a polypeptide conjugate as defined
above, wherein at least one of the amino acid residues selected
from the group consisting of K16, K23, K34 and K40 has been deleted
or substituted with another amino acid residue. Preferably, at
least K16 is substituted with another amino acid residue.
[0069] Examples of preferred amino acid substitutions include one
or more of Q70K, Q90K, T105K, Q120K, T133K, S159K and H170K/Q/R,
such as two, three, four or five of these substitutions, for
example: Q70K+Q90K, Q70K+T105K, Q70K+Q120K, Q70K+T133K, Q70K+S159K,
Q70K+H170K, Q90K+T105K, Q90K+Q120K, Q90K+T133K, Q90K+S159K,
Q90K+H170K, T105K+Q120K, T105K+T133K, T105K+S159K, T105K+H170K,
Q120K+T133K, Q120K+S159K, Q120K+H170K, T133K+S159K, T133K+H170K,
S159K+H170K, Q70K+Q90K+T105K, Q70K+Q90K+Q120K, Q70K+Q90K+T133K,
Q70K+Q90K+S159K, Q70K+Q90K+H170K, Q70K+T105K+Q120K,
Q70K+T105K+T133K, Q70K+T105K+S159K, Q70K+T105K+H170K,
Q70K+Q120K+T133K, Q70K+Q120K+S159K, Q70K+Q120K+H170K,
Q70K+T133K+S159K, Q70K+T133K+H170K, Q70K+S159K+H170K,
Q90K+T105K+Q120K, Q90K+T105K+T133K, Q90K+T105K+S159K,
Q90K+T105K+H170K, Q90K+Q120K+T133K, Q90K+Q120K+S159K,
Q90K+Q120K+H170K, Q90K+T133K+S159K, Q90K+T133K+H170K,
Q90+S159K+H170K, T105K+Q120K+T133K, T105K+Q120K+S159K,
T105K+Q120K+H170K, T105K+T133K+S159K, T105K+T133K+H170K,
T105K+S159K+H170K, Q120K+T133K+S159K, Q120K+T133K+H170K,
Q120K+S159K+H170K, T133K+S159K+H170K, Q70K+Q90K+T105K+Q120K,
Q70K+Q90K+T105K+T133K, Q70K+Q90K+T105K+S159K,
Q70K+Q90K+T105K+H170K, Q70K+Q90K+Q120K+T133K,
Q70K+Q90K+Q120K+S159K, Q70K+Q90K+Q120K+H170K,
Q70K+Q90K+T133K+S159K, Q70K+Q90K+T133K+H170K,
Q70K+Q90K+S159K+H170K, Q70K+T105K+Q120K+T133K,
Q70K+T105K+Q120K+S159K, Q70K+T105K+Q120K+H170K,
Q70K+T105K+T133K+S159K, Q70K+T105K+T133K+H170K,
Q70K+T105K+S159K+H170K, Q70K+Q120K+T133K+S159K,
Q70K+Q120K+T133K+H170K, Q70K+T133K+S159K+H170K,
Q90K+T105K+Q120K+T133K, Q90K+T105K+Q120K+S159K,
Q90K+T105K+Q120K+H170K, Q90K+T105+T133K+S159K,
Q90K+T105+T133K+H1.70K, Q90K+T105+S159K+H170K,
Q90K+Q120K+T133K+S159K, Q90K+Q120K+T133K+H170K,
Q90K+Q120K+S159K+H170K, Q90K+T133K+S159K+H170K,
T105K+Q120K+T133K+S159K, T105K+Q120K+T133K+H170K,
T105K+Q120K+S159K+H170K, T105K+T133K+S159K+H170K or
Q120K+T133K+S159K+H170K. In any of the variants listed above, the
substitution H170K may instead be H170Q or H170R.
[0070] The polypeptide of the conjugate according to this aspect of
the invention, i.e. having at least one introduced and one removed
lysine, preferably comprises at least one, such as one, two, three
or four, of the substitutions selected from the group consisting of
K16R, K16Q, K23R, K23Q, K34R, K34Q, K40R and K40Q, preferably at
least the substitution K16R, whereby conjugation of this residue
can be avoided. Preferably, the polypeptide comprises at least one
substitution selected from the group consisting of K16R+K23R,
K16R+K34R, K16R+K40R, K23R+K34R, K23R+K40R, K34R+K40R,
K16R+K23R+K34R, K16R+K23R+K40R, K23R+K34R+K40R, K16R+K34R+K40R and
K16R+K23R+K34R+K40R. In one preferred embodiment, the polypeptide
includes the substitutions K16R+K34R+K40R, while the lysine in
position 23 is left unaltered. As indicated above, it is
contemplated that any of the individual substitutions or
combinations listed in this paragraph for removal of a lysine
residue may suitably be used with any of the other substitutions
disclosed herein for introduction of lysine residues, in particular
the substitutions listed in the paragraph above.
[0071] In a particular embodiment the polypeptide includes the
substitutions K16R, K34R, K40R, T105K and S159K and is conjugated
to 2-6, typically 3-6 polyethylene glycol moieties with a molecular
weight of about 1000-10,000 Da.
[0072] In one embodiment the conjugate of the invention has a
glycosylation in T133, i.e. this position is unaltered from the
wild-type hG-CSF. This is the natural glycosylation site.
Alternatively, the conjugate may be non-glycosylated, although
glycosylated conjugates are preferred.
[0073] In particular, the conjugate may have 2-6, typically 3-6
polyethylene glycol moieties with a molecular weight of about
5000-6000 Da attached, e.g. mPEG with a molecular weight of about 5
kDa. Preferably, the conjugate has 4-5 polyethylene glycol moieties
with a molecular weight of about 5000-6000 Da attached, e.g. 5 kDa
mPEG.
[0074] In another embodiment, the conjugate may be produced so as
to have only a single number of PEG moieties attached, e.g. either
2, 3, 4, 5 or 6 PEG moieties per polypeptide, or to have a desired
mix of polypeptide conjugates with different numbers of PEG
moieties attached, e.g. a mix having 2-5, 2-4, 3-5, 3-4, 4-6, 4-5
or 5-6 attached PEG moieties. As indicated above, an example of a
preferred conjugate mix is one having 4-5 PEG moieties of about 5
kDa.
[0075] It will be understood that a conjugate having a specific
number of attached PEG moieties, or a mix of conjugates having a
defined range of numbers of attached PEG moieties, may be obtained
by choosing suitable PEGylation conditions and optionally by using
subsequent purification to separate conjugates having the desired
number of PEG moieties. Examples of methods for separation of G-CSF
molecules with different numbers of PEG moieties attached are
provided below. Determination of the number of attached PEG
moieties may e.g. be performed using SDS-PAGE. For purposes of the
present invention, a polypeptide conjugate may be considered to
have a given number of attached PEG moieties if separation on an
SDS-PAGE gel shows no or only insignificant bands other than the
band(s) corresponding to the given number(s) of PEG moieties. For
example, a sample of a polypeptide conjugate is considered to have
4-5 attached PEG groups if an SDS-PAGE gel on which the sample has
been run shows bands corresponding to 4 and 5 PEG groups,
respectively, and only insignificant bands or, preferably, no bands
corresponding to 3 or 6 PEG groups.
[0076] While the non-polypeptide moiety of the conjugate according
to this aspect of the invention may be any molecule which, when
using the given conjugation method has lysine as an attachment
group such as a carbohydrate moiety, it is preferred that the
non-polypeptide moiety is a polymer molecule. The polymer molecule
may be any of the molecules mentioned in the section entitled
"Conjugation to a polymer molecule", but is preferably selected
from the group consisting of linear or branched polyethylene glycol
or another polyalkylene oxide. Preferred polymer molecules are e.g.
mPEG-SPA (in particular SPA-mPEG 5000) from Shearwater Corp. or
oxycarbonyl-oxy-N-dicarboxyimide PEG (U.S. Pat. No. 5,122,614).
[0077] It will be understood that any of the amino acid changes, in
particular substitutions, specified in this section can be combined
with any of the amino acid changes, preferably substitutions,
specified in the other sections herein disclosing specific amino
acid modifications, including introduction and/or removal of
glycosylation sites.
[0078] Conjugate of the Invention Wherein the Non-Polypeptide
Moiety is a Molecule which has Cysteine as an Attachment Group
[0079] In another aspect the invention relates to a conjugate
comprising
[0080] i) a polypeptide exhibiting G-CSF activity, which comprises
an amino acid sequence that differs from the amino acid sequence of
hG-CSF shown in SEQ ID NO:1 in at least one substitution selected
from the group consisting of TIC, P2C, L3C, G4C, P5C, A6C, S7C,
S8C, L9C, P1.degree. C., Q11C, S12C, F13C, L14C, L15C, E19C, Q20C,
V21C, R22C, Q25C, G26C, D27C, A29C, A30C, E33C, A37C, T38C, Y39C,
L41C, H43C, P44C, E45C, E46C, V48C, L49C, L50C, H52C, S53C, L54C,
I56C, P57C, P60C, L61C, S62C, S63C, P65C, S66C, Q67C, A68C, L69C,
Q70C, L71C, A72C, G73C, S76C, Q77C, L78C, S80C, F83C, Q86C, G87C,
Q90C, E93C, G94C, S96C, P97C, E98C, L99C, G100C, P101C, T102C,
D104C, T105C, Q107C, L108C, D109C, A111C, D112C, F113C, T115C,
T116C, W118C, Q119C, Q120C, M121C, E122C, E123C, L124C, M126C,
A127C, P128C, A129C, L130C, Q131C, P132C, T133C, Q134C, G135C,
A136C, M137C, P138C, A139C, A141C, S142C, A143C, F144C, Q145C,
R146C, R147C, S155C, H156C, Q158C, S159C, L161C, E162C, V163C,
S164C, Y165C, R166C, V167C, L168C, R169C, H170C, L171C, A172C,
Q173C and P174C, and
[0081] ii) at least one non-polypeptide moiety attached to a
cysteine residue of the polypeptide.
[0082] The receptor-binding domain of hG-CSF contains a cysteine
residue in position 17 which does not take part in a cystine and
which may advantageously be removed in order to avoid conjugation
of a non-polypeptide moiety to said cysteine. Accordingly, in
another, more preferred aspect the invention relates to a conjugate
comprising
[0083] i) a polypeptide exhibiting G-CSF activity, which comprises
an amino acid sequence that differs from the amino acid sequence
shown in SEQ ID NO:1 in at least one substitution selected from the
group consisting of TIC, P2C, L3C, G4C, P5C, A6C, S7C, S8C, L9C,
P10C, Q11C, S12C, F13C, L14C, L15C, E19C, Q20C, V21C, R22C, Q25C,
G26C, D27C, A29C, A30C, E33C, A37C, T38C, Y39C, L41C, H43C, P44C,
E45C, E46C, V48C, L49C, L50C, H52C, S53C, L54C, I56C, P57C, P60C,
L61C, S62C, S63C, P65C, S66C, Q67C, A68C, L69C, Q70C, L71C, A72C,
G73C, S76C, Q77C, L78C, S80C, F83C, Q86C, G87C, Q90C, E93C, G94C,
S96C, P97C, E98C, L99C, G100C, P101C, T102C, D104C, T105C, Q107C,
L108C, D109C, A111C, D112C, F113C, T115C, T116C, W118C, Q119C,
Q120C, M121C, E122C, E123C, L124C, M126C, A127C, P128C, A129C,
L130C, Q131C, P132C, T133C, Q134C, G135C, A136C, M137C, P138C,
A139C, A141C, S142C, A143C, F144C, Q145C, R146C, R147C, S155C,
H156C, Q158C, S159C, L161C, E162C, V163C, S164C, Y165C, R166C,
V167C, L168C, R169C, H170C, L171C, A172C, Q173C and P174C, in
combination with removal of C17, preferably substitution of C17
with any other amino acid residue, e.g. with a serine residue,
and
[0084] ii) a non-polypeptide moiety which has a cysteine residue as
an attachment group.
[0085] Preferred substitutions according to this aspect of the
invention are substitutions of arginine with cysteine, for example
one or more of R146C, R147C, R166C and R169C.
[0086] It will be understood that any of the amino acid
modifications, in particular substitutions, specified in this
section can be combined with any of the amino acid changes, in
particular substitutions, specified in the other sections herein
disclosing specific amino acid modifications, including
introduction and/or removal of glycosylation sites.
[0087] Conjugate of the Invention Wherein the Non-Polypeptide
Moiety Binds to an Acid Group or the C-Terminal Amino Acid
Residue
[0088] In a still further aspect the invention relates to a
conjugate comprising
[0089] i) a polypeptide exhibiting G-CSF activity, which comprises
an amino acid sequence that differs from the amino acid sequence
shown in SEQ ID NO:1 in at least one substitution selected from the
group consisting of T1D, P2D, L3D, G4D, P5D, A6D, S7D, S8D, L9D,
P10D, Q11D, S12D, F13D, L14D, L15D, K16D, Q20D, V21D, R22D, K23D,
Q25D, G26D, A29D, A30D, K34D, A37D, T38D, Y39D, K40D, L41D, H43D,
P44D, V48D, L49D, L50D, H52D, S53D, L54D, I56D, P57D, P60D, L61D,
S62D, S63D, P65D, S66D, Q67D, A68D, L69D, Q70D, L71D, A72D, G73D,
S76D, Q77D, L78D, S80D, F83D, Q86D, G87D, Q90D, G94D, S96D, P97D,
L99D, G100D, P101D, T102D, T105D, Q107D, L108D, A111D, F113D,
T115D, T116D, W118D, Q119D, Q120D, M121D, L124D, M126D, A127D,
P128D, A129D, L130D, Q131D, P132D, T133D, Q134D, G135D, A136D,
M137D, P138D, A139D, A141D, S142D, A143D, F144D, Q145D, R146D,
R147D, S155D, H156D, Q158D, S159D, L161D, V163D, S164D, Y165D,
R166D, V167D, L168D, R169D, H170D, L171D, A172D, Q173D and P174D;
or at least one substitution selected from the group consisting of
TIE, P2E, L3E, G4E, P5E, A6E, S7E, S8E, L9E, P10E, Q11E, S12E,
F13E, L14E, L15E, K16E, Q20E, V21E, R22E, K23E, Q25E, G26E, A29E,
A30E, K34E, A37E, T38E, Y39E, K40E, L41E, H43E, P44E, V48E, L49E,
L50E, H52E, S53E, L54E, I56E, P57E, P60E, L61E, S62E, S63E, P65E,
S66E, Q67E, A68E, L69E, Q70E, L71E, A72E, G73E, S76E, Q77E, L78E,
S80E, F83E, Q86E, G87E, Q90E, G94E, S96E, P97E, L99E, G100E, P101E,
T102E, T105E, Q107E, L108E, A111E, F113E, T15E, T116E, W118E,
Q119E, Q120E, M121E, L124E, M126E, A127E, P128E, A129E, L130E,
Q131E, P132E, T133E, Q134E, G135E, A136E, M137E, P138E, A139E,
A141E, S142E, A143E, F144E, Q145E, R146E, R147E, S155E, H156E,
Q158E, S159E, L161E, V163E, S164E, Y165E, R166E, V167E, L168E,
R169E, H170E, L171E, A172E, Q173E and P174E; and
[0090] ii) a non-polypeptide moiety having an aspartic acid or a
glutamic acid residue as an attachment group.
[0091] Examples of preferred substitutions according to this aspect
of the invention include Q67D/E, Q70D/E, Q77D/E, Q86D/E, Q90D/E,
Q120D/E, Q131D/E, Q134D/E, Q145D/E and Q173D/E.
[0092] In addition to the above listed substitutions, the
polypeptide of the conjugate according to any of the above aspects
may comprise removal, preferably by substitution, of at least one
of the amino acid residues selected from the group consisting of
D27, D104, D109, D112, E19, E33, E45, E46, E93, E98, E122, E123,
and E163. The substitution may be for any other amino acid residue,
in particular for an asparagine or a glutamine residue, whereby
conjugation of these residues can be avoided. In particular, the
polypeptide may comprise at least one of the following
substitutions: D27N, D104N, D109N, D112N, E19Q, E33Q, E45Q, E46Q,
E93Q, E98Q, E122Q, E123Q and E163Q. Preferably, the amino acid
substitution in one or more of the above positions may in addition
be combined with at least one of the following substitutions:
D109N, D112N, E19Q, E122Q and E123Q.
[0093] While the non-polypeptide moiety of the conjugate according
to this aspect of the invention, which has an acid group as an
attachment group, can be any non-polypeptide moiety with such
property, it is presently preferred that the non-polypeptide moiety
is a polymer molecule or an organic derivatizing agent, in
particular a polymer molecule, and the conjugate is prepared e.g.
as described by Sakane and Pardridge, Pharmaceutical Research, Vol.
14, No. 8, 1997, pp 1085-1091.
[0094] It will be understood that any of the amino acid changes, in
particular substitutions, specified in this section can be combined
with any of the amino acid changes, in particular substitutions
specified in the other sections herein disclosing specific amino
acid changes, including introduction and/or removal of
glycosylation sites.
[0095] Other Conjugates of the Invention
[0096] In addition to the non-polypeptide moieties specified above
e.g. in the sections entitled "Conjugate of the invention . . . "
the conjugate of the invention may contain one or more carbohydrate
moieties as a consequence of the polypeptide being expressed in a
glycosylating host cell to result in glycosylation at the natural
glycosylation site of hG-CSF (T133) and/or at introduced
glycosylation site(s).
[0097] Conjugate of the Invention Wherein the Non-Polypeptide
Moiety is a Carbohydrate Moiety
[0098] In a further aspect the invention relates to a conjugate
comprising a glycosylated polypeptide exhibiting G-CSF activity,
which comprises an amino acid sequence that differs from that shown
in SEQ ID NO:1 in that at least one non-naturally occurring
glycosylation site has been introduced into the amino acid sequence
by way of at least one substitution selected from the group
consisting of L3N+P5S/T, P5N, A6N, S8N+P10S/T, P10N, Q11N+F13S/T,
S12N+L14S/T, F13N+L15S/T, L14N+K16S/T, K16N+L18S/T, E19N+V21S/T,
Q20N+R22S/T, V21N+K23S/T, R22N+I24S/T, K23N+Q25S/T, Q25N+D27S/T,
G26N+G28S/T, D27N+A29S/T, A29N+L31S/T, A30N+Q32S/T, E33N+L35S/T,
A37N+Y39S/T, T38N+K40S/T, Y39N+L41S/T, P44N+E46S/T, E45N+L47S/T,
E46N+V48S/T, V48N+L50S/T, L49N+G51S/T, L50N+H52S/T, H52N+L54S/T,
S53N+G55S/T, P60N, L61N, S63N+P65S/T, P65N+Q67S/T, S66N+A68S/T,
Q67N+L69S/T, A68N+Q70S/T, L69N+L71S/T, Q70N+A72S/T, L71N+G73S/T,
G73N+L75S/T, S76N+L78S/T, Q77N+H79S/T, L78N, S80N+L82S/T,
F83N+Y85S/T, Q86N+L88S/T, G87N+L89S/T, Q90N+L92S/T, E93N+I95S/T,
P97N+L99S/T, L99N+P101S/T, P101N+L103S/T, T102N+D104S/T,
D104N+L106S/T, T105N+Q107S/T, Q107N+D109S/T, L108N+V110S/T,
D109N+A111S/T, A111N+F113S/T, D1112N+A114S/T, F113N, T115N+I17S/T,
T116N+W118S/T, W118N+Q120S/T, Q119N+M121S/T, Q120N+E122S/T,
M121N+E123S/T, E122N+L124S/T, E123N+G125S/T, L124N+M126S/T,
M126N+P128S/T, P128N+L130S/T, L130N+P132S/T, P132N+Q134S/T,
T133N+G135S/T, Q134N+A136S/T, A136N+P138S/T, P138N+F140S/T,
A139N+A141S/T, A141N+A143S/T, S142N+F144S/T, A143N+Q145S/T,
F144N+R146S/T, Q145N+R147S/T, R146N+A148S/T, R147N+G149S/T,
S155N+L157S/T, H156N+Q158S/T, S159N+L161S/T, L161N+V163S/T, E162N,
V163N+Y165S/T, S164N+R166S/T, Y165N+V167S/T, R166N+L168S/T,
V167N+R169S/T, L168N+H170S/T, R169N+L171S/T and H170N+A172S/T,
wherein S/T indicates an S or a T residue, preferably a T
residue.
[0099] It will be understood that in order to prepare a conjugate
according to this aspect the polypeptide must be expressed in a
glycosylating host cell capable of attaching oligosaccharide
moieties at the glycosylation site(s) or alternatively subjected to
in vitro glycosylation. Examples of glycosylating host cells are
given in the section further below entitled "Coupling to an
oligosaccharide moiety".
[0100] Alternatively, the conjugate according to this aspect
comprises a polypeptide exhibiting G-CSF activity, which comprises
an amino acid sequence that differs from that shown in SEQ ID NO:1
in at least one substitution selected from the group consisting of
P5N, A6N, P10N, P60N, L61N, L78N, F113N and E162N, in particular
from the group consisting of P5N, A6N, P10N, P60N, L61N, F113N and
E162N, such as from the group consisting of P60N, L61N, F113N and
E162N.
[0101] Alternatively, the conjugate according to this aspect
comprises a polypeptide exhibiting G-CSF activity, which comprises
an amino acid sequence that differs from that shown in SEQ ID NO:1
in at least one substitution selected from the group consisting of
D27N+A29S, D27N+A29T, D104N+L106S, D104N+L106T, D109N+A111S,
D109N+A111T, D112N+A114S and D112N+A114T, more preferably from the
group consisting of D27N+A29S, D27N+A29T, D104N+L106S, D104N+L106T,
D112N+A114S and D112N+A114T, such as from the group consisting of
D27N+A29S, D27N+A29T, D104N+L106S and D104N+L106T.
[0102] In addition to a carbohydrate molecule, the conjugate
according to the aspect of the invention described in the present
section may contain additional non-polypeptide moieties, in
particular a polymer molecule, as described in the present
application, conjugated to one or more attachment groups present in
the polypeptide part of the conjugate.
[0103] It will be understood that any of the amino acid changes, in
particular substitutions, specified in this section can be combined
with any of the amino acid changes, in particular substitutions,
specified in the other sections herein disclosing specific amino
acid changes.
[0104] Circularly Permuted Variants
[0105] In a further embodiment, the polypeptide part of the
polypeptide conjugate of the invention may be in the form of a
circularly permuted variant of a polypeptide sequence otherwise
disclosed herein. In such a circularly permuted polypeptide, the
original N-terminus and C-terminus are joined together either
directly by a peptide bond or indirectly via a peptide linker,
while new N- and C-termini are formed between two adjacent amino
acid residues that originally were joined by a peptide bond. Since
the original N- and C-termini will normally be located at some
distance from each other, they will typically be linked by means of
a peptide linker having a suitable length and composition so that
the structure and activity of the conjugate is not adversely
affected. It will be clear that the new N-terminus and C-terminus
should not be formed between an amino acid residue pair where this
would interfere with the activity of the polypeptide. Circularly
permuted G-CSF receptor agonists are disclosed in U.S. Pat. No.
6,100,070, to which reference is made for further information on
selecting peptide linkers and the location of the new N-terminus
and C-terminus as well as methods for producing them such
variants.
[0106] White Blood Cell and Neutrophil Formation of Conjugates of
the Invention
[0107] In a further embodiment, the polypeptide conjugate of the
invention may be characterized as being a conjugate exhibiting
G-CSF activity and comprising a polypeptide with an amino acid
sequence that differs in at least one amino acid residue from the
amino acid sequence shown in SEQ ID NO:1 and having at least one
non-polypeptide moiety attached to an attachment group of the
polypeptide, the polypeptide conjugate further fulfilling at least
one of the following criteria (A)-(D):
[0108] (A) after one subcutaneous administration of 100 microgram
per kg body weight to rats (based on the weight of the polypeptide
part of the conjugate) it:
[0109] i) increases formation of white blood cells with at least
about the same rate and to at least about the same level (measured
as number of cells per liter of blood) as administration of 100
microgram of non-conjugated hG-CSF per kg body weight for a period
of 6 hours, preferably 12 hours after administration, and
[0110] ii) increases the level of white blood cells (measured as
number of cells per liter blood) above the level of white blood
cells prior to administration for a period of at least about 96
hours, preferably for at least about 120 hours;
[0111] (B) after one subcutaneous administration of 25 microgram
per kg body weight to rats (based on the weight of the polypeptide
part of the conjugate) it:
[0112] i) increases formation of white blood cells with at least
about the same rate and to at least about the same level (measured
as number of cells per liter of blood) as administration of 100
microgram of non-conjugated hG-CSF per kg body weight for a period
of 6 hours, preferably 12 hours after administration, and
[0113] ii) increases the level of white blood cells (measured as
number of cells per liter blood) above the level of white blood
cells prior to administration for a period of at least about 72
hours, preferably at least about 96 hours, more preferably at least
about 120 hours;
[0114] (C) after one subcutaneous administration of 100 microgram
per kg body weight to rats (based on the weight of the polypeptide
part of the conjugate) it:
[0115] i) increases formation of neutrophils with at least about
the same rate and to at least about the same level (measured as
number of cells per liter of blood) as administration of 100
microgram of non-conjugated hG-CSF per kg body weight for a period
of 6 hours, preferably 12 hours after administration, and
[0116] ii) increases the level of neutrophils (measured as number
of cells per liter blood) above the level of neutrophils prior to
administration for a period of at least about 96 hours, preferably
at least about 120 hours;
[0117] (D) after one subcutaneous administration of 25 microgram
per kg body weight to rats (based on the weight of the polypeptide
part of the conjugate) it:
[0118] i) increases formation of neutrophils with at least about
the same rate and to at least about the same level (measured as
number of cells per liter of blood) as administration of 100
microgram of non-conjugated hG-CSF per kg body weight for a period
of 6 hours, preferably 12 hours after administration, and
[0119] ii) increases the level of neutrophils (measured as number
of cells per liter blood) above the level of neutrophils prior to
administration for a period of at least about 72 hours, preferably
at least about 96 hours, more preferably at least about 120
hours.
[0120] Non-Polypeptide Moiety of the Conjugate of the Invention
[0121] As indicated further above the non-polypeptide moiety of the
conjugate of the invention is preferably selected from the group
consisting of a polymer molecule, a lipophilic compound, a
carbohydrate moiety (e.g. by way of in vivo glycosylation) and an
organic derivatizing agent. All of these agents may confer
desirable properties to the polypeptide part of the conjugate, in
particular increased functional in vivo half-life and/or increased
serum half-life. The polypeptide part of the conjugate is normally
conjugated to only one type of non-polypeptide moiety, but may also
be conjugated to two or more different types of non-polypeptide
moieties, e.g. to a polymer molecule and an oligosaccharide moiety,
to a lipophilic group and an oligosaccharide moiety, to an organic
derivatizing agent and an oligosaccharide moiety, to a lipophilic
group and a polymer molecule, etc. The conjugation to two or more
different non-polypeptide moieties may be done simultaneously or
sequentially.
[0122] Methods for Preparing a Conjugate of the Invention
[0123] In the following sections "Conjugation to a lipophilic
compound", "Conjugation to a polymer molecule", "Conjugation to an
oligosaccharide moiety" and "Conjugation to an organic derivatizing
agent" conjugation to specific types of non-polypeptide moieties is
described. In general, a polypeptide conjugate according to the
invention may be produced by culturing an appropriate host cell
under conditions conducive for expression of the polypeptide, and
recovering the polypeptide, wherein a) the polypeptide comprises at
least one N- or O-glycosylation site and the host cell is a
eukaryotic host cell capable of in vivo glycosylation, and/or b)
the polypeptide is subjected to conjugation to a non-polypeptide
moiety in vitro.
[0124] Conjugation to a Lipophilic Compound
[0125] The polypeptide and the lipophilic compound may be
conjugated to each other, either directly or by use of a linker.
The lipophilic compound may be a natural compound such as a
saturated or unsaturated fatty acid, a fatty acid diketone, a
terpene, a prostaglandin, a vitamin, a carotenoid or steroid, or a
synthetic compound such as a carbon acid, an alcohol, an amine and
sulphonic acid with one or more alkyl, aryl, alkenyl or other
multiple unsaturated compounds. The conjugation between the
polypeptide and the lipophilic compound, optionally through a
linker, may be done according to methods known in the art, e.g. as
described by Bodanszky in Peptide Synthesis, John Wiley, New York,
1976 and in WO 96/12505.
[0126] Conjugation to a Polymer Molecule
[0127] The polymer molecule to be coupled to the polypeptide may be
any suitable polymer molecule, such as a natural or synthetic
homo-polymer or heteropolymer, typically with a molecular weight in
the range of about 300-100,000 Da, such as about 500-20,000 Da,
more preferably in the range of about 1000-15,000 Da, even more
preferably in the range of about 2000-12,000 Da, such as about
3000-10,000. When used about polymer molecules herein, the word
"about" indicates an approximate average molecular weight and
reflects the fact that there will normally be a certain molecular
weight distribution in a given polymer preparation.
[0128] Examples of homo-polymers include a polyol (i.e. poly-OH), a
polyamine (i.e. poly-NH.sub.2) and a polycarboxylic acid (i.e.
poly-COOH). A hetero-polymer is a polymer which comprises different
coupling groups, such as a hydroxyl group and an amine group.
[0129] Examples of suitable polymer molecules include polymer
molecules selected from the group consisting of polyalkylene oxide
(PAO), including polyalkylene glycol (PAG), such as linear or
branched polyethylene glycol (PEG) and polypropylene glycol (PPG),
poly-vinyl alcohol (PVA), poly-carboxylate, poly-(vinylpyrolidone),
polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid
anhydride, dextran, including carboxymethyl-dextran, or any other
biopolymer suitable for reducing immunogenicity and/or increasing
functional in vivo half-life and/or serum half-life. Another
example of a polymer molecule is human albumin or another abundant
plasma protein. Generally, polyalkylene glycol-derived polymers are
biocompatible, non-toxic, non-antigenic, non-immunogenic, have
various water solubility properties, and are easily excreted from
living organisms.
[0130] PEG is the preferred polymer molecule, since it has only few
reactive groups capable of cross-linking compared to
polysaccharides such as dextran. In particular, monofunctional PEG,
e.g. methoxypolyethylene glycol (mPEG), is of interest since its
coupling chemistry is relatively simple (only one reactive group is
available for conjugating with attachment groups on the
polypeptide). Consequently, the risk of cross-linking is
eliminated, the resulting polypeptide conjugates are more
homogeneous and the reaction of the polymer molecules with the
polypeptide is easier to control.
[0131] To effect covalent attachment of the polymer molecule(s) to
the polypeptide, the hydroxyl end groups of the polymer molecule
are provided in activated form, i.e. with reactive functional
groups. Suitable activated polymer molecules are commercially
available, e.g. from Shearwater Corp., Huntsville, Ala., USA, or
from PolyMASC Pharmaceuticals plc, UK. Alternatively, the polymer
molecules can be activated by conventional methods known in the
art, e.g. as disclosed in WO 90/13540. Specific examples of
activated linear or branched polymer molecules for use in the
present invention are described in the Shearwater Corp. 1997 and
2000 Catalogs (Functionalized Biocompatible Polymers for Research
and pharmaceuticals, Polyethylene Glycol and Derivatives,
incorporated herein by reference). Specific examples of activated
PEG polymers include the following linear PEGs: NHS-PEG (e.g.
SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and
SCM-PEG), and NOR-PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG,
CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and
branched PEGs such as PEG2-NHS and those disclosed in U.S. Pat. No.
5,932,462 and U.S. Pat. No. 5,643,575, both of which are
incorporated herein by reference. Furthermore, the following
publications, incorporated herein by reference, disclose useful
polymer molecules and/or PEGylation chemistries: U.S. Pat. No.
5,824,778, U.S. Pat. No. 5,476,653, WO 97/32607, EP 229,108, EP
402,378, U.S. Pat. No. 4,902,502, U.S. Pat. No. 5,281,698, U.S.
Pat. No. 5,122,614, U.S. Pat. No. 5,219,564, WO 92/16555, WO
94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO
95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO
97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO
99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO
97/03106, WO 96/21469, WO 95/13312, EP 921 131, U.S. Pat. No.
5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No. 5,629,384, WO
96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat. No.
5,516,673, EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400
472, EP 183 503 and EP 154 316.
[0132] The conjugation of the polypeptide and the activated polymer
molecules is conducted by use of any conventional method, e.g. as
described in the following references (which also describe suitable
methods for activation of polymer molecules): R. F. Taylor, (1991),
"Protein immobilisation. Fundamental and applications", Marcel
Dekker, N.Y.; S. S. Wong, (1992), "Chemistry of Protein Conjugation
and Crosslinking", CRC Press, Boca Raton; G. T. Hermanson et al.,
(1993), "Immobilized Affinity Ligand Techniques", Academic Press,
N.Y.). The skilled person will be aware that the activation method
and/or conjugation chemistry to be used depends on the attachment
group(s) of the polypeptide (examples of which are given further
above), as well as the functional groups of the polymer (e.g. being
amine, hydroxyl, carboxyl, aldehyde, sulfydryl, succinimidyl,
maleimide, vinysulfone or haloacetate). The PEGylation may be
directed towards conjugation to all available attachment groups on
the polypeptide (i.e. such attachment groups that are exposed at
the surface of the polypeptide) or may be directed towards one or
more specific attachment groups, e.g. the N-terminal amino group
(U.S. Pat. No. 5,985,265). Furthermore, the conjugation may be
achieved in one step or in a stepwise manner (e.g. as described in
WO 99/55377).
[0133] It will be understood that the PEGylation is designed so as
to produce the optimal molecule with respect to the number of PEG
molecules attached, the size and form of such molecules (e.g.
whether they are linear or branched), and where in the polypeptide
such molecules are attached. The molecular weight of the polymer to
be used will be chosen taking into consideration the desired effect
to be achieved. For instance, if the primary purpose of the
conjugation is to achieve a conjugate having a high molecular
weight and larger size (e.g. to reduce renal clearance), one may
choose to conjugate either one or a few high molecular weight
polymer molecules or a number of polymer molecules with a smaller
molecular weight to obtain the desired effect. Preferably, however,
several polymer molecules with a lower molecular weight will be
used. This is also the case if a high degree of epitope shielding
is desired. In such cases, 2-8 polymers with a molecular weight of
e.g. about 5,000 Da, such as 3-6 such polymers, may for example be
used. As the examples below illustrate, it may be advantageous to
have a larger number of polymer molecules with a lower molecular
weight (e.g. 4-6 with a MW of 5000) compared to a smaller number of
polymer molecules with a higher molecular weight (e.g. 1-3 with a
MW of 12,000-20,000) in terms of improving the functional in vivo
half-life of the polypeptide conjugate, even where the total
molecular weight of the attached polymer molecules in the two cases
is the same or similar. It is believed that the presence of a
larger number of smaller polymer molecules provides the polypeptide
with a larger diameter or apparent size than e.g. a single yet
larger polymer molecule, at least when the polymer molecules are
relatively uniformly distributed on the polypeptide surface.
[0134] It has further been found that advantageous results are
obtained when the apparent size (also referred to as the "apparent
molecular weight" or "apparent mass") of at least a major portion
of the conjugate of the invention is at least about 50 kDa,
preferably at least about 55 kDa, more preferably at least about 60
kDa, e.g. at least about 66 kDa. This is believed to be due to the
fact that renal clearance is substantially eliminated for
conjugates having a sufficiently large apparent size. In the
present context, the "apparent size" of a G-CSF conjugate or
polypeptide is determined by the SDS-PAGE method described in the
examples section below.
[0135] The use of the term "major portion" is related to the fact
that the polypeptide conjugates of the invention will typically
comprise individual conjugates having varying numbers of
non-polypeptide moieties attached. For example, a given polypeptide
subjected to PEGylation under a given set of PEGylation conditions
may result in a composition in which most of the individual
polypeptide conjugates have e.g. between 3 and 5 PEG groups
attached, with a majority of the conjugates having 4 PEG groups
attached. It will be clear that the apparent molecular weight of
these individual conjugate molecules will vary. In this example, if
we assume that a G-CSF polypeptide is conjugated to PEG groups with
a MW of 5 kDa, conjugates having only 3 PEG groups attached will be
seen on an SDS-PAGE gel as a band that is likely to have an
apparent molecular weight of less than about 50 kDa, while
conjugates having 4 or 5 PEG groups attached will result in bands
with progressively higher apparent molecular weights that most
likely all are greater than about 50 kDa. Thus, in this example
there would be 3 major bands on an SDS-PAGE gel, corresponding to
conjugates with 3, 4 or 5 attached PEG groups, respectively. The
term "major portion" in the context of the present specification
and claims is therefore intended to refer to the fact that at least
one of these major bands on an SDS-PAGE gel will correspond to the
indicated minimum apparent molecular weight.
[0136] Preferably, at least 50% of the individual conjugate
molecules will have a minimum apparent size as described above.
More preferably, at least 60% of the individual conjugate molecules
with have such a minimum apparent size, still more preferably at
least 70%, 75%, 80% or 85%. Most preferably, at least 90% of the
individual conjugate molecules will have a minimum apparent size as
described above, i.e. at least 50 kDa and preferably higher, such
as at least 55 kDa or 60 kDa.
[0137] It will be understood that the apparent size in kDa of a
conjugate or polypeptide is not necessarily the same as the actual
molecular weight of the conjugate or polypeptide. Rather, the
apparent size is a reflection of both the actual molecular weight
and the overall bulk. Since, in most cases, attachment of one or
more PEG groups or other non-polypeptide moieties will result in a
relatively large increase of the bulk of the polypeptide to which
such moieties are attached, the polypeptide conjugates of the
invention will normally have an apparent size that exceeds the
actual molecular weight of the conjugate. Therefore, in connection
with renal clearance, a conjugate of the invention can easily
exhibit properties characteristic of a polypeptide with a molecular
weight above e.g. 66 kDa (corresponding to the apparent size) but
have an actual molecular weight well below 66 kDa. This effect on
apparent size is believed to be responsible for the observation
that attachment of, for example, four PEG groups each having a
molecular weight of 5 kDa provides results that are superior to a
corresponding polypeptide with a single 20 kDa PEG group
attached.
[0138] While conjugation of only a single polymer molecule to a
single attachment group on the protein is not preferred, in the
event that only one polymer molecule is attached, it will generally
be advantageous that the polymer molecule, which may be linear or
branched, has a relatively high molecular weight, e.g. about 20
kDa.
[0139] In a further preferred embodiment, the conjugates of the
invention have 1) at least a major portion with an apparent
molecular weight of at least about 50 kDa and 2) a reduced in vitro
bioactivity (reduced receptor binding affinity) compared to hG-CSF
as described above. It has been found that such conjugates have
both a low renal clearance as a result of the large apparent size
and a low receptor-mediated clearance as a result of the low in
vitro bioactivity (low receptor binding affinity). The overall
result is excellent performance in terms of effective stimulation
of neutrophils together with a significantly increased in vivo
half-life and thus a long duration of action that provides
important clinical advantages.
[0140] Normally, the polymer conjugation is performed under
conditions aiming at reacting as many of the available polymer
attachment groups as possible with polymer molecules. This is
achieved by means of a suitable molar excess of the polymer in
relation to the polypeptide (number of attachment sites). Typical
molar ratios of activated polymer molecules to polypeptide
attachment sites are up to about 1000-1, such as up to about 200-1
or up to about 100-1. In some cases, the ratio may be somewhat
lower, however, such as up to about 50-1, 10-1 or 5-1, e.g. if a
lower degree of polymer attachment is desired.
[0141] It is also contemplated according to the invention to couple
the polymer molecules to the polypeptide through a linker. Suitable
linkers are well known to the skilled person. A preferred example
is cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem.,
252, 3578-3581; U.S. Pat. No. 4,179,337; Shafer et al., (1986), J.
Polym. Sci. Polym. Chem. Ed., 24, 375-378.
[0142] Subsequent to the conjugation residual activated polymer
molecules are blocked according to methods known in the art, e.g.
by addition of primary amine to the reaction mixture, and the
resulting inactivated polymer molecules are removed by a suitable
method (see Materials and Methods).
[0143] In a preferred embodiment, the polypeptide conjugate of the
invention comprises a PEG molecule attached to some, most or
preferably substantially all of the lysine residues in the
polypeptide available for PEGylation, in particular a linear or
branched PEG molecule, e.g. with a molecular weight of about 1-15
kDa, typically about 2-12 kDa, such as about 3-10 kDa, e.g. about 5
or 6 kDa.
[0144] It will be understood that depending on the circumstances,
e.g. the amino acid sequence of the polypeptide, the nature of the
activated PEG compound being used and the specific PEGylation
conditions, including the molar ratio of PEG to polypeptide,
varying degrees of PEGylation may be obtained, with a higher degree
of PEGylation generally being obtained with a higher ratio of PEG
to polypeptide. The PEGylated polypeptides resulting from any given
PEGylation process will, however, normally comprise a stochastic
distribution of polypeptide conjugates having slightly different
degrees of PEGylation. If desired, such a mixture of polypeptide
species having different numbers of PEG moieties attached may be
subjected to purification, e.g. using the methods described in the
examples below, to obtain a product having a more uniform degree of
PEGylation.
[0145] In yet another embodiment, the polypeptide conjugate of the
invention may comprise a PEG molecule attached to the lysine
residues in the polypeptide available for PEGylation, and in
addition to the N-terminal amino acid residue of the
polypeptide.
[0146] Coupling to an Oligosaccharide Moiety
[0147] The conjugation to an oligosaccharide moiety may take place
in vivo or in vitro. In order to achieve in vivo glycosylation of a
G-CSF molecule comprising one or more glycosylation sites the
nucleotide sequence encoding the polypeptide must be inserted in a
glycosylating, eukaryotic expression host. The expression host cell
may be selected from fungal (filamentous fungal or yeast), insect
or animal cells or from transgenic plant cells. In one embodiment
the host cell is a mammalian cell, such as a CHO cell, BHK or HEK,
e.g. HEK 293, cell, or an insect cell, such as an SF9 cell, or a
yeast cell, e.g. S. cerevisiae or Pichia pastoris, or any of the
host cells mentioned hereinafter. Covalent in vitro coupling of
glycosides (such as dextran) to amino acid residues of the
polypeptide may also be used, e.g. as described in WO 87/05330 and
in Aplin et al., CRC Crit Rev. Biochem., pp. 259-306, 1981.
[0148] The in vitro coupling of oligosaccharide moieties or PEG to
protein- and peptide-bound Gln-residues can be carried out by
transglutaminases (TG'ases). Transglutaminases catalyze the
transfer of donor amine-groups to protein- and peptide-bound
Gln-residues in a so-called cross-linking reaction. The donor-amine
groups can be protein- or peptide-bound e.g. as the
.epsilon.-amino-group in Lys-residues or can be part of a small or
large organic molecule. An example of a small organic molecule
functioning as an amino-donor in TG'ase-catalyzed cross-linking is
putrescine (1,4-diaminobutane). An example of a larger organic
molecule functioning as an amino-donor in TG'ase-catalyzed
cross-linking is an amine-containing PEG (Sato et al., Biochemistry
35, 13072-13080).
[0149] TG'ases are in general highly specific enzymes, and not
every Gln-residue exposed on the surface of a protein is accessible
to TG'ase-catalyzed cross-linking to amino-containing substances.
On the contrary, only a few Gln-residues function naturally as
TG'ase substrates, but the exact parameters governing which
Gln-residues are good TG'ase substrates remain unknown. Thus, in
order to render a protein susceptible to TG'ase-catalyzed
cross-linking reactions it is often a prerequisite to add at
convenient positions stretches of amino acid sequence known to
function very well as TG'ase substrates. Several amino acid
sequences are known to be or to contain excellent natural TG'ase
substrates e.g. substance P, elafin, fibrinogen, fibronectin,
.alpha..sub.2-plasmin inhibitor, .alpha.-caseins, and
.beta.-caseins.
[0150] Coupling to an Organic Derivatizing Agent
[0151] Covalent modification of the polypeptide exhibiting G-CSF
activity may be performed by reacting one or more attachment groups
of the polypeptide with an organic derivatizing agent. Suitable
derivatizing agents and methods are well known in the art. For
example, cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(4-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues are
derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0
because this agent is relatively specific for the histidyl side
chain. Para-bromophenacyl bromide is also useful. The reaction is
preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl
and amino terminal residues are reacted with succinic or other
carboxylic acid anhydrides. Derivatization with these agents has
the effect of reversing the charge of the lysinyl residues. Other
suitable reagents for derivatizing .alpha.-amino-containing
residues include imidoesters such as methyl picolinimidate,
pyridoxal phosphate, pyridoxal, chloroborohydride,
trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione and
transaminase-catalyzed reaction with glyoxylate. Arginyl residues
are modified by reaction with one or several conventional reagents,
among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione,
and ninhydrin. Derivatization of arginine residues requires that
the reaction be performed in alkaline conditions because of the
high pKa of the guanidine functional group.
[0152] Furthermore, these reagents may react with the groups of
lysine as well as the arginine guanidino group. Carboxyl side
groups (aspartyl or glutamyl) are selectively modified by reaction
with carbodiimides (R--N.dbd.C.dbd.N--R'), where R and R' are
different alkyl groups, such as
1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0153] Blocking of the Functional Site
[0154] It has been reported that excessive polymer conjugation can
lead to a loss of activity of the polypeptide to which the polymer
is conjugated. This problem can be eliminated by e.g. removal of
attachment groups located at the functional site or by blocking the
functional site prior to conjugation so that the functional site is
blocked during conjugation. The latter strategy constitutes a
further embodiment of the invention (the first strategy being
exemplified further above, e.g. by removal of lysine residues which
may be located close to the functional site). More specifically,
according to the second strategy the conjugation between the
polypeptide and the non-polypeptide moiety is conducted under
conditions where the functional site of the polypeptide is blocked
by a helper molecule capable of binding to the functional site of
the polypeptide.
[0155] Preferably, the helper molecule is one which specifically
recognizes a functional site of the polypeptide, such as a
receptor, in particular the G-CSF receptor or a part of the G-CSF
receptor.
[0156] Alternatively, the helper molecule may be an antibody, in
particular a monoclonal antibody recognizing the polypeptide
exhibiting G-CSF activity. In particular, the helper molecule may
be a neutralizing monoclonal antibody.
[0157] The polypeptide is allowed to interact with the helper
molecule before effecting conjugation. This ensures that the
functional site of the polypeptide is shielded or protected and
consequently unavailable for derivatization by the non-polypeptide
moiety such as a polymer. Following its elution from the helper
molecule, the conjugate between the non-polypeptide moiety and the
polypeptide can be recovered with at least a partially preserved
functional site.
[0158] The subsequent conjugation of the polypeptide having a
blocked functional site to a polymer, a lipophilic compound, an
oligosaccharide moiety, an organic derivatizing agent or any other
compound is conducted in the normal way, e.g. as described in the
sections above entitled "Conjugation to . . . ".
[0159] Irrespective of the nature of the helper molecule to be used
to shield the functional site of the polypeptide from conjugation,
it is desirable that the helper molecule is free of or comprises
only a few attachment groups for the non-polypeptide moiety of
choice in part(s) of the molecule where the conjugation to such
groups would hamper desorption of the conjugated polypeptide from
the helper molecule. Hereby, selective conjugation to attachment
groups present in non-shielded parts of the polypeptide can be
obtained and it is possible to reuse the helper molecule for
repeated cycles of conjugation. For instance, if the
non-polypeptide moiety is a polymer molecule such as PEG, which has
the epsilon amino group of a lysine or N-terminal amino acid
residue as an attachment group, it is desirable that the helper
molecule is substantially free of conjugatable epsilon amino
groups, preferably free of any epsilon amino groups. Accordingly,
in a preferred embodiment the helper molecule is a protein or
peptide capable of binding to the functional site of the
polypeptide, which protein or peptide is free of any conjugatable
attachment groups for the non-polypeptide moiety of choice.
[0160] Of particular interest in connection with the embodiment of
the present invention wherein the polypeptide conjugates are
prepared from a diversified population of nucleotide sequences
encoding a polypeptide of interest, the blocking of the functional
group is effected in microtiter plates prior to conjugation, for
instance by plating the expressed polypeptide variant in a
microtiter plate containing an immobilized blocking group such as a
receptor, an antibody or the like.
[0161] In a further embodiment the helper molecule is first
covalently linked to a solid phase such as column packing
materials, for instance Sephadex or agarose beads, or a surface,
e.g. a reaction vessel. Subsequently, the polypeptide is loaded
onto the column material carrying the helper molecule and
conjugation carried out according to methods known in the art, e.g.
as described in the sections above entitled "Conjugation to . . .
". This procedure allows the polypeptide conjugate to be separated
from the helper molecule by elution. The polypeptide conjugate is
eluted by conventional techniques under physico-chemical conditions
that do not lead to a substantive degradation of the polypeptide
conjugate. The fluid phase containing the polypeptide conjugate is
separated from the solid phase to which the helper molecule remains
covalently linked. The separation can be achieved in other ways:
For instance, the helper molecule may be derivatized with a second
molecule (e.g. biotin) that can be recognized by a specific binder
(e.g. streptavidin). The specific binder may be linked to a solid
phase, thereby allowing the separation of the polypeptide conjugate
from the helper molecule-second molecule complex through passage
over a second helper-solid phase column which will retain, upon
subsequent elution, the helper molecule-second molecule complex,
but not the polypeptide conjugate. The polypeptide conjugate may be
released from the helper molecule in any appropriate fashion.
Deprotection may be achieved by providing conditions in which the
helper molecule dissociates from the functional site of the G-CSF
to which it is bound. For instance, a complex between an antibody
to which a polymer is conjugated and an anti-idiotypic antibody can
be dissociated by adjusting the pH to an acid or alkaline pH.
[0162] Conjugation of a Tagged Polypeptide
[0163] In an alternative embodiment the polypeptide is expressed as
a fusion protein with a tag, i.e. an amino acid sequence or peptide
stretch made up of typically 1-30, such as 1-20 amino acid
residues. Besides allowing for fast and easy purification, the tag
is a convenient tool for achieving conjugation between the tagged
polypeptide and the non-polypeptide moiety. In particular, the tag
may be used for achieving conjugation in microtiter plates or other
carriers, such as paramagnetic beads, to which the tagged
polypeptide can be immobilized via the tag. The conjugation to the
tagged polypeptide in e.g. microtiter plates has the advantage that
the tagged polypeptide can be immobilized in the microtiter plates
directly from the culture broth (in principle without any
purification) and subjected to conjugation. Thereby, the total
number of process steps (from expression to conjugation) can be
reduced. Furthermore, the tag may function as a spacer molecule,
ensuring an improved accessibility to the immobilized polypeptide
to be conjugated. The conjugation using a tagged polypeptide may be
to any of the non-polypeptide moieties disclosed herein, e.g. to a
polymer molecule such as PEG.
[0164] The identity of the specific tag to be used is not critical
as long as the tag is capable of being expressed with the
polypeptide and is capable of being immobilized on a suitable
surface or carrier material. A number of suitable tags are
commercially available, e.g. from Unizyme Laboratories, Denmark.
For instance, the tag may consist of any of the following
sequences:
3 Met-Lys-His-Gln-His-Gln-His-Gln-His- (SEQ ID NO:5)
Gln-His-Gln-His-Gln-Gln His-His-His-His-His-His (SEQ ID NO:9)
Met-Lys-His-His-His-His-His-His (SEQ ID NO:10)
Met-Lys-His-His-Ala-His-His-Gln-His- (SEQ ID NO:11) His
Met-Lys-His-Gln-His-Gln-His-Gln-His- (SEQ ID NO:12)
Gln-His-Gln-His-Gln
[0165] or any of the following:
4 (SEQ ID NO:13) EQKLISEEDL; a C-terminal tag described in Mol.
Cell. Biol. 5:3610-16, 1985 (SEQ ID NO:14) DYKDDDDK; a C- or
N-terminal tag (SEQ ID NO:15) YPYDVPDYA
[0166] Antibodies against the above tags are commercially
available, e.g. from ADI, Aves Lab and Research Diagnostics.
[0167] A convenient method for using a tagged polypeptide for
PEGylation is given in the Materials and Methods section below. The
subsequent cleavage of the tag from the polypeptide may be achieved
by use of commercially available enzymes.
[0168] Methods for Preparing a Polypeptide of the Invention or the
Polypeptide Part of the Conjugate of the Invention
[0169] The polypeptide of the present invention or the polypeptide
part of a conjugate of the invention, optionally in glycosylated
form, may be produced by any suitable method known in the art. Such
methods include constructing a nucleotide sequence encoding the
polypeptide and expressing the sequence in a suitable transformed
or transfected host. However, polypeptides of the invention may be
produced, albeit less efficiently, by chemical synthesis or a
combination of chemical synthesis or a combination of chemical
synthesis and recombinant DNA technology.
[0170] A nucleotide sequence encoding a polypeptide or the
polypeptide part of a conjugate of the invention may be constructed
by isolating or synthesizing a nucleotide sequence encoding the
parent G-CSF, such as hG-CSF with the amino acid sequence shown in
SEQ ID NO:1, and then changing the nucleotide sequence so as to
effect introduction (i.e. insertion or substitution) or deletion
(i.e. removal or substitution) of the relevant amino acid
residue(s).
[0171] The nucleotide sequence is conveniently modified by
site-directed mutagenesis in accordance with conventional methods.
Alternatively, the nucleotide sequence is prepared by chemical
synthesis, e.g. by using an oligonucleotide synthesizer, wherein
oligonucleotides are designed based on the amino acid sequence of
the desired polypeptide, and preferably selecting those codons that
are favored in the host cell in which the recombinant polypeptide
will be produced. For example, several small oligonucleotides
coding for portions of the desired polypeptide may be synthesized
and assembled by PCR, ligation or ligation chain reaction (LCR)
(Barany, PNAS 88:189-193, 1991). The individual oligonucleotides
typically contain 5' or 3' overhangs for complementary
assembly.
[0172] Alternative nucleotide sequence modification methods are
available for producing polypeptide variants for high throughput
screening, for instance methods which involve homologous cross-over
such as disclosed in U.S. Pat. No. 5,093,257, and methods which
involve gene shuffling, i.e. recombination between two or more
homologous nucleotide sequences resulting in new nucleotide
sequences having a number of nucleotide alterations when compared
to the starting nucleotide sequences. Gene shuffling (also known as
DNA shuffling) involves one or more cycles of random fragmentation
and reassembly of the nucleotide sequences, followed by screening
to select nucleotide sequences encoding polypeptides with desired
properties. In order for homology-based nucleic acid shuffling to
take place, the relevant parts of the nucleotide sequences are
preferably at least 50% identical, such as at least 60% identical,
more preferably at least 70% identical, such as at least 80%
identical. The recombination can be performed in vitro or in
vivo.
[0173] Examples of suitable in vitro gene shuffling methods are
disclosed by Stemmer et al. (1994), Proc. Natl. Acad. Sci. USA;
vol. 91, pp. 10747-10751; Stemmer (1994), Nature, vol. 370, pp.
389-391; Smith (1994), Nature vol. 370, pp. 324-325; Zhao et al.,
Nat. Biotechnol. 1998, March; 16(3): 258-61; Zhao H. and Arnold, F
B, Nucleic Acids Research, 1997, Vol. 25. No. 6 pp. 1307-1308; Shao
et al., Nucleic Acids Research 1998, Jan. 15; 26(2): pp. 681-83;
and WO 95/17413. An example of a suitable in vivo shuffling method
is disclosed in WO 97/07205. Other techniques for mutagenesis of
nucleic acid sequences by in vitro or in vivo recombination are
disclosed e.g. in WO 97/20078 and U.S. Pat. No. 5,837,458. Examples
of specific shuffling techniques include "family shuffling",
"synthetic shuffling" and "in silico shuffling". Family shuffling
involves subjecting a family of homologous genes from different
species to one or more cycles of shuffling and subsequent screening
or selection. Family shuffling techniques are disclosed e.g. by
Crameri et al. (1998), Nature, vol. 391, pp. 288-291; Christians et
al. (1999), Nature Biotechnology, vol. 17, pp. 259-264; Chang et
al. (1999), Nature Biotechnology, vol. 17, pp. 793-797; and Ness et
al. (1999), Nature Biotechnology, vol. 17, 893-896. Synthetic
shuffling involves providing libraries of overlapping synthetic
oligonucleotides based e.g. on a sequence alignment of homologous
genes of interest. The synthetically generated oligonucleotides are
recombined, and the resulting recombinant nucleic acid sequences
are screened and if desired used for further shuffling cycles.
Synthetic shuffling techniques are disclosed in WO 00/42561. In
silico shuffling refers to a DNA shuffling procedure which is
performed or modelled using a computer system, thereby partly or
entirely avoiding the need for physically manipulating nucleic
acids. Techniques for in silico shuffling are disclosed in WO
00/42560.
[0174] Once assembled (by synthesis, site-directed mutagenesis or
another method), the nucleotide sequence encoding the polypeptide
is inserted into a recombinant vector and operably linked to
control sequences necessary for expression of the G-CSF in the
desired transformed host cell.
[0175] It should of course be understood that not all vectors and
expression control sequences function equally well to express the
nucleotide sequence encoding a polypeptide described herein.
Neither will all hosts function equally well with the same
expression system. However, one of skill in the art may make a
selection among these vectors, expression control sequences and
hosts without undue experimentation. For example, in selecting a
vector, the host must be considered because the vector must
replicate in it or be able to integrate into the chromosome. The
vector's copy number, the ability to control that copy number, and
the expression of any other proteins encoded by the vector, such as
antibiotic markers, should also be considered. In selecting an
expression control sequence, a variety of factors should also be
considered. These include, for example, the relative strength of
the sequence, its controllability, and its compatibility with the
nucleotide sequence encoding the polypeptide, particularly as
regards potential secondary structures. Hosts should be selected by
consideration of their compatibility with the chosen vector, the
toxicity of the product coded for by the nucleotide sequence, their
secretion characteristics, their ability to fold the polypeptide
correctly, their fermentation or culture requirements, and the ease
of purification of the products coded for by the nucleotide
sequence.
[0176] The recombinant vector may be an autonomously replicating
vector, i.e. a vector, which exists as an extrachromosomal entity,
the replication of which is independent of chromosomal replication,
e.g. a plasmid. Alternatively, the vector is one which, when
introduced into a host cell, is integrated into the host cell
genome and replicated together with the chromosome(s) into which it
has been integrated.
[0177] The vector is preferably an expression vector in which the
nucleotide sequence encoding the polypeptide of the invention is
operably linked to additional segments required for transcription
of the nucleotide sequence. The vector is typically derived from
plasmid or viral DNA. A number of suitable expression vectors for
expression in the host cells mentioned herein are commercially
available or described in the literature. Useful expression vectors
for eukaryotic hosts include, for example, vectors comprising
expression control sequences from SV40, bovine papilloma virus,
adenovirus and cytomegalovirus. Specific vectors are, e.g.,
pcDNA3.1 (+).backslash.Hyg (Invitrogen, Carlsbad, Calif., USA) and
pCI-neo (Stratagene, La Jolla, Calif., USA). Useful expression
vectors for yeast cells include the 2.mu. plasmid and derivatives
thereof, the POT1 vector (U.S. Pat. No. 4,931,373), the pJSO37
vector described in Okkels, Ann. New York Acad. Sci. 782, 202-207,
1996, and pPICZ A, B or C (Invitrogen). Useful vectors for insect
cells include pVL941, pBG311 (Cate et al., "Isolation of the Bovine
and Human Genes for Mullerian Inhibiting Substance And Expression
of the Human Gene In Animal Cells", Cell, 45, pp. 685-98 (1986),
pBluebac 4.5 and pMelbac (both available from Invitrogen). Useful
expression vectors for bacterial hosts include known bacterial
plasmids, such as plasmids from E. coli, including pBR322, pET3a
and pET12a (both from Novagen Inc., WI, USA), wider host range
plasmids, such as RP4, phage DNAs, e.g. the numerous derivatives of
phage lambda, e.g. NM989, and other DNA phages, such as M13 and
filamentous single stranded DNA phages.
[0178] Other vectors for use in this invention include those that
allow the nucleotide sequence encoding the polypeptide to be
amplified in copy number. Such amplifiable vectors are well known
in the art. They include, for example, vectors able to be amplified
by DHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461,
Kaufman and Sharp, "Construction Of A Modular Dihydrafolate
Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient
Expression", Mol. Cell. Biol., 2, pp. 1304-19 (1982)) and glutamine
synthetase ("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464
and EP 338,841).
[0179] The recombinant vector may further comprise a DNA sequence
enabling the vector to replicate in the host cell in question. An
example of such a sequence (when the host cell is a mammalian cell)
is the SV40 origin of replication. When the host cell is a yeast
cell, suitable sequences enabling the vector to replicate are the
yeast plasmid 2.mu. replication genes REP 1-3 and origin of
replication.
[0180] The vector may also comprise a selectable marker, e.g. a
gene whose product complements a defect in the host cell, such as
the gene coding for dihydrofolate reductase (DHFR) or the
Schizosaccharomyces pombe TPI gene (described by P. R. Russell,
Gene 40, 1985, pp. 125-130), or one which confers resistance to a
drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol,
neomycin, hygromycin or methotrexate. For Saccharomyces cerevisiae,
selectable markers include ura3 and leu2. For filamentous fungi,
selectable markers include amdS, pyrG, arcB, niaD and sC.
[0181] The term "control sequences" is defined herein to include
all components which are necessary or advantageous for the
expression of the polypeptide of the invention. Each control
sequence may be native or foreign to the nucleic acid sequence
encoding the polypeptide. Such control sequences include, but are
not limited to, a leader sequence, polyadenylation sequence,
propeptide sequence, promoter, enhancer or upstream activating
sequence, signal peptide sequence, and transcription terminator. At
a minimum, the control sequences include a promoter.
[0182] A wide variety of expression control sequences may be used
in the present invention. Such useful expression control sequences
include the expression control sequences associated with structural
genes of the foregoing expression vectors as well as any sequence
known to control the expression of genes of prokaryotic or
eukaryotic cells or their viruses, and various combinations
thereof.
[0183] Examples of suitable control sequences for directing
transcription in mammalian cells include the early and late
promoters of SV40 and adenovirus, e.g. the adenovirus 2 major late
promoter, the MT-1 (metallothionein gene) promoter, the human
cytomegalovirus immediate-early gene promoter (CMV), the human
elongation factor 1.alpha. (EF-1.alpha.) promoter, the Drosophila
minimal heat shock protein 70 promoter, the Rous Sarcoma Virus
(RSV) promoter, the human ubiquitin C (UbC) promoter, the human
growth hormone terminator, SV40 or adenovirus E1b region
polyadenylation signals and the Kozak consensus sequence (Kozak, M.
J Mol Biol 1987 Aug. 20;196(4):947-50).
[0184] In order to improve expression in mammalian cells a
synthetic intron may be inserted in the 5' untranslated region of
the nucleotide sequence encoding the polypeptide. An example of a
synthetic intron is the synthetic intron from the plasmid pCI-Neo
(available from Promega Corporation, WI, USA).
[0185] Examples of suitable control sequences for directing
transcription in insect cells include the polyhedrin promoter, the
P10 promoter, the Autographa californica polyhedrosis virus basic
protein promoter, the baculovirus immediate early gene 1 promoter,
the baculovirus 39K delayed-early gene promoter, and the SV40
polyadenylation sequence. Examples of suitable control sequences
for use in yeast host cells include the promoters of the yeast
.alpha.-mating system, the yeast triose phosphate isomerase (TPI)
promoter, promoters from yeast glycolytic genes or is alcohol
dehydrogenase genes, the ADH2-4c promoter, and the inducible GAL
promoter. Examples of suitable control sequences for use in
filamentous fungal host cells include the ADH3 promoter and
terminator, a promoter derived from the genes encoding Aspergillus
oryzae TAKA amylase triose phosphate isomerase or alkaline
protease, an A. niger .alpha.-amylase, A. niger or A. nidulans
glucoamylase, A. nidulans acetamidase, Rhizomucor miehei aspartic
proteinase or lipase, the TPI1 terminator and the ADH3 terminator.
Examples of suitable control sequences for use in bacterial host
cells include promoters of the lac system, the trp system, the TAC
or TRC system, and the major promoter regions of phage lambda.
[0186] The presence or absence of a signal peptide will, e.g.,
depend on the expression host cell used for the production of the
polypeptide to be expressed (whether it is an intracellular or
extracellular polypeptide) and whether it is desirable to obtain
secretion. For use in filamentous fungi, the signal peptide may
conveniently be derived from a gene encoding an Aspergillus sp.
amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase
or protease or a Humicola lanuginosa lipase. The signal peptide is
preferably derived from a gene encoding A. oryzae TAKA amylase, A.
niger neutral .alpha.-amylase, A. niger acid-stable amylase, or A.
niger glucoamylase. For use in insect cells, the signal peptide may
conveniently be derived from an insect gene (cf. WO 90/05783), such
as the Lepidopteran manduca sexta adipokinetic hormone precursor,
(cf. U.S. Pat. No. 5,023,328), the honeybee melittin (Invitrogen),
ecdysteroid UDPglucosyltransferase (egt) (Murphy et al., Protein
Expression and Purification 4, 349-357 (1993) or human pancreatic
lipase (hpl) (Methods in Enzymology 284, pp. 262-272, 1997). A
preferred signal peptide for use in mammalian cells is that of
hG-CSF or the murine Ig kappa light chain signal peptide (Coloma, M
(1992) J. I=m. Methods 152:89-104). For use in yeast cells suitable
signal peptides have been found to be the .alpha.-factor signal
peptide from S. cereviciae (cf. U.S. Pat. No. 4,870,008), a
modified carboxypeptidase signal peptide (cf. L. A. Valls et al.,
Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO
87/02670), the yeast aspartic protease 3 (YAP3) signal peptide (cf.
M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137), and the
synthetic leader sequence TA57 (WO98/32867). For use in E. coli
cells a suitable signal peptide has been found to be the signal
peptide ompA.
[0187] The nucleotide sequence of the invention encoding a
polypeptide exhibiting G-CSF activity, whether prepared by
site-directed mutagenesis, synthesis, PCR or other methods, may
optionally also include a nucleotide sequence that encodes a signal
peptide. The signal peptide is present when the polypeptide is to
be secreted from the cells in which it is expressed. Such signal
peptide, if present, should be one recognized by the cell chosen
for expression of the polypeptide. The signal peptide may be
homologous (e.g. be that normally associated with hG-CSF) or
heterologous (i.e. originating from another source than hG-CSF) to
the polypeptide or may be homologous or heterologous to the host
cell, i.e. be a signal peptide normally expressed from the host
cell or one which is not normally expressed from the host cell.
Accordingly, the signal peptide may be prokaryotic, e.g. derived
from a bacterium such as E. coli, or eukaryotic, e.g. derived from
a mammalian, or insect or yeast cell.
[0188] Any suitable host may be used to produce the polypeptide or
polypeptide part of the conjugate of the invention, including
bacteria, fungi (including yeasts), plant, insect, mammal, or other
appropriate animal cells or cell lines, as well as transgenic
animals or plants. Examples of bacterial host cells include
gram-positive bacteria such as strains of Bacillus, e.g. B. brevis
or B. subtilis, or Streptomyces, or gram-negative bacteria, such as
strains of E. coli or Pseudomonas. The introduction of a vector
into a bacterial host cell may, for instance, be effected by
protoplast transformation (see, e.g., Chang and Cohen, 1979,
Molecular General Genetics 168: 111-115), using competent cells
(see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:
823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular
Biology 56: 209-221), electroporation (see, e.g., Shigekawa and
Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g.,
Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).
Examples of suitable filamentous fungal host cells include strains
of Aspergillus, e.g. A. oryzae, A. niger, or A. nidulans, Fusarium
or Trichoderma. Fungal cells may be transformed by a process
involving protoplast formation, transformation of the protoplasts,
and regeneration of the cell wall in a manner known per se.
Suitable procedures for transformation of Aspergillus host cells
are described in EP 238 023 and U.S. Pat. No. 5,679,543. Suitable
methods for transforming Fusarium species are described by
Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Examples
of suitable yeast host cells include strains of Saccharomyces, e.g.
S. cerevisiae, Schizosaccharomyces, Klyveromyces, Pichia, such as
P. pastoris or P. methanolica, Hansenula, such as H. polymorpha or
Yarrowia. Yeast may be transformed using the procedures described
by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,
editors, Guide to Yeast Genetics and Molecular Biology, Methods in
Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York;
Ito et al., 1983, Journal of Bacteriology 153: 163; Hinnen et al.,
1978, Proceedings of the National Academy of Sciences USA 75: 1920:
and as disclosed by Clontech Laboratories, Inc, Palo Alto, Calif.,
USA (in the product protocol for the Yeastmaker.TM. Yeast
Transformation System Kit). Examples of suitable insect host cells
include a Lepidoptora cell line, such as Spodoptera frugiperda (Sf9
or Sf21) or Trichoplusioa ni cells (High Five) (U.S. Pat. No.
5,077,214). Transformation of insect cells and production of
heterologous polypeptides therein may be performed as described by
Invitrogen. Examples of suitable mammalian host cells include
Chinese hamster ovary (CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61),
Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7
(ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney
(BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human
cells (e.g. HEK 293 (ATCC CRL-1573)), as well as plant cells in
tissue culture. Additional suitable cell lines are known in the art
and available from public depositories such as the American Type
Culture Collection, Rockville, Md. Methods for introducing
exogeneous DNA into mammalian host cells include calcium
phosphate-mediated transfection, electroporation, DEAE-dextran
mediated transfection, liposome-mediated transfection, viral
vectors and the transfection method described by Life Technologies
Ltd, Paisley, UK using Lipofectamin 2000. These methods are well
known in the art and e.g. described by Ausbel et al. (eds.), 1996,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, USA. The cultivation of mammalian cells is conducted
according to established methods, e.g. as disclosed in (Animal Cell
Biotechnology, Methods and Protocols, Edited by Nigel Jenkins,
1999, Human Press Inc, Totowa, N.J., USA and Harrison M A and Rae I
F, General Techniques of Cell Culture, Cambridge University Press
1997).
[0189] In the production methods of the present invention, the
cells are cultivated in a nutrient medium suitable for production
of the polypeptide using methods known in the art. For example, the
cell may be cultivated by shake flask cultivation, small-scale or
large-scale fermentation (including continuous, batch, fed-batch,
or solid state fermentations) in laboratory or industrial
fermenters performed in a suitable medium and under conditions
allowing the polypeptide to be expressed and/or isolated. The
cultivation takes place in a suitable nutrient medium comprising
carbon and nitrogen sources and inorganic salts, using procedures
known in the art. Suitable media are available from commercial
suppliers or may be prepared according to published compositions
(e.g., in catalogues of the American Type Culture Collection). If
the polypeptide is secreted into the nutrient medium, the
polypeptide can be recovered directly from the medium. If the
polypeptide is not secreted, it can be recovered from cell
lysates.
[0190] The resulting polypeptide may be recovered by methods known
in the art. For example, the polypeptide may be recovered from the
nutrient medium by conventional procedures including, but not
limited to, centrifugation, filtration, extraction, spray drying,
evaporation, or precipitation.
[0191] The polypeptides may be purified by a variety of procedures
known in the art including, but not limited to, chromatography
(e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and
size exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing), differential solubility (e.g., ammonium
sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein
Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989). Specific methods for purifying polypeptides
exhibiting G-CSF activity are described by D. Metcalf and N. A.
Nicola in The hemopoietic colony-stimulating factors, p. 50-51,
Cambridge University Press (1995), by C. S. Bae et al., Appl.
Microbiol. Biotechnol, 52:338-344 (1999) and in U.S. Pat. No.
4,810,643.
[0192] Pharmaceutical Composition of the Invention and its Use
[0193] In a further aspect, the present invention comprises a
composition comprising a polypeptide or conjugate as described
herein and at least one pharmaceutically acceptable carrier or
excipient.
[0194] The polypeptide, the conjugate or the pharmaceutical
composition according to the invention may be used for the
manufacture of a medicament for treatment of diseases, in
particular prevention of infection in cancer patients undergoing
certain types of chemotherapy, radiation therapy and bone marrow
transplantations, mobilisation of progenitor cells for collection
in peripheral blood progenitor cell transplantations, treatment of
severe chronic or relative leukopenia, treatment of patients with
acute myeloid leukaemia, treatment of AIDS or other
immunodeficiency diseases, and for antifungal therapy, in
particular for treatment of systemic or invasive candidiasis.
[0195] In another aspect the polypeptide, the conjugate or the
pharmaceutical composition according to the invention is used in a
method of treating a mammal having a general haematopoietic
disorder, including those arising from radiation therapy or from
chemotherapy, in particular neutropenia or leukopenia, AIDS or
other immunodeficiency diseases, comprising administering to a
mammal in need thereof such a polypeptide, conjugate or
pharmaceutical composition. In particular, the method is aimed at
increasing the level of neutrophils in a patient suffering from an
insufficient neutrophil level, for example due to chemotherapy,
radiation therapy, or HIV or another viral infection.
[0196] The polypeptides and conjugates of the invention will be
administered to patients in a "therapeutically effective" dose,
i.e. a dose that is sufficient to produced the desired effects in
relation to the condition for which it is administered. The exact
dose will depend on the disorder to be treated, and will be
ascertainable by one skilled in the art using known techniques. The
polypeptides or conjugates of the invention may e.g. be
administered at a dose similar to that employed in therapy with
rhG-CSF such as Neupogen.RTM.. A suitable dose of a conjugate of
the invention is contemplated to be in the range of about 5-300
microgram/kg body weight (based on the weight of the protein part
of the conjugate), e.g. 10-200 microgram/kg, such as 25-100
microgram/kg. It will be apparent to those of skill in the art that
an effective amount of a polypeptide, conjugate or composition of
the invention depends, inter alia, upon the disease, the dose, the
administration schedule, whether the polypeptide or conjugate or
composition is administered alone or in conjunction with other
therapeutic agents, the serum half-life of the compositions, the
general health of the patient, and the frequency of administration.
Preferably, the polypeptide, conjugate, preparation or composition
of the invention is administered in an effective dose, in
particular a dose which is sufficient to normalize the number of
leukocytes, in particular neutrophils, in the patient in question.
Normalization of the number of leukocytes may be determined by
simply counting the number of leukocytes at regular intervals in
accordance with established practice.
[0197] The polypeptide or conjugate of the invention is preferably
administered in a composition including one or more
pharmaceutically acceptable carriers or excipients. The polypeptide
or conjugate can be formulated into pharmaceutical compositions in
a manner known per se in the art to result in a polypeptide
pharmaceutical that is sufficiently storage-stable and is suitable
for administration to humans or animals. The pharmaceutical
composition may be formulated in a variety of forms, including as a
liquid or gel, or lyophilized, or any other suitable form. The
preferred form will depend upon the particular indication being
treated and will be apparent to one of skill in the art.
[0198] Accordingly, this invention provides compositions and
methods for treating various forms of leukopenia or neutropenia. In
particular the polypeptide, conjugate or composition of the
invention may be used to prevent infection in cancer patients
undergoing certain types of radiation therapy chemotherapy and bone
marrow transplantations, to mobilize progenitor cells for
collection in peripheral blood progenitor cell transplantations,
for treatment of severe chronic or relative leukopenia and to
support treatment of patients with acute myeloid leukaemia.
Additionally, the polypeptide, conjugate or composition of the
invention may be used for treatment of AIDS or other
immunodeficiency diseases and for antifungal therapy, in particular
for treament of systemic or invasive candidiasis, and for the
treatment of bacterial infections.
[0199] Since the polypeptide conjugates of the invention have a
long in vivo half-life and have been found to reduce the duration
of neutropenia and leukopenia by administration of a single dose,
in contrast to hG-CSF which must be administered daily, the
conjugates of the invention are well-suited for administration e.g.
on a weekly basis for the prevention and/or treatment of
neutropenia. In one embodiment, the polypeptide conjugate or
pharmaceutical composition of the invention is for the prevention
and/or treatment of neutropenia due to chemotherapy. In the case of
chemotherapy administered at intervals, e.g. on a weekly basis by
intravenous injection or by another type of injection, such as
subcutaneous or intramuscular injection, it will normally be
sufficient to administer the conjugate of the invention in a single
dose per chemotherapy treatment, i.e. given either before, after or
simultaneously with the chemotherapy. In other cases where the
chemotherapy is administered differently, for example orally on a
daily basis or over an extended period of time by means of an
infusion pump, the conjugates of the invention may be administered
in a similar manner, e.g. once a week, or, in the case of
chemotherapy sessions given less frequently than once a week, once
per session.
[0200] Drug Form
[0201] The polypeptide or conjugate of the invention can be used
"as is" and/or in a salt form thereof. Suitable salts include, but
are not limited to, salts with alkali metals or alkaline earth
metals, such as sodium, potassium, calcium and magnesium, as well
as e.g. zinc salts. These salts or complexes may by present as a
crystalline and/or amorphous structure.
[0202] Excipients
[0203] "Pharmaceutically acceptable" means a carrier or excipient
that at the dosages and concentrations employed does not cause any
untoward effects in the patients to whom it is administered. Such
pharmaceutically acceptable carriers and excipients are well known
in the art (see Remington's Pharmaceutical Sciences, 18th edition,
A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical
Formulation Development of Peptides and Proteins, S. Frokjaer and
L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of
Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed.,
Pharmaceutical Press [2000]).
[0204] Mix of Drugs
[0205] The pharmaceutical composition of the invention may be
administered alone or in conjunction with other therapeutic agents.
These agents may be incorporated as part of the same pharmaceutical
composition or may be administered separately from the polypeptide
or conjugate of the invention, either concurrently or in accordance
with another treatment schedule. In addition, the polypeptide,
conjugate or pharmaceutical composition of the invention may be
used as an adjuvant to other therapies.
[0206] Patients
[0207] A "patient" for the purposes of the present invention
includes both humans and other mammals. Thus the methods are
applicable to both human therapy and veterinary applications.
[0208] Administration Route
[0209] The administration of the formulations of the present
invention can be performed in a variety of ways, including, but not
limited to, orally, subcutaneously, intravenously, intracerebrally,
intranasally, transdermally, intraperitoneally, intramuscularly,
intrapulmonary, vaginally, rectally, intraocularly, or in any other
acceptable manner. The formulations can be administered
continuously by infusion, although bolus injection is acceptable,
using techniques well known in the art. Typically, the formulation
will designed for parenteral administration, e.g. by the
subcutaneous route.
[0210] Parenterals
[0211] An example of a pharmaceutical composition is a solution
designed for parenteral administration. Although in many cases
pharmaceutical solution formulations are provided in liquid form,
appropriate for immediate use, such parenteral formulations may
also be provided in frozen or in lyophilized form. In the former
case, the composition must be thawed prior to use. The latter form
is often used to enhance the stability of the active compound
contained in the composition under a wider variety of storage
conditions, as it is recognized by those skilled in the art that
lyophilized preparations are generally more stable than their
liquid counterparts. Such lyophilized preparations are
reconstituted prior to use by the addition of one or more suitable
pharmaceutically acceptable diluents such as sterile water for
injection or sterile physiological saline solution.
[0212] In case of parenterals, they are prepared for storage as
lyophilized formulations or aqueous solutions by mixing, as
appropriate, the polypeptide having the desired degree of purity
with one or more pharmaceutically acceptable carriers, excipients
or stabilizers typically employed in the art (all of which are
termed "excipients"), for example buffering agents, stabilizing
agents, preservatives, isotonifiers, non-ionic detergents,
antioxidants and/or other miscellaneous additives.
[0213] Buffering agents help to maintain the pH in the range which
approximates physiological conditions. They are typically present
at a concentration ranging from about 2 mM to about 50 mM Suitable
buffering agents for use with the present invention include both
organic and inorganic acids and salts thereof such as citrate
buffers (e.g., monosodium citrate-disodium citrate mixture, citric
acid-trisodium citrate mixture, citric acid-monosodium citrate
mixture, etc.), succinate buffers (e.g., succinic acid-monosodium
succinate mixture, succinic acid-sodium hydroxide mixture, succinic
acid-disodium succinate mixture, etc.), tartrate buffers (e.g.,
tartaric acid-sodium tartrate mixture, tartaric acid-potassium
tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.),
fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,
fumaric acid-disodium fumarate mixture, monosodium
fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g.,
gluconic acid-sodium glyconate mixture, gluconic acid-sodium
hydroxide mixture, gluconic acid-potassium glyuconate mixture,
etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture,
oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate
mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate
mixture, lactic acid-sodium hydroxide mixture, lactic
acid-potassium lactate mixture, etc.) and acetate buffers (e.g.,
acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide
mixture, etc.). Additional possibilities are phosphate buffers,
histidine buffers and trimethylamine salts such as Tris.
[0214] Preservatives are added to retard microbial growth, and are
typically added in amounts of about 0.2%-1% (w/v). Suitable
preservatives for use with the present invention include phenol,
benzyl alcohol, meta-cresol, methyl paraben, propyl paraben,
octadecyldimethylbenzyl ammonium chloride, benzalkonium halides
(e.g. benzalkonium chloride, bromide or iodide), hexamethonium
chloride, alkyl parabens such as methyl or propyl paraben,
catechol, resorcinol, cyclohexanol and 3-pentanol.
[0215] Isotonicifiers are added to ensure isotonicity of liquid
compositions and include polyhydric sugar alcohols, preferably
trihydric or higher sugar alcohols, such as glycerin, erythritol,
arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can
be present in an amount between 0.1% and 25% by weight, typically
1% to 5%, taking into account the relative amounts of the other
ingredients.
[0216] Stabilizers refer to a broad category of excipients which
can range in function from a bulking agent to an additive which
solubilizes the therapeutic agent or helps to prevent denaturation
or adherence to the container wall. Typical stabilizers can be
polyhydric sugar alcohols (enumerated above); amino acids such as
arginine, lysine, glycine, glutamine, asparagine, histidine,
alanine, omithine, L-leucine, 2-phenylalanine, glutamic acid,
threonine, etc., organic sugars or sugar alcohols, such as lactose,
trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol,
myoinisitol, galactitol, glycerol and the like, including cyclitols
such as inositol; polyethylene glycol; amino acid polymers;
sulfur-containing reducing agents, such as urea, glutathione,
thioctic acid, sodium thioglycolate, thioglycerol,
.alpha.-monothioglycerol and sodium thiosulfate; low molecular
weight polypeptides (i.e. <10 residues); proteins such as human
serum albumin, bovine serum albumin, gelatin or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides
such as xylose, mannose, fructose and glucose; disaccharides such
as lactose, maltose and sucrose; trisaccharides such as raffinose,
and polysaccharides such as dextran. Stabilizers are typically
present in the range of from 0.1 to 10,000 parts by weight based on
the active protein weight.
[0217] Non-ionic surfactants or detergents (also known as "wetting
agents") may be present to help solubilize the therapeutic agent as
well as to protect the therapeutic polypeptide against
agitation-induced aggregation, which also permits the formulation
to be exposed to shear surface stress without causing denaturation
of the polypeptide. Suitable non-ionic surfactants include
polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.),
Pluronic.RTM. polyols, polyoxyethylene sorbitan monoethers
(Tween.RTM.-20, Tween.RTM.-80, etc.).
[0218] Additional miscellaneous excipients include bulking agents
or fillers (e.g. starch), chelating agents (e.g. EDTA),
antioxidants (e.g., ascorbic acid, methionine, vitamin E) and
cosolvents.
[0219] The active ingredient may also be entrapped in microcapsules
prepared, for example, by coascervation techniques or by
interfacial polymerization, for example hydroxymethylcellulose,
gelatin or poly-(methylmethacylate) microcapsules, in colloidal
drug delivery systems (for example liposomes, albumin microspheres,
microemulsions, nano-particles and nanocapsules) or in
macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences, supra.
[0220] Parenteral formulations to be used for in vivo
administration must be sterile. This is readily accomplished, for
example, by filtration through sterile filtration membranes.
[0221] Sustained Release Preparations
[0222] Suitable examples of sustained-release preparations include
semi-permeable matrices of solid hydrophobic polymers containing
the polypeptide or conjugate, the matrices having a suitable form
such as a film or microcapsules. Examples of sustained-release
matrices include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)),
polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate,
non-degradable ethylene-vinyl acetate, degradable lactic
acid-glycolic acid copolymers such as the ProLease.RTM. technology
or Lupron Depot.RTM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for long periods such as up to or over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated polypeptides remain in the body for a long time, they
may denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for stabilization depending on the mechanism involved. For
example, if the aggregation mechanism is discovered to be
intermolecular S--S bond formation through thio-disulfide
interchange, stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions.
[0223] All references cited herein are hereby incorporated by
reference in their entirety for all purposes.
[0224] The invention is further described in the non-limiting
examples below.
DESCRIPTION OF THE DRAWINGS
[0225] FIG. 1: The in vivo half-lives of rhG-CSF (Neupogen.RTM.)
and SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70K Q90K
Q120K
[0226] FIG. 2: The in vivo half-lives of rhG-CSF (Neupogen.RTM.)
and SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q90K T105K
Q159K
[0227] FIG. 3: The in vivo biological activities in healthy rats of
rhG-CSF (Neupogen.RTM.), SPA-PEG 5000-conjugated hG-CSF K16R K34R
K40R Q70K Q120K and SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R
Q70K Q90K Q120K.
[0228] FIG. 4: The in vivo biological activities in healthy rats of
rhG-CSF (Neupogen.RTM.), SPA-PEG 5000-conjugated hG-CSF K16R K34R
K40R Q70K Q120K T133K and SPA-PEG 5000-conjugated hG-CSF K16R K34R
K40R Q90K Q120K T133K.
[0229] FIG. 5: The in vivo biological activities in healthy rats of
rhG-CSF (Neupogen.RTM.), SPA-PEG 12000-conjugated hG-CSF K16R K34R
K40R and different doses of SPA-PEG 5000-conjugated hG-CSF K16R
K34R K40R Q70K Q90K Q120K.
[0230] FIG. 6: The in vivo biological activities in healthy rats of
rhG-CSF (Neupogen.RTM.), SPA-PEG 5000-conjugated hG-CSF K16R K34R
K40R Q70K Q90K Q120K, SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R
Q90K T105K S159K and SPA-PEG 20000-conjugated hG-CSF K16R K34R K40R
T105K S159K.
[0231] FIG. 7: The in vivo biological activities in rats with
chemotherapy-induced neutropenia of rhG-CSF (Neupogen.RTM.),
SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K,
SPA-PEG 20000-conjugated hG-CSF K16R K34R K40R Q90K.
[0232] FIG. 8: The in vivo biological activities (white blood cell
count) in rats with chemotherapy-induced neutropenia of rhG-CSF
(Neupogen.RTM.), SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R
T105K S159K, and SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q90K
T105K S159K.
[0233] FIG. 9: The in vivo biological activities (absolute
neutrophil count) in rats with chemotherapy-induced neutropenia of
rhG-CSF (Neupogen.RTM.) and SPA-PEG 5000-conjugated hG-CSF K16R
K34R K40R T105K S159K.
[0234] FIG. 10: The in vivo biological activities (white blood cell
count) in rats with chemotherapy-induced neutropenia of rhG-CSF
(Neupogen.RTM.), rhG-CSF with a 20 kDa N-terminal PEG group
(Neulasta.TM.), and SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R
T105K S159K produced in yeast and in CHO cells.
[0235] FIG. 11: The in vivo biological activities (absolute
neutrophil count) in rats with chemotherapy-induced neutropenia of
rhG-CSF (Neupogen.RTM.), rhG-CSF with a 20 kDa N-terminal PEG group
(Neulasta.TM.), and SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R
T105K S159 K produced in yeast and in CHO cells.
SEQUENCE LISTING
[0236] The appended sequence listing contains the following
sequences:
[0237] SEQ ID NO:1: The amino acid sequence of human G-CSF.
[0238] SEQ ID NO:2: A synthetic DNA sequence encoding human G-CSF,
with codon usage optimized for expression in E. coli.
[0239] SEQ ID NO:3: The amino acid sequence of the OmpA signal
sequence.
[0240] SEQ ID NO:4: A synthetic DNA sequence encoding the OmpA
signal sequence.
[0241] SEQ ID NO:5: A synthetic histidine tag.
[0242] SEQ ID NO:6: A synthetic DNA sequence encoding the histidine
tag of SEQ ID NO:5.
[0243] SEQ ID NO:7: The amino acid sequence of a human G-CSF signal
peptide.
[0244] SEQ ID NO:8: A synthetic DNA sequence encoding human G-CSF,
including the signal peptide of SEQ ID NO:7, with codon usage
optimized for expression in CHO cells.
[0245] SEQ ID NO:9-15: Various synthetic tags
[0246] Materials and Methods
[0247] Methods Used to Determine the Amino Acids to be Modified
[0248] Accessible Surface Area (ASA)
[0249] A 3D ensemble of 10 structures determined by NMR
spectroscopy (Zink et al. (1994) Biochemistry 33: 8453-8463) is
available from the Protein Data Bank (PDB) (www.rcsb.org/pdb/).
This information can be entered into the computer program Access
(B. Lee and F. M. Richards, J. Mol. Biol. 55: 379-400 (1971))
version 2 (.COPYRGT.1983 Yale University) and used to compute the
accessible surface area (ASA) of the individual atoms in the
structure. This method typically uses a probe size of 1.4 .ANG. and
defines the Accessible Surface Area (ASA) as the area formed by the
centre of the probe. Prior to this calculation all water molecules
and all hydrogen atoms should be removed from the coordinate set as
should other atoms not directly related to the protein.
[0250] Fractional ASA of Side Chain
[0251] The fractional ASA of the side chain atoms is computed by
division of the sum of the ASA of the atoms in the side chain with
a value representing the ASA of the side chain atoms of that
residue type in an extended ALA-x-ALA tripeptide. See Hubbard,
Campbell & Thornton (1991) J. Mol. Biol. 220, 507-530. For this
example the CA atom is regarded as a part of the side chain of
glycine residues but not for the remaining residues. The values in
the following table are used as standard 100% ASA for the side
chain:
5 Ala 69.23 .ANG..sup.2 Arg 200.35 .ANG..sup.2 Asn 106.25
.ANG..sup.2 Asp 102.06 .ANG..sup.2 Cys 96.69 .ANG..sup.2 Gln 140.58
.ANG..sup.2 Glu 134.61 .ANG..sup.2 Gly 32.28 .ANG..sup.2 His 147.00
.ANG..sup.2 Ile 137.91 .ANG..sup.2 Leu 140.76 .ANG..sup.2 Lys
162.50 .ANG..sup.2 Met 156.08 .ANG..sup.2 Phe 163.90 .ANG..sup.2
Pro 119.65 .ANG..sup.2 Ser 78.16 .ANG..sup.2 Thr 101.67 .ANG..sup.2
Trp 210.89 .ANG..sup.2 Tyr 176.61 .ANG..sup.2 Val 114.14
.ANG..sup.2
[0252] Residues not detected in the structure are defined as having
100% exposure as they are thought to reside in flexible
regions.
[0253] Determining Distances Between Atoms:
[0254] The distance between atoms is most easily determined using
molecular graphics software, e.g. InsightII.RTM. v. 98.0, MSI
INC.
[0255] General Considerations Regarding Amino Acid Residues to be
Modified
[0256] As explained above, amino acid residues to be modified in
accordance with the present invention are preferably those whose
side chains are surface exposed, in particular those with more than
about 25% of the side chain exposed at the surface of the molecule,
and more preferably those with more than 50% side chain exposure.
Another consideration is that residues located in receptor
interfaces are preferably excluded so as to avoid or at least
minimize possible interference with receptor binding or activation.
A further consideration is that residues that are less than 10
.ANG. from the nearest Lys (Glu, Asp) CB-CB (CA for Gly) should
also be excluded. Finally, preferred positions for modification are
in particular those that have a hydrophilic and/or charged residue,
i.e. Asp, Asn, Glu, Gln, Arg, His, Tyr, Ser and Thr, positions that
have an arginine residue being especially preferred.
[0257] Identifying G-CSF Amino Acid Residues for Modification
[0258] The information below illustrates the factors that generally
should be taken into consideration when identifying amino acid
residues to be modified in accordance with the present
invention.
[0259] Three-dimensional structures have been reported for human
G-CSF by X-ray crystallography (Hill et al. (1993) Proc. Natl.
Acad. Sci. USA 90: 5167-5171) and by NMR spectroscopy (Zink et al.
(1994) Biochemistry 33: 8453-8463). As mentioned above, Aritomi et
al. (Nature 401:713-717, 1999) have identified the following hG-CSF
residues as being part of the receptor binding interfaces: G4, P5,
A6, S7, S8, L9, P10, Q11, S12, L15, K16, E19, Q20, L108, D109,
D112, T115, T116, Q119, E122, E123, and L124. Thus, although it is
possible to modify these residues, it is preferred that these
residues are excluded from modification.
[0260] Using the 0.10 NMR structures of G-CSF identified by Zink et
al. (1994) as input structures followed by a computation of the
average ASA of the side chain, the following residues have been
identified as having more than 25% ASA: M0, T1, P2, L3, G4, P5, A6,
S7, S8, L9, P10, Q11, S12, F13, L14, L15, K16, C17, E19, Q20, V21,
R22, K23, Q25, G26, D27, A29, A30, E33, K34, C36, A37, T38, Y39,
K40, L41, H43, P44, E45, E46, V48, L49, L50, H52, S53, L54, I56,
P57, P60, L61, S62, S63, P65, S66, Q67, A68, L69, Q70, L71, A72,
G73, C74, S76, Q77, L78, S80, F83, Q86, G87, Q90, E93, G94, S96,
P97, E98, L99, G100, P101, T102, D104, T105, Q107, L108, D109,
A111, D112, F113, T115, T116, W118, Q119, Q120, M121, E122, E123,
L124, M126, A127, P128, A129, L130, Q131, P132, T133, Q134, G135,
A136, M137, P138, A139, A141, S142, A143, F144, Q145, R146, R147,
S155, H156, Q158, S159, L161, E162, V163, S164, Y165, R166, V167,
L168, R169, H170, L171, A172, Q173, P174.
[0261] Similarly, the following residues have more than 50% ASA:
M0, T1, P2, L3, G4, P5, A6, S7, S8, L9, P10, Q11, S12, F13, L14,
L15, K16, C17, E19, Q20, R22, K23, G26, D27, A30, E33, K34, T38,
K40, L41, H43, P44, E45, E46, L49, L50, S53, P57, P60, L61, S62,
S63, P65, S66, Q67, A68, L69, Q70, L71, A72, G73, S80, F83, Q90,
G94, P97, E98, P101, D104, T105, L108, D112, F113, T115, T116,
Q119, Q120, E122, E123, L124, M126, P128, A129, L130, Q131, P132,
T133, Q134, G135, A136, A139, A141, S142, A143, F144, R147, S15S,
S159, E162, R166, V167, R169, H170, L171, A172, Q173, P174.
[0262] The molecular graphics program InsightII.RTM. v.98.0 was
used to determine residues having their CB atom (CA in the case of
glycine) at a distance of more than 15 .ANG. from the nearest amine
group, defined as the NZ atoms of lysine and the N atom of the
N-terminal residue T1. The following list includes the residues
that fulfill this criteria in at least one of the 10 NMR
structures. G4, P5, A6, S7, S8, L9, P10, Q11, L14, L15, L18, V21,
R22, Q25, G26, D27, G28, A29, Q32, L35, C36, T38, Y39, C42, H43,
P44, E45, E46, L47, V48, L49, L50, G51, H52, S53, L54, G55, I56,
P57, W58, A59, P60, L61, S62, S63, C64, P65, S66, Q67, A68, L69,
Q70, L71, A72, G73, C74, L75, S76, Q77, L78, H79, S80, G81, L82,
F83, L84, Y85, Q86, G87, L88, L89, Q90, A91, L92, E93, G94, I95,
S96, P97, E98, L99, G100, P101, T102, L103, D104, T105, L106, Q107,
L108, D109, V110, A111, D112, F113, A114, T115, T116, I117, W118,
Q119, Q120, M121, E122, E123, L124, G125, M126, A127, P128, A129,
L130, Q131, P132, T133, Q134, G135, A136, M137, P138, A139, F140,
A141, S142, A143, F144, Q145, R146, R147, A148, G149, G150, V151,
L152, V153, A154, S155, H156, L157, Q158, S159, F160, L161, E162,
V163, S164, Y165, R166, V167, L168, R169, H170, L171, A172, Q173,
P174.
[0263] The InsightII.RTM. v.98.0 program was similarly used to
determine residues having their CB atom (CA atom in the case of
glycine) at a distance of more than 10 .ANG. from the nearest
acidic group, defined as the CG atoms of aspartic acid, the CD
atoms of glutamic acid and the C atom of the C-terminal residue
P1174. The following list includes the residues that fulfill this
criteria in at least one of the 10 NMR structures. M0, T1, P2, L3,
G4, P5, A6, S7, S8, L9, P10, Q11, S12, F13, L14, T38, Y39, K40,
L41, C42, L50, G51, H52, S53, L54, G55, I56, P57, W58, A59, P60,
L61, S62, S63, C64, P65, S66, Q67, A68, L69, Q70, L71, A72, G73,
C74, L75, S76, Q77, L78, H79, S80, G81, L82, F83, L84, Y85, Q86,
G87, L88, I117, M126, A127, P128, A129, L130, Q131, P132, T133,
Q134, G135, A136, M137, P138, A139, F140, A141, S142, A143, F144,
Q145, R146, R147, A148, G149, G150, V151, L152, V153, A154, S155,
H156, L157, V167, L168, R169, H170, L171.
[0264] By combining and comparing the above lists, it is possible
to select individual amino acid residues for modification to result
in a list containing a limited number of amino acid residues whose
modification in a given G-CSF polypeptide is likely to result in
desired properties.
[0265] Methods for PEGylation of hG-CSF and Variants Thereof
[0266] PEGylation of hG-CSF and Variants Thereof in Solution
[0267] Human G-CSF and variants thereof are PEGylated at a
concentration of 250 .mu.g/ml in 50 mM sodium phosphate, 100 mM
NaCl, pH 8.5. The molar surplus of PEG is 50-100 times with respect
to PEGylation sites on the protein. The reaction mixture is placed
in a thermo mixer for 30 minutes at 37.degree. C. at 1200 rpm.
After 30 minutes, quenching of the reaction is obtained by adding a
molar excess of glycine.
[0268] Cation exchange chromatography is applied to remove excess
PEG, glycine and other by-products from the reaction mixture. The
PEGylation reaction mixture is diluted with 20 mM sodium citrate pH
2.5 until the ionic strength is less than 7 mS/cm. pH is adjusted
to 2.5 using 5 N HCl. The mixture is applied to a SP-sepharose FF
column equilibrated with 20 mM sodium citrate pH 2.5. Unbound
material is washed off the column using 4 column volumes of
equilibration buffer. PEGylated protein is eluted in three column
volumes by adding 20 mM sodium citrate, 750 mM sodium chloride.
Pure PEGylated G-CSF is concentrated and buffer exchange is
performed using VivaSpin concentration devices, molecular weight
cut-off (mwco): 10 kDa.
[0269] PEGylation in Microtiter Plates of a Tagged Polypeptide with
G-CSF Activity
[0270] A polypeptide exhibiting G-CSF activity is expressed with a
suitable tag, e.g. any of the tags exemplified in the general
description above, and culture broth is transferred to one or more
wells of a microtiter plate capable of immobilising the tagged
polypeptide. When the tag is
Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-Gln (SEQ ID
NO:5), a nickel-nitrilotriacetic acid (Ni-NTA) HisSorb microtiter
plate commercially available from QIAGEN can be used.
[0271] After immobilization of the tagged polypeptide to the
microtiter plate, the wells are washed in a buffer suitable for
binding and subsequent PEGylation followed by incubating the wells
with the activated PEG of choice. As an example, M-SPA-5000 from
Shearwater Corp. is used. The molar ratio of activated PEG to
polypeptide should be optimized, but will typically be greater than
10:1, e.g. up to about 100:1 or higher. After a suitable reaction
time at ambient temperature, typically around 1 hour, the reaction
is stopped by removal of the activated PEG solution. The conjugated
protein is eluted from the plate by incubation with a suitable
buffer. Suitable elution buffers may contain imidazole, excess NTA
or another chelating compound. The conjugated protein is assayed
for biological activity and immunogenicity as appropriate. The tag
may optionally be cleaved off using a method known in the art, e.g.
using diaminopeptidase the Gin in pos -1 can be converted to
pyroglutamyl with GCT (glutamylcyclotransferase) and finally
cleaved off with PGAP (pyro-glutamyl-aminopeptidase), giving the
untagged protein. The process involves several steps of metal
chelate affinity chromatography. Alternatively, the tagged
polypeptide may be conjugated.
[0272] PEGylation of a Polypeptide Exhibiting hG-CSF Activity and
Having a Blocked Receptor-Binding Site
[0273] In order to optimize PEGylation of hG-CSF in a manner
excluding PEGylation of lysines involved in receptor recognition,
the following method has been developed:
[0274] Purified hG-CSF is obtained as described in Example 4. A
homodimer complex consisting of an hG-CSF polypeptide and the
soluble domain of the G-CSF receptor in a 2:2 stochiometry is
formed in a phosphate-buffered saline solution (PBS) buffer at pH
7. The concentration of hG-CSF polypeptide is approximately 20
.mu.g/ml or 1 .mu.M and the receptor is present at an equimolar
concentration.
[0275] M-SPA-5000 from Shearwater Corp. is added at 3 different
concentration levels corresponding to a 10, 25 and 50 molar excess
to the number of attachment sites in hG-CSF polypeptide. The
reaction time is 30 min at room temperature. After the 30 min
reaction period, the pH of the reaction mixture is adjusted to pH
2.0 and the reaction mixture is applied to a Vydac C18 column and
eluted with an acetonitrile gradient essentially as described
(Utsumi et al., J. Biochem., Vol. 101, 1199-1208, (1987).
Alternatively, an isopropanol gradient can be used.
[0276] Fractions are analyzed using the primary screening assay
described herein and active PEGylated hG-CSF polypeptide obtained
by this method is stored at -80.degree. C. in PBS, pH 7 containing
1 mg/ml human serum albumin (HSA).
[0277] Methods Used to Characterize Conjugated and Non-Conjugated
hG-CSF and Variants Thereof.
[0278] Determination of the Molecular Size of hG-CSF and Variants
Thereof
[0279] The molecular weight of conjugated or non-conjugated hG-CSF
or variants thereof is determined by either SDS-PAGE, gel
filtration, matrix assisted laser desorption mass spectrometry or
equilibrium centrifugation. As explained above, SDS-PAGE provides
information on the "apparent molecular weight". The actual
molecular weight can advantageously be determined using mass
spectrometry. SDS-PAGE is carried out using the NuPAGE.RTM. kit
(Novex high-performance pre-cast gels) from Invitrogen.TM.. 15
.mu.l of the samples are loaded onto NuPAGE 4-12% Bis-Tris gels
(Cat. Nr. NPO321) and eluted in NuPAGE MES SDS running buffer (Cat.
Nr. NPO002-O.sub.2) for 35 minutes at 200 V and 120 mA.
[0280] Determination of Polypeptide Concentration
[0281] The concentration of a polypeptide can be measured using
optical density measurements at 280 nm, an enzyme-linked
immunoadsorption assay (ELISA), a radio-immunoassay (RIA), or other
such immunodetection techniques well known in the art. Furthermore,
the polypeptide concentration in a sample can be measured with the
Biacore.RTM. instrument using a Biacore.RTM. chip coated with an
antibody specific for the polypeptide.
[0282] Such an antibody can be coupled covalently to the
Biacore.RTM. chip by various chemistries. Alternatively, the
antibody can be bound non-covalently e.g. by means of an antibody
specific for the Fc portion of the anti-polypeptide antibody. The
Fc specific antibody is first coupled covalently to the chip. The
anti-polypeptide antibody is then flowed over the chip and is bound
by the first antibody in a directed fashion. Furthermore,
biotinylated antibodies can be immobilized using a streptavidin
coated surface (e.g. Biacore Sensor Chip SA.RTM.) (Real-Time
Analysis of Biomolecular Interactions, Nagata and Handa (Eds.),
2000, Springer Verlag, Tokyo; Biacore 2000 Instrument Handbook,
1999, Biacore AB).
[0283] When the sample is flowed over the chip the polypeptide will
bind to the coated antibody and the increase in mass can be
measured. By using a preparation of the polypeptide in a known
concentration, a standard curve can be established and subsequently
the concentration of the polypeptide in the sample can be
determined. After each injection of sample the sensor chip is
regenerated by a suitable eluent (e.g. a low pH buffer) that
removes the bound analyte.
[0284] Generally, the applied antibodies will be monoclonal
antibodies raised against the wild type polypeptide. Introduction
of mutations or other manipulations of the wild type polypeptide
(extra glycosylations or polymer conjugations) may alter the
recognition by such antibodies. Furthermore, such manipulations
that give rise to an increased molecular weight of the polypeptide
will result in an increased plasmon resonance signal. Consequently,
it is necessary to establish a standard curve for every molecule to
be tested.
[0285] Methods Used to Determine the In Vitro and In Vivo Activity
of Conjugated and Non-Conjugated hG-CSF and Variants Thereof.
[0286] Primary Assay 1--In Vitro G-CSF Activity Assay
[0287] Proliferation of the murine cell line NFS-60 (obtained from
Dr. J. Ihle, St. Jude Children's Research Hospital, Tennessee, USA)
is dependent on the presence of active G-SCF in the growth medium.
Thus, the in vitro biological activity of hG-CSF and variants
thereof can be determined by measuring the number of dividing
NFS-60 cells after addition of a G-CSF sample to the growth medium
followed by incubation over a fixed period of time.
[0288] NFS-60 cells are maintained in Iscoves DME Medium containing
10% w/w FBS (fetal bovine serum), 1% w/w Pen/Strep, 10 .mu.g per
litre hG-CSF and 2 mM Glutamax. Prior to sample addition, cells are
washed twice in growth medium without hG-CSF and diluted to a
concentration of 2.2.times.10 cells per ml. 100 .mu.l of the cell
suspension is added to each well of a 96 well microtiter plate
(Corning).
[0289] Samples containing conjugated or non-conjugated G-CSF or
variants thereof are diluted to concentrations between
1.1.times.10.sup.-6 M and 1.1.times.10.sup.-13 M in the growth
medium. 10 .mu.l of each sample is added to 3 wells containing
NFS-60 cells. A control consisting of 10 .mu.l of mammalian growth
medium is added to 8 wells on each microtiter plate. The cells are
incubated for 48 hours (37.degree. C., 5% CO.sub.2) and the number
of dividing cells in each well is quantified using the WST-1 cell
proliferation agent (Roche Diagnostics GmbH, Mannheim, Germany).
0.01 ml WST-1 is added to the wells followed by incubation for 150
min. at 37.degree. C. in a 5% CO.sub.2 air atmosphere. The cleavage
of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in
viable cells results in the formation of formazan that is
quantified by measuring the absorbance at 450 nm. Hereby, the
number of viable cells in each well is quantified.
[0290] Based on these measurements, dose-response curves for each
conjugated and non-conjugated G-CSF molecule or variants thereof
are calculated, after which the EC50 value for each molecule can be
determined. This value is equal to the amount of active G-CSF
protein that is necessary to obtain 50% of the maximum
proliferation activity of non-conjugated human G-CSF. Thus, the
EC50 value is a direct measurement of the in vitro activity of the
given protein, a lower EC50 value indicating a higher activity.
[0291] Primary Assay 2--In Vitro G-CSF Activity Assay
[0292] The murine hematopoietic cell line BaF3 is transfected with
a plasmid carrying the human G-CSF receptor and the promoter of the
transcription regulator, fos, in front of the luciferase reporter
gene. Upon stimulation of such a cell line with a G-CSF sample, a
number of intracellular reactions lead to stimulation of fos
expression, and consequently to expression of luciferase. This
stimulation is monitored by the Steady-G Luciferase Assay System
(Promega, Cat. No. E2510) whereby the in vitro activity of the
G-CSF sample may be quantified.
[0293] BaF3/hGCSF-R/pfos-lux cells are maintained at 37.degree. C.
in a humidified 5% CO.sub.2 atmosphere in complete culture media
(RPMI-1640/HEPES (Gibco/BRL, Cat. No. 22400), 10% FBS (HyClone,
characterized), 1.times. Penicillin/Streptomycin (Gibco/BRL, Cat.
No. 15140-122), 1.times. L-Glutamine (Gibco/BRL, Cat. No.
25030-081), 10% WEHI-3 conditioned media (source of muIL-3), and
grown to a density of 5.times.10.sup.5 cells/mL (confluent). The
cells are reseeded at about 2.times.10.sup.4 cells/mL every 2-3
days.
[0294] One day prior to the assay, log-phase cells are resuspended
at 2.times.1 cells/mL in starving media (DMEM/F-12 (Gibco/BRL, Cat.
No. 11039), 1% BSA (Sigma, Cat. No. A3675), 1.times.
Penicillin/Streptomycin (Gibco/BRL, Cat. No. 15140-122), 1.times.
L-Glutamine (Gibco/BRL, Cat. No. 25030-081), 0.1% WEHI-3
conditioned media) and starved for 20 hours. The cells are washed
twice with PBS and tested for viability using Trypan Blue viability
staining. The cells are resuspended in assay media (RPMI-1640
(phenol-red free, Gibco/BRL, Cat. No. 11835), 25 mM HEPES, 1% BSA
(Sigma, Cat. No. A3675), 1.times. Penicillin/Streptomycin
(Gibco/BRL, Cat. No. 15140-122), 1.times. L-Glutamine (Gibco/BRL,
Cat. No. 25030-081) at 4.times.10.sup.6 cells/mL, and 50 .mu.L are
aliquotted into each well of a 96-well microtiter plate (Corning).
Samples containing conjugated or non-conjugated G-CSF or variants
thereof are diluted to concentrations between 1.1.times.10.sup.-7 M
and 1.1.times.10.sup.-12 M in the assay medium. 50 .mu.l of each
sample is added to 3 wells containing BaF3/hGCSF-R/pfos-lux cells.
A negative control consisting of 50 .mu.l of medium is added to 8
wells on each microtiter plate. The plates are mixed gently and
incubated for 2 hours at 37.degree. C. The luciferase activity is
measured by following the Promega Steady-Glo.TM. protocol (Promega
Steady-Glo.TM. Luciferase Assay System, Cat. No. E2510). 100 .mu.L
of substrate is added per well followed by gentle mixing.
Luminescence is measured on a TopCount luminometer (Packard) in SPC
(single photon counting) mode.
[0295] Based on these measurements, dose-response curves for each
conjugated and non-conjugated G-CSF molecule or variants thereof
are calculated, after which the EC50 value for each molecule can be
determined.
[0296] Secondary Assay--Binding Affinity of G-CSF or Variants
Thereof to the hG-CSF Receptor
[0297] Binding of rhG-CSF or variants thereof to the hG-CSF
receptor is studied using standard binding assays. The receptors
may be purified extracellular receptor domains, receptors bound to
purified cellular plasma membranes, or whole cells--the cellular
sources being either cell lines that inherently express G-CSF
receptors (e.g. NFS-60) or cells transfected with cDNAs encoding
the receptors. The ability of rhG-CSF or variants thereof to
compete for the binding sites with native G-CSF is analyzed by
incubating with a labeled G-CSF-analog, for instance biotinylated
hG-CSF or radioiodinated hG-CSF. An example of such an assay is
described by Yamasaki et al. (Drugs. Exptl. Clin. Res. 24:191-196
(1998)).
[0298] The extracellular domains of the hG-CSF receptor can
optionally be coupled to Fc and immobilized in 96 well plates.
RhG-CSF or variants thereof are subsequently added and the binding
of these is detected using either specific anti-hG-CSF antibodies
or biotinylated or radioiodinated hG-CSF.
[0299] Measurement of the In Vivo Half-Life of Conjugated and
Non-Conjugated rhG-CSF and Variants Thereof
[0300] An important aspect of the invention is the prolonged
biological half-life that is obtained by construction of a hG-CSF
with or without conjugation of the polypeptide to the polymer
moiety. The rapid decrease of hG-CSF serum concentrations has made
it important to evaluate biological responses to treatment with
conjugated and non-conjugated hG-CSF and variants thereof.
Preferably, the conjugated and non-conjugated hG-CSF and variants
thereof of the present invention have prolonged serum half-lives
also after i.v. administration, making it possible to measure by
e.g. an ELISA method or by the primary screening assay. Measurement
of in vivo biological half-life was carried out as described
below.
[0301] Male Sprague Dawley rats (7 weeks old) were used. On the day
of administration, the weights of the animals were measured
(280-310 gram per animal). 100 .mu.g per kg body weight of the
non-conjugated and conjugated hG-CSF samples were each injected
intravenously into the tail vein of three rats. At 1 minute, 30
minutes, 1, 2, 4, 6, and 24 hours after the injection, 500 .mu.l of
blood was withdrawn from the eyes of each rat while under
CO.sub.2-anaesthesia. The blood samples were stored at room
temperature for 11/2 hours followed by isolation of serum by
centrifugation (4.degree. C., 18000.times.g for 5 minutes). The
serum samples were stored at -80.degree. C. until the day of
analysis. The amount of active G-CSF in the serum samples was
quantified by the G-CSF in vitro activity assay (see primary assay
2) after thawing the samples on ice.
[0302] Another example of an assay for the measurement of in vivo
half-life of G-CSF or variants thereof is described in U.S. Pat.
No. 5,824,778, the content of which is hereby incorporated by
reference.
[0303] Measurement of the In Vivo Biological Activity in Healthy
Rats of Conjugated and Non-Conjugated hG-CSF and Variants
Thereof
[0304] Measurement of the in vivo biological effects of hG-CSF in
SPF Sprague Dawley rats (purchased from M & B A/S, Denmark) is
used to evaluate the biological efficacy of conjugated and
non-conjugated G-CSF and variants thereof.
[0305] On the day of arrival the rats are randomly allocated into
groups of 6. The animals are acclimatized for a period of 7 days
wherein individuals in poor condition or at extreme weights are
rejected. The weight range of the rats at the start of the
acclimatization period is 250-270 g.
[0306] On the day of administration the rats are fasted for 16
hours followed by subcutaneous injection of 100 .mu.g per kg body
weight of hG-CSF or a variant thereof. Each hG-CSF sample is
injected into a group of 6 randomized rats. Blood samples of 300
.mu.l EDTA stabilized blood are drawn from a tail vein of the rats
prior to dosing and at 6, 12, 24, 36, 48, 72, 96, 120 and 144 hours
after dosing. The blood samples are analyzed for the following
haematological parameters: Haemoglobin, red blood cell count,
haematocrit, mean cell volume, mean cell haemoglobin concentration,
mean cell haemoglobin, white blood cell count, differential
leucocyte count (neutrophils, lymphocytes, eosinophils, basophils,
monocytes). On the basis of these measurements the biological
efficacy of conjugated and non-conjugated hG-CSF and variants
thereof is evaluated. Further examples of assays for the
measurement of in vivo biological activity of hG-CSF or variants
thereof are described in U.S. Pat. No. 5,681,720, U.S. Pat. No.
5,795,968, U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,985,265 and by
Bowen et al., Experimental Hematology 27:425-432 (1999).
[0307] Measurement of the In Vivo Biological Activity in Rats with
Chemotherapy-Induced Neutropenia of Conjugated and Non-Conjugated
hG-CSF and Variants Thereof
[0308] SPF Sprague Dawley rats were purchased from M & B A/S,
Denmark. On the day of arrival the rats are randomly allocated into
groups of 6. The animals are acclimatized for a period of 7 days
wherein individuals in poor condition or at extreme weights are
rejected. The weight range of the rats at the start of the
acclimatization period is 250-270 g.
[0309] 24 hours before administration of the hG-CSF samples the
rats are injected i.p. with 50 or 90 mg per kg body weight of
cyclophosphamide (CPA). The PEGylated hG-CSF variants are given as
a single dose injected s.c. at day 0, while non-conjugated hG-CSF
is injected s.c. either in a single dose at day 0 or on a daily
basis. For hG-CSF or variants given in a single dose at day 0, the
dosage is 100 .mu.g per kg body weight. For non-conjugated hG-CSF
(Neupogen.RTM.) given on a daily basis, the dosage varied and is
given in the examples below. Each hG-CSF sample is injected into a
group of 6 randomized rats. Blood samples of 300 .mu.l EDTA
stabilized blood are drawn from a tail vein of the rats prior to
dosing and at 6, 12, 24, 36, 48, 72, 96, 120, 144 and 168 hours
after dosing. The blood samples are analyzed for the following
haematological parameters: hemoglobin, red blood cell count,
haematocrit, mean cell volume, mean cell haemoglobin concentration,
mean cell haemoglobin, white blood cell count, differential
leucocyte count (neutrophils, lymphocytes, eosinophils, basophils,
monocytes). On the basis of these measurements the biological
efficacy of conjugated and non-conjugated hG-CSF and variants
thereof is evaluated.
[0310] Determination of Polypeptide Receptor-Binding Affinity (On-
and Off-Rate)
[0311] The strength of the binding between a receptor and ligand
can be measured using an enzyme-linked immunoadsorption assay
(ELISA), a radio-immunoassay (RIA), or other such immunodetection
techniques well known in the art. The ligand-receptor binding
interaction may also be measured with the Biacore.RTM. instrument,
which exploits plasmon resonance detection (Zhou et al.,
Biochemistry, 1993, 32, 8193-98; Faegerstram and O'Shannessy, 1993,
In Handbook of Affinity Chromatography, 229-52, Marcel Dekker,
Inc., NY).
[0312] The Biacore.RTM. technology allows one to bind receptor to a
gold surface and to flow ligand over it. Plasmon resonance
detection gives direct quantification of the amount of mass bound
to the surface in real time. This technique yields both on- and
off-rate constants and thus a ligand-receptor dissociation constant
and an affinity constant can be directly determined.
[0313] In Vitro Immunogenicity Test of hG-CSF Conjugates
[0314] The reduced immunogenicity of a conjugate of the invention
can be determined by use of an ELISA method measuring the
immunoreactivity of the conjugate relative to a reference molecule
or preparation. The reference molecule or preparation is normally a
recombinant human G-CSF preparation such as Neupogen.RTM. or
another recombinant human G-CSF preparation, e.g. an N-terminally
PEGylated rhG-CSF molecule as described in U.S. Pat. No. 5,824,784.
The ELISA method is based on antibodies from patients treated with
one of these recombinant G-CSF preparations. The immunogenicity is
considered to be reduced when the conjugate of the invention has a
statistically significant lower response in the assay than the
reference molecule or preparation.
[0315] Neutralisation of Activity in G-CSF Bioassay
[0316] The neutralisation of hG-CSF conjugates by anti-G-CSF sera
is analyzed using the G-CSF bioassay described above.
[0317] Sera from patients treated with the G-CSF reference molecule
or from immunized animals are used. Sera are added either in a
fixed concentration (dilution 1:20-1:500 (pt sera) or 20-1000 ng/ml
(animal sera)) or in five-fold serial dilutions of sera starting at
1:20 (pt sera) or 1000 ng/ml (animal sera). HG-CSF conjugate is
added either in seven fold-dilutions starting at 10 nM or in a
fixed concentration (1-100 pM) in a total volume of 80 .mu.l DMEM
medium+10% FCS. The sera are incubated for 1 hr. at 37.degree. C.
with hG-CSF conjugate.
[0318] The samples (0.01 ml) are then transferred to 96 well tissue
culture plates containing NFS-60 cells in 0.1 ml DMEM media. The
cultures are incubated for 48 hours at 37.degree. C. in a 5%
CO.sub.2 air atmosphere. 0.01 ml WST-1 (WST-1 cell proliferation
agent, Roche Diagnostics GmbH, Mannheim, Germany) is added to the
cultures and incubated for 150 min. at 37.degree. C. in a 5%
CO.sub.2 air atmosphere. The cleavage of the tetrazolium salt WST-1
by mitochondrial dehydrogenases in viable cells results in the
formation of formazan that is quantified by measuring the
absorbance at 450 nm.
[0319] When hG-CSF conjugate samples are titrated in the presence
of a fixed amount of serum, the neutralising effect is defined as
fold inhibition (FI) quantified as EC50 (with serum)/EC50 (without
serum). The reduction of antibody neutralisation of G-CSF variant
proteins is defined as 1 ( 1 - ( FI variant - 1 ) ( FI wt - 1 ) )
.times. 100 %
EXAMPLE 1
[0320] Construction and Cloning of Synthetic Genes Encoding
hG-CSF
[0321] The following DNA fragments were synthesized following the
general procedure described by Stemmer et al. (1995), Gene 164, pp.
49-53:
[0322] Fragment 1, consisting of a Bam HI digestion site, a
sequence encoding the YAP3 signal peptide (WO 98/32867), a sequence
encoding the TA57 leader sequence (WO 98/32867), a sequence
encoding a KEX2 protease recognition site (AAAAGA), a sequence
encoding hG-CSF with its codon usage optimized for expression in E.
coli, (SEQ ID NO:2) and a Xba I digestion site.
[0323] Fragment 2, consisting of a Bam HI digestion site, a
sequence encoding the YAP3 signal peptide (WO 98/32867), a sequence
encoding the TA57 leader sequence (WO 98/32867), a sequence
encoding a histidine tag (SEQ ID NO:5), a sequence encoding a KEX2
protease recognition site (AAAAGA), a sequence encoding hG-CSF with
its codon usage optimized for expression in E. coli, (SEQ ID NO:2)
and a Xba I digestion site.
[0324] Fragment 3, consisting of a Nde I digestion site, a sequence
encoding the OmpA signal peptide (SEQ ID NO:3), a sequence encoding
hG-CSF with its codon usage optimized for expression in E. coli,
(SEQ ID NO:2) and a Bam HI digestion site.
[0325] Fragment 4, consisting of a Bam HI digestion site, the Kozak
consensus sequence (Kozak, M. J Mol Biol 1987 Aug.
20;196(4):947-50), a sequence encoding the hG-CSF signal peptide
(SEQ ID NO:7) and hG-CSF with its codon usage optimized for
expression in CHO cells (SEQ ID NO:8) and a Xba I digestion
site.
[0326] DNA fragment 1 and 2 were inserted into the Bam HI and Xba I
digestion sites in plasmid pJSO37 (Okkels, Ann. New York Acad. Sci.
782:202-207, 1996) using standard DNA techniques. This resulted in
plasmids pG-CSFcerevisiae and pHISG-CSFcerevisiae.
[0327] DNA fragment 3 was inserted into the Nde I and Bam HI
digestion sites in plasmid pET12a (Invitrogen) using standard DNA
techniques. This resulted in plasmid pG-CSFcoli.
[0328] DNA fragment 4 was inserted into the Bam HI and Xba I
digestion sites in plasmid pcDNA3.1 (+) (Invitrogen) using standard
DNA techniques. This resulted in plasmid pG-CSFCHO.
EXAMPLE 2
[0329] Expression of hG-CSF in S. cerevisiae and E. coli
[0330] Transformation of Saccharomyces cerevisiae YNG318 (available
from the American Type Culture Collection, VA, USA as ATCC 208973)
with either plasmid pG-CSFcerevisiae or pHISG-CSFcerevisiae,
isolation of transformants containing either of the two plasmids,
and subsequent extracellular expression of hG-CSF without and with
the HIS tag, respectively, was performed using standard techniques
described in the literature. Transformation of E. coli BL21 (DE3)
(Novagen, Cat. No. 69387-3) with pG-CSFcoli, isolation of
transformants containing the plasmid and subsequent expression of
hG-CSF in the supernatant and in the periplasm of the cell was
performed as described in the pET System Manual (8.sup.th edition)
from Novagen.
[0331] Expression of hG-CSF by S. cerevisiae and E. coli was
verified by Western Blot analysis using the ImmunoPure
Ultra-Sensitive ABC Rabbit IgG Staining kit (Pierce) and a
polyclonal antibody against hG-CSF (Pepro Tech EC Ltd.). It was
observed that the protein had the correct size.
[0332] The expression levels of hG-CSF with and without the
N-terminal histidine tag in S. cerevisiae and E. coli were
quantified using a commercially available G-CSF specific ELISA kit
(Quantikine Human G-CSF Immunoassay, R&D Systems Cat. No.
DCS50). The measured values are listed below.
6 Expression level Expression system (mg G-CSF per L) hG-CSF in S.
cerevisiae 30 hG-CSF with histidine tag in S. cerevisiae 25 hG-CSF
in E. coli 0.05
EXAMPLE 3
[0333] Generation of a Stable CHO-K1 G-CSF Producer
[0334] The day before transfection the CHO K1 cell line (ATCC
#CCl-61) is seeded in a T-25 flask in 5 ml DMEM/F-12 medium (Gibco
# 31330-038) supplemented with 10% FBS and penicillin/streptomycin.
The following day (at nearly 100% confluency) the transfection is
prepared: 90 .mu.l DMEM medium without supplements is aliquoted
into a 14 ml polypropylene tube (Corning). 10 .mu.l Fugene 6
(Roche) is added directly into the medium and incubated for 5 min
at room temperature. In the meantime 5 .mu.g plasmid pG-CSFCHO is
aliquoted into another 14 ml polypropylene tube. After incubation
the Fugene 6 mix is added directly to the DNA solution and
incubated for 15 min at room temperature. After incubation the
whole volume is added drop-wise to the cell medium.
[0335] The next day the medium is exchanged with fresh medium
containing 360 .mu.g/ml hygromycin (Gibco). Every day hereafter the
selection medium is renewed until the primary transfection pool has
reached 100% confluency. The primary transfection pool is
sub-cloned by limited dilution (300 cells seeded in five 96-well
plates).
EXAMPLE 4
[0336] Purification of hG-CSF and Variants Thereof from S.
cerevisiae Culture Supernatants
[0337] Purification of hG-CSF was Performed as Follows:
[0338] Cells are removed by centrifugation. Cell depleted
supernatant is then filter sterilized through a 0.22 .mu.m filter.
Filter sterilized supernatant is diluted 5 fold in 10 mM sodium
acetate pH 4.5. pH is adjusted by addition of 10 ml concentrated
acetic acid per 5 liters of diluted supernatant. The ionic strength
should be below 8 mS/cm before application to the cation exchange
column.
[0339] Diluted supernatant is loaded at a linear flow rate of 90
cm/h onto a SP-sepharose FF (Pharmacia) column equilibrated with 50
mM sodium acetate, pH 4.5 until the effluent from the column
reaches a stable UV and conductivity baseline. To remove any
unbound material, the column is washed using the equilibration
buffer until the effluent from the column reaches a stable level
with respect to UV absorbance and conductivity. The bound G-CSF
protein is eluted from the column using a linear gradient; 30
column volumes; 0-80% buffer B (SOM NaAc, pH 4.5, 750 mM NaCl) at a
flow rate of 45 cm/h. Based on SDS-polyacryl amide gel
electrophoresis, fractions containing G-CSF are pooled. Sodium
chloride is added until the ionic strength of the solution is more
than 80 mS/cm.
[0340] The protein solution is applied onto a Phenyl Toyo Pearl
650S column equilibrated with 50 mM NaAc, pH 4.5, 750 mM NaCl. Any
unbound material is washed off the column using the equilibration
buffer. Elution of G-CSF is performed by applying a step gradient
of MilliQ water. Fractions containing G-CSF are pooled. By using
this 2-step down stream processing strategy, more than 90% pure
G-CSF can be obtained. The purified protein is then quantified
using spectrophotometric measurements at 280 nm and/or by amino
acid analysis.
[0341] Fractions containing G-CSF are pooled. Buffer exchange and
concentration is performed using VivaSpin concentrators (mwco: 5
kDa).
EXAMPLE 5
[0342] Identification and Quantification of Non-Conjugated and
Conjugated hG-CSF and Variants Thereof
[0343] SDS-Polyacryl Amide Gel Electrophoresis
[0344] The purified, concentrated G-CSF was analyzed by SDS-PAGE. A
single band having an apparent molecular weight of approx. 17 kDa
was dominant.
[0345] Absorbance
[0346] An estimate of the G-CSF concentration is obtained by
spectrophotometric methods. By measuring the absorbance at 280 nm
and using a theoretically extinction coefficient of 0.83, the
protein concentration can be calculated.
[0347] Amino Acid Analysis
[0348] A more accurate protein determination can be obtained by
amino acid analysis. Amino acid analysis performed on a purified
G-CSF revealed that the experimentally determined amino acid
composition is in agreement with the expected amino acid
composition based on the DNA sequence.
EXAMPLE 6
[0349] MALDI-TOF mass spectrometry of PEGylated wt G-CSF and G-CSF
variants MALDI-TOF mass spectrometry was used to evaluate the
number of PEG-groups attached to PEGylated wt G-CSF and to selected
PEGylated G-CSF variants.
[0350] Wt G-CSF contains 5 primary amines that are the expected
attachment sites for SPA-PEG (the N-terminal amino-group and the
.epsilon.-amino-group on K16, K23, K34 and K40). Following
PEGylation of wt G-CSF with SPA-PEG 5000, MALDI-TOF mass
spectrometry showed the presence of species of wt G-CSF with mainly
4, 5 and 6 PEG-groups attached. In addition, wt G-CSF with 7
PEG-groups attached was clearly seen although in minor amounts.
[0351] The G-CSF variant having the substitutions K16R, K34R, K40R,
Q70K, Q90K, and Q120K also contains 5 primary amines (the
N-terminal amino-group and the .epsilon.-amino-group on K23, K70,
K90 and K120). Following PEGylation of this G-CSF variant with
SPA-PEG5000, MALDI-TOF mass spectrometry showed the presence of
species of the G-CSF variant with mainly 4, 5 and 6 PEG-groups
attached. In addition, the G-CSF variant with 7 PEG-groups attached
was clearly seen although in minor amounts.
[0352] The G-CSF variant having the substitutions K16R, K34R, and
K40R contains 2 primary amines (the N-terminal amino-group and the
.epsilon.-amino-group on K23). Following PEGylation of this G-CSF
variant with SPA-PEG 12000, MALDI-TOF mass spectrometry showed the
presence of species of the G-CSF variant with mainly 2 and 3
PEG-groups attached. In addition, the G-CSF variant with 4
PEG-groups attached was clearly seen although in minor amounts.
[0353] These observations clearly show that in addition to amino
acid residues containing amine groups, other amino acid residues
are sometimes PEGylated under the PEGylation conditions used. It
also shows that it is of some importance for the PEGylation where
amine groups are introduced. This has also been observed using
SDS-PAGE analysis of wt G-CSF and G-CSF variants.
[0354] As described in Example 12, it has been shown that histidine
170 is fully PEGylated when the SPA-PEG chemistry is used.
Furthermore, K23 and S159 are partly PEGylated. This explains the
presence of 1-2 extra PEGylation sites besides the primary amines
in hG-CSF and the variants that have been made.
EXAMPLE 7
[0355] Peptide Mapping of PEGylated and Non-PEGylated G-CSF
Variants
[0356] In order to map the additional attachment sites for SPA-PEG
on G-CSF and G-CSF variants the following strategy was used.
[0357] A G-CSF variant with a low number of amine groups was chosen
in order to reduce the number of expected PEGylation sites to a
minimum. The G-CSF variant chosen has the substitutions K16R, K34R,
K40R and H170Q. Apart from the .epsilon.-amino-group on K23 that
previous data had shown not to be PEGylated to any large extent,
this variant only contains one primary amine at the N-terminal.
Thus, the background PEGylation on amine groups is significantly
reduced in this G-CSF variant. The G-CSF variant was PEGylated
using SPA-PEG 5000. Following PEGylation, the G-CSF variant was
denatured, the disulphide bonds reduced, the resulting thiol groups
alkylated, and the alkylated and PEGylated protein degraded with a
glutamic acid-specific protease. Finally, the resulting peptides
were separated by reversed phase HPLC.
[0358] Parallel with this, the non-PEGylated version of the G-CSF
variant with the substitutions K16R, K34R, and K40R was treated
identically in order to create a reference HPLC chromatogram.
[0359] Comparison of the HPLC chromatograms of the degradation of
the PEGylated G-CSF variant and the non-PEGylated G-CSF variant
should then reveal which peptides disappear upon PEGylation.
Identification of these peptides by N-terminal amino acid
sequencing of the peptide from the non-PEGylated G-CSF variant then
indirectly points to the positions that are PEGylated.
[0360] In principle, it would have been preferable to use the
non-PEGylated version of the G-CSF variant having all the
substitutions K16R, K34R, K40R and H170Q, but for all practical
purposes this does not matter.
[0361] More specifically, approximately 1 mg of the PEGylated G-CSF
variant having the substitutions K16R, K34R, K40R and H170Q and
approximately 500 g of the non-PEGylated G-CSF variant having the
substitutions K16R, K34R, and K40R were dried in a SpeedVac
concentrator. The two samples were each dissolved in 400 .mu.l 6 M
guanidinium, 0.3 M Tris-HCl, pH 8.3 and denatured overnight at
37.degree. C. Following denaturation, the disulfide bonds in the
proteins were reduced by addition of 50 .mu.l 0.1 M DTT in 6 M
guanidinium, 0.3 M Tris-HCl, pH 8.3. After 2 h of incubation at
ambient temperature the thiol groups present were alkylated by
addition of 50 .mu.l 0.6 M iodoacetamid in 6 M guanidinium, 0.3 M
Tris-HCl, pH 8.3. Alkylation took place for 30 min at ambient
temperature before the reduced and alkylated proteins were buffer
changed into 50 mM NH.sub.4HCO.sub.3 using NAP5 columns. The
volumes of the samples were reduced to approximately 200 .mu.l in a
SpeedVac concentrator before addition of 20 .mu.g and 10 .mu.g
glutamic acid-specific protease, respectively. The degradations
with glutamic acid-specific protease were carried out for 16 h at
37.degree. C. The resulting peptides were separated by reversed
phase HPLC employing a Phenomenex Jupiter C18 column (0.2*5 cm)
eluted with a linear gradient of acetonitrile in 0.1% aqueous TFA.
The collected fractions were analyzed by MALDI-TOF mass
spectrometry and subsequently selected peptides were subjected to
N-terminal amino acid sequence analysis.
[0362] Comparison of the HPLC chromatograms of the degradations of
the PEGylated G-CSF variant and the non-PEGylated G-CSF variant
revealed that only two fractions disappear upon PEGylation.
N-terminal amino acid sequence analysis of the two fractions from
the non-PEGylated G-CSF variant showed that the peptides both were
derived from the N-terminal of G-CSF. One peptide consisted of
amino acid residues 1-11 generated by an unexpected cleavage
following Gln11. The other peptide consisted of amino acid residues
1-19 generated by an expected cleavage following Glu19.
[0363] It was expected that the N-terminal peptide of G-CSF would
be identified using this approach, as the N-terminal amino group is
easily PEGylated. However, none of the additional attachment sites
for SPA-PEG 5000 were identified using this approach.
[0364] An alternative to the indirect identification of PEG 5000
attachment sites is direct identification of the attachment sites
in PEGylated peptides. However, the fractions containing the
PEGylated peptides in the HPLC separation of the degraded PEGylated
G-CSF variant are poorly separated from each other and from several
fractions containing non-PEGylated peptides. Thus, N-terminal amino
acid sequence analysis of these fractions did not result in any
useful data except for an indication that K23 could be partially
PEGylated.
[0365] To overcome these problems, two pools of PEGylated peptides
were made from the fractions from the first HPLC separation. These
two pools were dried in a SpeedVac concentrator, dissolved in 200%1
freshly prepared 50 mM NH.sub.4HCO.sub.3 and further degraded with
1 .mu.g of chymotrypsin. The resulting peptides were separated by
reversed phase HPLC employing a Phenomenex Jupiter C.sub.18 column
(0.2*5 cm) eluted with a linear gradient of acetonitrile in 0.1%
aqueous TFA. The collected fractions were analyzed by MALDI-TOF
mass spectrometry and subsequently selected peptides were subjected
to N-terminal amino acid sequence analysis.
[0366] From the N-terminal amino acid sequence determinations it
could be determined that K23 as well as S159 are partially
PEGylated. It was not possible to determine the exact degree of
PEGylation at these two positions, but the PEGylation is only
partial as peptides where K23 and S159 are unmodified were
identified and sequenced from the initial HPLC separation.
EXAMPLE 8
[0367] Glycosylation of wt G-CSF and G-CSF Variants
[0368] A consistent observation when analyzing purified wt G-CSF
and G-CSF variants by MALDI-TOF mass spectrometry is the presence
of an additional component with a mass approximately 324 Da larger
than the mass of the G-CSF molecule analyzed. As the component with
the lowest mass invariantly has the mass of the G-CSF molecule and
because the G-CSF molecules have the correct N-terminal amino acid
sequence, it was concluded that the additional component is a
modified G-CSF molecule carrying two hexose residues. In many cases
the unmodified G-CSF molecule gives rise to the most intense signal
but in some cases the intensity of the signal for the modified
G-CSF molecule is the most intense.
[0369] During the analysis of the peptides generated with the aim
of identifying the additional PEGylation sites, two peptides of
interest for identifying the site of glycosylation were identified
in each of the degradations.
[0370] In both HPLC separations, the two peptides elute next to
each other and MALDI-TOF mass spectrometry shows a mass difference
between the two peptides of approximately 324 Da. The mass
spectrometry data indicates that the peptide covers amino acid
residues 124-162. N-terminal amino acid sequence analysis of all
four peptides showed that this assignment is correct and that
Thr133 is the only site of modification. In the peptides with the
mass of the unmodified peptide, Thr133 is clearly seen in the
sequence, while no amino acid residue can be assigned at position
133 in the peptides with an additional mass of 324 Da. As all other
amino acid residues could be assigned in the sequence, it was
concluded that Thr133 is the only site of modification. This
glycosylation site was previously reported to be used in
recombinant G-CSF expressed in CHO cells, although the glycan is
different from the one attached by yeast.
[0371] The non-glycosylated wt G-CSF has been separated from the
glycosylated wt G-CSF, employing reversed phase HPLC using a Vydac
C18 column (0.21*5 cm) isocratically eluted with 51% acetonitrile
in 0.1% TFA, as a fraction shown by MALDI-TOF mass spectrometry
only to contain the non-glycosylated form of wt G-CSF.
EXAMPLE 9
[0372] Separation of G-CSF Molecules with Different Numbers of PEG
Molecules Covalently Attached
[0373] Separation of G-CSF molecules covalently attached to 4, 5 or
6 PEG-groups was obtained as follows. PEGylated protein in 20 mM
sodium citrate, pH 2.5 was applied to an SP-sepharose FF column
equilibrated with 20 mM sodium citrate pH 2.5. Any unbound material
was washed off the column. Elution was performed using a pH
gradient. PEGylated G-CSF began to elute from the column at approx.
pH 3.8 and continued to elute in fractions covering a pH span from
3.8 to 4.5.
[0374] The fractions were subjected to SDS-PAGE and mass
spectrometric analysis. These analyses indicate that G-CSF having
the highest degree of PEGylation is located in the "low pH
fractions". PEGylated G-CSF having a lower degree of PEGylation is
eluted in the "high pH fractions".
[0375] Amino acid analysis performed on PEGylated G-CSF showed good
consistency between the theoretically and the experimentally
determined extinction coefficient.
EXAMPLE 10
[0376] Construction of hG-CSF Variants
[0377] Specific substitutions of existing amino acids in hG-CSF to
other amino acid residues, e.g. the specific substitutions
discussed above in the general description, were introduced using
standard DNA techniques known in the art. The new G-CSF variants
were made using plasmid pG-CSFcerevisiae containing the gene,
encoding hG-CSF without the HIS tag, as DNA template in the PCR
reactions. The variants were expressed in S. cerevisiae and
purified as described in Example 4. Some of the constructed G-CSF
variants are listed below (see Examples 12 and 13).
EXAMPLE 11
[0378] Covalent Attachment of SPA-PEG to hG-CSF or Variants
Thereof
[0379] Human G-CSF and variants thereof were covalently linked to
SPA-PEG 5000, SPA-PEG 12000 and SPA-PEG 20000 (Shearwater) as
described above ("PEGylation of hG-CSF and variants thereof in
solution"). The in vitro activities of the conjugates are listed in
Example 13.
EXAMPLE 12
[0380] Identification of SPA-PEG Attachment Sites in G-CSF by
Site-Directed Mutagenesis Followed by PEGylation of the Purified
Variants
[0381] SPA-PEG may be attached to other amino acid residues than
lysine in G-CSF. In order to determine whether SPA-PEG was attached
to histidines, serines, threonines and arginines, variants were
made in which these amino acids were substituted to lysine, alanine
or glutamine. The variants were expressed in S. cerevisiae,
purified and PEGylated followed by analysis of the number of
attached SPA-PEG molecules on SDS-PAGE. This analysis was performed
by visual inspection of the SDS-PAGE gels, all of which contained
three major bands. The degree of PEGylation was estimated to the
nearest 5% for each band based on the relative size of the bands. A
reduction in the number of attached SPA-PEG molecules after
substitution of a given amino acid with glutamine or alanine
strongly indicates that this amino acid is PEGylated by SPA-PEG,
and this observation is further supported by an unchanged degree of
PEGylation after substitution of the amino acid to lysine. The
analyzed variants are listed below.
7 G-CSF variant No. of attached PEG groups hG-CSF 10% 4 PEG, 75% 5
PEG, 15% 6 PEG K23R 10% 4 PEG, 85% 5 PEG, 5% 6 PEG H43Q 10% 4 PEG,
75% 5 PEG, 15% 6 PEG H43K 10% 5 PEG, 75% 6 PEG, 15% 7 PEG H52Q 10%
4 PEG, 75% 5 PEG, 15% 6 PEG H52K 10% 5 PEG, 75% 6 PEG, 15% 7 PEG
H156Q 10% 4 PEG, 75% 5 PEG, 15% 6 PEG H156K 10% 5 PEG, 75% 6 PEG,
15% 7 PEG H170Q 10% 3 PEG, 75% 4 PEG, 15% 5 PEG H170K 10% 4 PEG,
75% 5 PEG, 15% 6 PEG K16/34R 10% 2 PEG, 75% 3 PEG, 15% 4 PEG
K16/34R R22K 10% 3 PEG, 75% 4 PEG, 15% 5 PEG K16/34R R22Q 10% 2
PEG, 75% 3 PEG, 15% 4 PEG K16/34R S142A 10% 2 PEG, 75% 3 PEG, 15% 4
PEG K16/34/40R 10% 1 PEG, 75% 2 PEG, 15% 3 PEG K16/34/40R S53K 10%
2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S53A 10% 2 PEG, 75% 3 PEG,
15% 4 PEG K16/34/40R S62K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG
K16/34/40R S66K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S80K 10%
2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R T105K 10% 2 PEG, 75% 3 PEG,
15% 4 PEG K16/34/40R T133K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG
K16/34/40R S142K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R R147K
10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S155K 10% 2 PEG, 75% 3
PEG, 15% 4 PEG K16/34/40R S159K 10% 2 PEG, 85% 3 PEG, 5% 4 PEG
K16/34/40R S170K 10% 1 PEG, 75% 2 PEG, 15% 3 PEG
[0382] The data show that besides the N-terminus, K16, K34 and K40,
SPA-PEG also is covalently bound to H170. Furthermore, the data
show that only 10% of the available K23 amino acid residues are
PEGylated, and that approximately 10% of S159 is PEGylated.
EXAMPLE 13
[0383] In Vitro Biological Activity of Non-Conjugated and
Conjugated hG-CSF and Variants Thereof
[0384] The in vitro biological activities of conjugated and
non-conjugated hG-CSF and variants thereof were measured as
described above in "Primary assay 2--in vitro hG-CSF activity
assay". The in vitro bioactivities, represented by the measured
EC50 values for each variant with and without conjugation of
SPA-PEG 5000 to the available PEGylation sites, are listed below.
The values have been normalized with respect to the EC50 value of
non-conjugated hG-CSF (Neupogen.RTM.), i.e. the values in the table
indicate % activity relative to the activity of non-conjugated
hG-CSF. This value was measured simultaneously with the variants
each time under identical assay conditions. The EC50 value of
hG-CSF in the described assay is 30 pM.
8 EC50 (% of hG-CSF) EC50 (% of hG-CSF) conjugated to G-CSF variant
non-conjugated SPA-PEG 5000 G-CSF with N-terminal Histidine tag 10
Not determined G-CSF without N-terminal Histidine tag 100 0.1 16R
100 1 16Q 80 1 23Q 80 0.1 23R 100 0.1 34R 100 1 34A 80 1 34Q 70 1
40R 50 1 K16/23R 100 1 K16/23Q 80 1 K34/40R 50 5 K16/34R 100 10
K16/40R 50 5 K16/23/34R 50 10 K16/23/40R 50 5 K16/34/40R 35 30
K16/23/34/40R 20 15 K16/34/40R L3K 50 25 K16/34/40R E45K Expressed
at low levels Not determined K16/34/40R E46K 10 1 K16/34/40R S53K 5
0.5 K16/34/40R S62K 10 0.5 K16/34/40R S66K 20 2 K16/34/40R Q67K 10
0.2 K16/34/40R Q70K 30 20 K16/34/40R S76 50 20 K16/34/40R Q77 1 0
K16/34/40R S80K 10 0.2 K16/34/40RQ90K 30 20 K16/34/40R E98K
Expressed at low levels Not determined K16/34/40R D104K 10 0.9
K16/34/40R T105K 30 10 K16/34/40R Q120K 30 20 K16/34/40R Q131K
Expressed at low levels Not determined K16/34/40R T133K 30 10
K16/34/40R Q134K 10 0.2 K16/34/40R S142K 20 7 K16/34/40R R147K 20 1
K16/34/40R S155K 20 1 K16/34/40R Q158 20 5 K16/34/40R S159K 20 3
K16/34/40R Q70K Q90K Not determined 20 K16/34/40R Q70K Q120K 25 25
K16/34/40R Q90K T105K 40 10 K16/34/40R Q90K Q120K 25 15 K16/34/40R
Q90K S159K 45 Not determined K16/34/40R T105K Q120K 20 8 K16/34/40R
T105K S159K 40 20 K16/34/40R Q120K T133K 20 8 K16/34/40R Q120K
S142K 10 2 K16/34/40R Q70K Q90K T105K 10 4 K16/34/40R Q70K Q90K
Q120K 20 12 K16/34/40R Q70K Q90K T133K 15 5 K16/34/40R Q70K T105K
Q120K 10 2 K16/34/40R Q70K Q120K T133K 15 2 K16/34/40R Q70K Q120K
S142K 10 1 K16/34/40R Q90K T105K Q120K 10 2 K16/34/40R Q90K T105K
T133K 10 2 K16/34/40R Q90K T105K S159K 55 5 K16/34/40R Q90K Q120K
T133K 15 2 K16/34/40R Q90K Q120K S142K 10 1 K16/34/40R T105K Q120K
T133K 10 1 K16/34/40R Q120K T133K S142K 10 1
[0385] The data show that substitution of K23 to arginine does not
increase the activity of the conjugated protein. This is due to the
fact that only 10% of K23 is PEGylated, whereby the conjugated K23R
variant has essentially the same number of PEG groups attached to
it and has the same location of the PEGylation sites as hG-CSF.
Removal of the remaining lysines at position K16, K34 and K40
resulted in a G-CSF variant with significant activity after
PEGylation. Conjugation of SPA-PEG 5000 to this variant does not
decrease the activity significantly as compared to the
non-conjugated variant. Thus, PEGylation of the N-terminus and H170
with SPA-PEG 5000 (see Example 12) does not decrease the activity
of hG-CSF. It was decided to use hG-CSF K16R K34R K40R as the
backbone for insertion of new PEGylation sites. 24 new PEGylation
sites between residues L3 and H159 were introduced in this
backbone. These residues are distributed over the parts of hG-CSF
that do not interact with the G-CSF receptor. Introduction of new
PEGylation sites at positions L3, Q70, S76, Q90, T105, Q120, T133
and S142 resulted in hG-CSF variants that retained a significant
amount of activity after PEGylation by SPA-PEG 5000. Thus, some of
these new PEGylation sites were combined in hG-CSF variants that
had 2 or 3 new PEGylation sites.
[0386] Furthermore, SPA-PEG 12000 and SPA-PEG 20000 were attached
to a group a selected hG-CSF variants. The in vitro activities are
listed below (% of Neupogen.RTM.).
9 EC50 EC50 (% of hG-CSF) (% of hG-CSF) conjugated to conjugated to
G-CSF variant SPA-PEG 12000 SPA-PEG 20000 K16/34/40R 10 1
K16/34/40R Q90K Not determined 7 K16/34/40R Q70K Q90K 8 Not
determined K16/34/40R Q90K T105K 1 <1 K16/34/40R T105K S159K 6 5
K16/34/40R Q90K T105K S159K 1 <1
EXAMPLE 14
[0387] In Vivo Half-Life of Non-Conjugated and Conjugated hG-CSF
and Variants Thereof
[0388] The in vivo half-lives of non-conjugated hG-CSF
(Neupogen.RTM.), SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K
Q90K Q120K and SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q90K
T105K S159K were measured as described above ("Measurement of the
in vivo half-life of conjugated and non-conjugated rhG-CSF and
variants thereof"). The results are shown in FIGS. 1 and 2. The in
vivo half-life of Neupogen.RTM. was determined to be 2.01 hours and
1.40 hours, respectively. In an earlier, similar experiment (U.S.
Pat. No. 5,824,778), the in vivo half-life of hG-CSF was determined
to be 1.79 hours. The results of the experiments described herein
can therefore be directly compared to that experiment. The in vivo
half-lives of SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K
Q90K Q120K and SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q90K
T105K S159K were determined to be 12.15 hours and 16.10 hours,
respectively. Thus, introducing new PEGylation sites in hG-CSF and
conjugating SPA-PEG 5000 to them has resulted in a significant
increase in the in vivo half-life.
[0389] In the earlier experiment described above (U.S. Pat. No.
5,824,778), the in vivo half-life of hG-CSF conjugated to a larger
N-terminally attached PEG molecule (10 kDa) was determined to be
7.05 hours. Thus, SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R
Q70K Q90K Q120K and SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R
Q90K T105K S159K have significantly longer half-lives than both
Neupogen.RTM. and hG-CSF with a 10 kDa N-terminally conjugated PEG
molecule. SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K
Q120K and SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q90K T105K
S159K both have three removed endogenous PEGylation sites and three
new introduced PEGylation sites and thus are identical in size. The
only difference between the two compounds is the in vitro activity,
which is 12% and 5%, respectively, of that of Neupogen.RTM.. This
difference results in a longer in vivo half-life of SPA-PEG 5000
conjugated K16R K34R K40R Q90K T105K S159K compared to SPA-PEG 5000
conjugated K16R K34R K40R Q70K Q90K Q120K. Since the in vitro
activities correlate with the receptor binding affinities of the
compounds, it can be concluded that the receptor-mediated clearance
of SPA-PEG 5000 conjugated K16R K34R K40R Q90K T105K S159K is
slower than that of SPA-PEG 5000 conjugated K16R K34R K40R Q70K
Q90K Q120K. Thus, a combination of increasing the size and reducing
the in vitro activity of G-CSF results in G-CSF compounds with
significantly longer in vivo half-lives than previously described
compounds.
EXAMPLE 15
[0390] In Vivo Biological Activity in Healthy Rats of
Non-Conjugated and Conjugated hG-CSF and Variants Thereof.
[0391] The in vivo biological activities of non-conjugated hG-CSF
(Neupogen.RTM.), SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K
Q120K, SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K
Q120K, SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q120K
T133K and SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q90K Q120K
T133K were measured as described above ("Measurement of the in vivo
biological activity in healthy rats of conjugated and
non-conjugated hG-CSF and variants thereof"). The results are shown
in FIGS. 3 and 4.
[0392] No activity of Neupogen.RTM. could be detected at 48 hours
after injection of 100 .mu.g per kg body weight at t=0 hours.
Activity of SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K
Q120K, SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q120K
T133K and SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q90K Q120K
T133K could be detected until 72 hours after the initial injection,
while SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K
remained active in vivo until 96 hours after the initial injection.
Thus, it was shown that all of these conjugated variants had a
longer in vivo biological activity than Neupogen.RTM. and that
SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K
remained active twice as long in vivo as Neupogen.RTM.. SPA-PEG
5000 conjugated hG-CSF K16R K34R K40R Q70K Q120K T133K and SPA-PEG
5000 conjugated hG-CSF K16R K34R K40R Q90K Q120K T133K, both with
an in vitro activity of 2% of Neupogen.RTM. (Example 13), did not
induce the same level of white blood cell formation during the
initial 12 hours after administration as observed after
administration of Neupogen.RTM., SPA-PEG 5000 conjugated hG-CSF
K16R K34R K40R Q70K Q120K and SPA-PEG 5000 conjugated hG-CSF K16R
K34R K40R Q70K Q90K Q120K. Thus, the compounds with an in vitro
activity of 2% or less compared to that of Neupogen.RTM. were
unable to stimulate full formation of white blood cells immediately
after administration.
[0393] Furthermore, the in vivo biological activities of
Neupogen.RTM., SPA-PEG 12000 conjugated hG-CSF K16R K34R K40R and
different doses of SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R
Q70K Q90K Q120K were measured as described above ("Measurement of
the in vivo biological activity in healthy rats of conjugated and
non-conjugated hG-CSF and variants thereof"). The results are shown
in FIG. 5. As observed earlier, no activity of Neupogen.RTM. could
be detected 48 hours after the initial injection of 100 .mu.g per
kg body weight. Administration of 5 .mu.g per kg body weight of
SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K
resulted in a slightly longer in vivo biological activity than
Neupogen.RTM., while administration of 25 .mu.g per kg body weight
and 100 .mu.g per kg body weight of this compound resulted in
hG-CSF activity until 72 and 96 hours, respectively, after the
initial injection. Thus, the duration of action of the SPA-PEG
conjugated hG-CSF compounds can be controlled by increasing or
decreasing the standard dosing regimen. SPA-PEG 12000 conjugated
hG-CSF K16R K34R K40R remained active in vivo until 72 hours after
administration of 100 .mu.g per kg body weight. As described in
Example 6, SPA-PEG 12000 conjugated hG-CSF K16R K34R K40R has 2 or
3 SPA-PEG 12000 groups attached while SPA-PEG 5000 conjugated
hG-CSF K16R K34R K40R Q70K Q90K Q120K has 5 or 6 SPA-PEG 5000
groups attached. Thus, the molecular weights of the two compounds
are 42-54 kDa and 43-48 kDa, respectively. The in vitro activities
of the two compounds are 30% and 12%, respectively, of that of
Neupogen.RTM.. The longer in vivo biological activity of SPA-PEG
5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K as compared
to SPA-PEG 12000 conjugated hG-CSF K16R K34R K40R with essentially
the same molecular weight suggests that when the size of the G-CSF
compounds is increased above a certain molecular weight through
PEGylation, the duration of action can only be increased further by
reducing the specific activity of the G-CSF compounds and thus, the
receptor-mediated clearance (see Example 14).
[0394] Furthermore, the in vivo biological activities of
Neupogen.RTM., SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K
Q90K Q120K, SPA-PEG 5000 conjugated hG-CSF K16R 34R 40R Q90K T105K
S159K and SPA-PEG 20000 conjugated hG-CSF K16R 34R 40R T105K S159K
were measured as described above ("Measurement of the in vivo
biological activity in healthy rats of conjugated and
non-conjugated hG-CSF and variants thereof"). The results are shown
in FIG. 6.
[0395] As observed earlier, the conjugated hG-CSF variants had a
significant longer duration of action than Neupogen.RTM..
Administration of each of these three conjugated hG-CSF variants
resulted in formation of white blood cells at the same rate and to
the same level as observed after administration of Neupogen.RTM.
during the initial 12 hours after administration. The in vitro
activities of SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K
Q90K Q120K, SPA-PEG 5000 conjugated hG-CSF K16R 34R 40R Q90K T105K
S159K and SPA-PEG 20000 conjugated hG-CSF K16R 34R 40R T105K S159K
are 12%, 5% and 5%, respectively, of that of Neupogen.RTM., and
thus, a hG-CSF compound with 5% of Neupogen.RTM. activity in vitro
is able to induce full white blood cell formation after
administration.
[0396] The apparent size on SDS-PAGE of Neupogen.RTM., SPA-PEG 5000
conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K, SPA-PEG 5000
conjugated hG-CSF K16R 34R 40R Q90K T105K S159K and SPA-PEG 20000
conjugated hG-CSF K16R 34R 40R T105K S159K is 18 kDa, 60 kDa, 60
kDa and >100 kDa, respectively. SPA-PEG 5000 conjugated hG-CSF
K16R 34R 40R Q90K T105K S159K and SPA-PEG 20000 conjugated hG-CSF
K16R 34R 40R T105K S159K have almost identical durations of action
in vivo, showing that the duration of action is not increased by
increasing the molecular size of the conjugated hG-CSF compounds
above an apparent size of about 60 kDa. Instead, when the apparent
size of the conjugated hG-CSF compounds is above about 60 kDa, the
duration of action may be increased be reducing the in vitro
activity and hence, the receptor binding affinity of the compound.
An additional example of this (see above) can be observed by
comparing the in vivo duration of action of SPA-PEG 5000 conjugated
hG-CSF K16R 34R 40R Q70K Q90K Q120K and SPA-PEG 5000 conjugated
hG-CSF K16R 34R 40R Q90K T105K S159K. The two compounds both have
an apparent size of 60 kDa, while the in vitro activities are 12%
and 5%, respectively. This difference is reflected directly in the
in vivo duration of action of the two compounds, which is 96 hours
and 144 hours, respectively.
EXAMPLE 16
[0397] In Vivo Biological Activity in Rats with
Chemotherapy-Induced Neutropenia of Non-Conjugated and Conjugated
hG-CSF and Variants Thereof.
[0398] The in vivo biological activities in rats with
chemotherapy-induced neutropenia of non-conjugated hG-CSF
(Neupogen.RTM.), SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K
Q90K T105K and SPA-PEG 20000 conjugated hG-CSF K16R K34R K40R Q90K
were measured as described above ("Measurement of the in vivo
biological activity in rats with chemotherapy-induced neutropenia
of conjugated and non-conjugated hG-CSF and variants thereof")
using 50 mg per kg body weight of CPA and a single dose (100 .mu.g
per kg body weight) of G-CSF. The results are shown in FIG. 7. The
three compounds induced an initial formation of white blood cells
with identical rates. Thus, an in vitro activity of 4% of that of
Neupogen.RTM. is sufficient for a conjugated hG-CSF compound to
give full stimulation of white blood cell formation in vivo
immediately after administration. After 36 hours the number of
white blood cells (WBC) in the Neupogen.RTM.-treated rats dropped
to the level that was observed in the untreated group
(<3.times.10.sup.9 cells per litre). At this point the rats were
neutropenic. The level of WBC in both groups reached normal levels
(9.times.10.sup.9 cells per litre) after 144 hours.
[0399] The level of WBC in the two groups treated with SPA-PEG 5000
conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K and SPA-PEG 20000
conjugated hG-CSF K16R K34R K40R Q90K dropped to a minimum of
4.times.10.sup.9 cells per litre after 48 hours and then
immediately started to increase. The WBC levels in both groups were
back to normal after 96 hours. Thus, the two conjugated hG-CSF
compounds were able to both relieve the degree of neutropenia and
to reduce the time until the WBC levels were back to normal (the
duration of neutropenia) significantly from 112 hours in the
Neupogen.RTM.-treated group to 48 hours in the groups treated with
either SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K
T105K and SPA-PEG 20000 conjugated hG-CSF K16R K34R K40R Q90K.
[0400] SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K
T105K more efficiently shortened the duration of neutropenia as
compared to SPA-PEG 20000 conjugated hG-CSF K16R K34R K40R Q90K.
Since the apparent size of both molecules is above 60 kDa (60 kDa
and 80 kDa, respectively) this cannot be explained by a lower renal
clearance of SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K
Q90K T105K than SPA-PEG 20000 conjugated hG-CSF K16R K34R K40R
Q90K. The in vitro activity of SPA-PEG 5000 conjugated hG-CSF K16R
K34R K40R Q70K Q90K T105K and SPA-PEG 20000 conjugated hG-CSF K16R
K34R K40R Q90K are 4% and 7% of Neupogen.RTM., respectively. This
means that the receptor binding affinity and thus, the
receptor-mediated clearance, of SPA-PEG 5000 conjugated hG-CSF K16R
K34R K40R Q70K Q90K T105K is lower than for SPA-PEG 20000
conjugated hG-CSF K16R K34R K40R Q90K in the initial 48 hours after
administration where the white blood cell levels are increased.
Hence, when the rats become neutropenic after 48 hours, the in vivo
concentration of SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K
Q90K T105K is higher than SPA-PEG 20000 conjugated hG-CSF K16R K34R
K40R Q90K. Since a relatively low in vitro G-CSF activity of 4-5%
of that of Neupogen.RTM. is sufficient to obtain full activation of
the G-CSF receptors on the neutrophil progenitor cells (see above),
this higher G-CSF concentration after 48 hours explains the faster
increase in WBC levels in the SPA-PEG 5000 conjugated hG-CSF K16R
K34R K40R Q70K Q90K T105K-treated group. Thus, in rats with
chemotherapy-induced neutropenia, a conjugated G-CSF compound of
the invention with an apparent size of at least about 60 kDa and an
in vitro activity of 4% of that of Neupogen.RTM. is superior to
similar size compounds with a higher in vitro activity.
EXAMPLE 17
[0401] Purification of G-CSF from S. cerevisiae Culture
Supernatants
[0402] This example provides an alternative purification procedure
to that of Example 4 for purification of hG-CSF and G-CSF
variants.
[0403] Cells are removed by centrifugation, 5000 rpm, 10 min,
4.degree. C., and the clarified supernatant is filtered through a
0.22 .mu.m filter. The clarified and filtered supernatant is
concentrated and diafiltered into 50 mM sodium acetate, pH 4.5, by
Tangential Flow Filtration using 10 kDa membranes.
[0404] The resulting ultra filtrate is applied onto an SP-sepharose
column (200 ml packed bed) equilibrated with at least 5 column
volumes of 50 mM sodium acetate. Samples are loaded at a flow rate
of approx. 20 ml/min. The column is washed using the equilibration
buffer until a stable effluent is obtained as determined by
absorbance at 280 nm. Using a stepwise buffer gradient (e.g. 10%,
20%, 30% and 35% buffer), G-CSF is eluted at 35% buffer at ambient
flow rate, where the buffer is 750 mM NaCl in 50 mM sodium
acetate.
[0405] This one-step method yields >95% pure G-CSF (as
determined by SDS-PAGE).
EXAMPLE 18
[0406] Separation of Multi-PEGylated Species of G-CSF
[0407] Example 9 above describes a method for separation of G-CSF
molecules with different numbers of PEG groups attached. This
example provides an alternative procedure for separation of such
multi-PEGylated G-CSF species in order to obtain a G-CSF product
with a desired degree of uniformity in terms of the number of
attached PEG groups.
[0408] A mixture of PEGylated G-CSF, covalently linked to e.g.
SPA-PEG 5000 (Shearwater) as described above ("PEGylation of hG-CSF
and variants thereof in solution"), is diluted with 20 mM citrate
buffer, pH 2.5. The conductivity should be <3.5 mS/cm. The pH is
adjusted to 2.5 as necessary using dilute HCl. The following
buffers are used for the separation:
[0409] Buffer A: 20 mM sodium citrate, pH 2.5 (equilibration and
washing buffer).
[0410] Buffer B: 20 mM sodium citrate, pH 2.5; 750 mM sodium
chloride (elution buffer).
[0411] The sample to be separated is loaded onto an equilibrated
SP-sepharose HP column (7 ml) at a flow rate of 2 ml/min. The
column is washed with Buffer A until a stable baseline is obtained
as monitored by A.sub.280.
[0412] Multi-PEGylated species are separated by applying a linear
gradient of 0-50% Buffer B for 180 minutes at a flow rate of 4
ml/min and collecting 2 ml fractions. The collected fraction are
analyzed by SDS-PAGE, and fractions having a desired number of
attached PEG groups are pooled. This allows purification of a
PEGylated G-CSF mixture comprising species initially having, e.g.,
3-6 attached PEG groups to result in a product having e.g. only 4
or 5 PEG groups attached, or a product having only a single number
of attached PEG groups.
EXAMPLE 19
[0413] Peptide Mapping
[0414] Using a similar procedure to that described above in Example
7, but based on degradation with trypsin, the PEGylation pattern of
a G-CSF conjugate of the invention was determined by peptide
mapping. In this case, the polypeptide was produced in CHO cells
(see Example 3) and had the substitutions K16R, K34R, K40R, T105K
and S159K relative to the sequence of native human G-CSF. It was
PEGylated with 5 kDa SPA-PEG as described above, resulting in
modified proteins carrying predominantly 3, 4 or 5 PEG moieties,
and to a small extent 6 PEG moieties. Five of the six possible PEG
attachment sites are known, these being the N-terminal amino group,
Lys23, Lys105, Lys159 and His170.
[0415] This peptide mapping analysis showed that the conjugated
protein was essentially fully PEGylated at the N-terminal and at
Lys105 and Lys159, while Lys23 was partially PEG-ylated. Although
His 170 has been shown to be partially PEGylated in previous
experiments, this was surprisingly not found in this experiment.
One possible explanation for this observation is that the bond
between the PEG and the His 170 residue may be unstable during the
sample preparation carried out prior to the peptide mapping. A
possible unstable PEGylation such as may be the case here may be
avoided by substituting the histidine residue with another residue,
in particular a lysine residue if a more stable PEGylation is
desired, or a glutamine or arginine residue if PEGylation is to be
avoided.
EXAMPLE 20
[0416] In Vivo Biological Activity in Rats with
Chemotherapy-Induced Neutropenia
[0417] The in vivo biological activity of two PEGylated G-CSF
variants of the invention was tested in rats with
chemotherapy-induced neutropenia. The variants had, relative to SEQ
ID NO:1, the amino acid substitutions K16R, K34R, K40R, T105K and
S159K (referred to below as "105/159") and K16R, K34R, K40R, Q90K,
T105K and S159K (referred to as "90/105/159"), respectively. Both
variants were produced in yeast (S. cerevisiae) and were conjugated
with SPA-PEG-5000 as described above. The in vivo biological
activity of a single dose of the two variants was tested against
the activity of daily doses of non-conjugated hG-CSF
(Neupogen.RTM.) and a control (vehicle).
[0418] 24 hours before administration of the G-CSF samples, the
rats were given 50 mg per kg body weight of CPA. The PEGylated
variants of the invention were administered as a single dose of 100
.mu.g per kg. body weight at time 0, while Neupogen.RTM. was
administered in daily doses of 30 .mu.g per kg body weight for 5
days (from 0 hours to 96 hours).
[0419] The in vivo biological activity was measured as described
above ("Measurement of the in vivo biological activity in rats with
chemotherapy-induced neutropenia of conjugated and non-conjugated
hG-CSF and variants thereof"). The results are shown in FIG. 8
(white blood cell count, WBC) and in FIG. 9 (absolute neutrophil
count, ANC).
[0420] As seen in FIG. 8, administration of 105/159, 90/105/159 and
Neupogen.RTM. all resulted in an initial increase in white blood
cell levels in the first 12 hours, after which the white blood cell
levels fell as a result of the chemotherapy, reaching a minimum
after about 48 hours. After 48 hours, the numbers of white blood
cells increased for all three treatment groups, although the rate
of increase was clearly greater for the group treated with the two
PEGylated variants of the invention than for the group treated with
Neupogen.RTM.. Treatment with the PEGylated variants 105/159 and
90/105/159 resulted in a normal level of white blood cells (over
10.times.10.sup.9/l) after 96 hours, while the Neupogen.RTM.
treated group still had a white blood cell level under
10.times.10.sup.9/l after 120 hours. Since the last of the five
daily Neupogen.RTM. treatments was given at 96 hours, the white
blood cell level in this group fell again after 120 hours. In
contrast, the white blood cell level in the two groups treated with
a single dose of the PEGylated variants of the invention was
relatively stable at just over 10.times.10.sup.9/l from 96 hours
and for the duration of the experiment until 216 hours.
[0421] A similar pattern for the numbers of neutrophils is seen in
FIG. 9, which shows that the neutrophil level for the group treated
with the PEGylated variant 105/159 increased significantly faster
than for the group treated with Neupogen.RTM. (ANC was not
determined for the 90/105/159 group).
EXAMPLE 21
[0422] In Vivo Biological Activity in Rats with
Chemotherapy-Induced Neutropenia
[0423] The in vivo biological activities of non-conjugated hG-CSF
(Neupogen.RTM.) and hG-CSF with a single N-terminally linked 20 kDa
PEG group (Neulasta.TM.) were compared to two PEGylated G-CSF
variants of the invention in rats with chemotherapy-induced
neutropenia. These two variants, which were produced in yeast (S.
cerevisiae) and CHO cells, respectively, had the same amino acid
substitutions relative to the sequence of hG-CSF, namely K16R,
K34R, K40R, T105K and S159K, and were conjugated to SPA-PEG 5000.
The PEGylated variants of the invention, which initially consisted
of multi-PEGylated species having 3-6 PEG moieties attached, were
separated to give a more uniform product having only 4-5 PEG
moieties attached. These variants are referred to below as "G20"
(produced in yeast) and "G21" (produced in CHO cells).
[0424] The G-CSF samples were administered 24 hours after
administration of CPA (90 mg per kg body weight). The PEGylated
variants, i.e. Neulasta.TM., G20 and G21, were administered as a
single dose of 100 .mu.g per kg body weight, while Neupogen.RTM.
was administered in daily doses of 10 .mu.g per kg body weight for
seven days.
[0425] The in vivo biological activity was measured as described
above ("Measurement of the in vivo biological activity in rats with
chemotherapy-induced neutropenia of conjugated and non-conjugated
hG-CSF and variants thereof"). The results are shown in FIG. 10
(white blood cell count, WBC) and FIG. 11 (absolute neutrophil
count, ANC).
[0426] FIGS. 10 and 11 show that all of the G-CSF compounds induced
an initial formation of white blood cells and neutrophils at
approximately identical rates during the first 12 hours, after
which the levels of white blood cells and neutrophils fell as a
result of the chemotherapy. After 96 hours, the levels of white
blood cells and neutrophils increased once again in all cases, but
the rate of increase was significantly higher for rats treated with
G20 or G21 than for rats treated with either Neupogen.RTM. or
Neulasta.TM.. FIG. 10 shows that the white blood cell levels of
rats treated with G20 or G21 reached a normal level of
approximately 10.sup.9/l after 144 hours, while the rats treated
with Neupogen.RTM. or Neulasta.TM. did not reach this level until
after 168 hours. As shown in FIG. 11, the same pattern is seen when
looking at the neutrophil count, i.e. the neutrophil count of rats
treated with G20 or G21 reach a normal level approximately 24 hours
before rats treated with Neupogen.RTM. or Neulasta.TM. reach a
similar level. It may thus be concluded that these PEGylated G-CSF
variants of the invention are able to reduce the duration of
chemotherapy-induced neutropenia in rats by about 24 hours compared
to treatment with the currently available G-CSF products
Neupogen.RTM. and Neulasta.TM..
[0427] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims. For example, all the techniques and
apparatus described above may be used in various combinations. All
publications, patents, patent applications, and/or other documents
cited in this application are incorporated herein by reference in
their entirety for all purposes to the same extent as if each
individual publication, patent, patent application, and/or other
document were individually indicated to be incorporated herein by
reference in its entirety for all purposes.
Sequence CWU 1
1
15 1 174 PRT Homo sapiens 1 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro
Gln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln Val Arg Lys Ile
Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 Glu Lys Leu Cys Ala Thr
Tyr Lys Leu Cys His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His
Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60 Pro Ser
Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 65 70 75 80
Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 85
90 95 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp 100 105 110 Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly
Met Ala Pro 115 120 125 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
Phe Ala Ser Ala Phe 130 135 140 Gln Arg Arg Ala Gly Gly Val Leu Val
Ala Ser His Leu Gln Ser Phe 145 150 155 160 Leu Glu Val Ser Tyr Arg
Val Leu Arg His Leu Ala Gln Pro 165 170 2 525 DNA Artificial
Sequence misc_feature DNA sequence encoding hG-CSF, with codon
usage for E. coli 2 acccctctgg gcccggccag cagtctgcct cagagttttt
tactgaaatg cttagaacag 60 gtgcgtaaaa tccagggcga tggcgcggcc
ctgcaggaaa aactgtgcgc gacctataaa 120 ctgtgccatc ctgaagaact
ggtcctgtta ggccatagct taggcatccc gtgggcgcct 180 ctgagtagct
gcccgagtca ggccctgcag ctggccggct gcctgagtca gttacatagt 240
ggcttatttt tatatcaggg cttactgcag gcgttagaag gcattagtcc ggaactgggc
300 ccgaccctgg ataccttaca gttagatgtc gcggattttg ccaccaccat
ttggcagcag 360 atggaagaat taggcatggc gcctgcgtta cagcctaccc
agggcgccat gcctgcgttt 420 gcgagtgcgt ttcagcgtcg cgccggcggc
gtgttagtgg ccagccatct gcagagcttt 480 ctggaagtga gttatcgtgt
gttacgccat ctggcccagc cttaa 525 3 21 PRT Escherichia coli 3 Met Lys
Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15
Thr Val Ala Gln Ala 20 4 63 DNA Artificial Sequence misc_feature
DNA sequence encoding the OmpA signal sequence 4 atgaaaaaga
cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtagcgcag 60 gcc 63 5
15 PRT Artificial Sequence PEPTIDE (1)..(15) Synthetic tag 5 Met
Lys His Gln His Gln His Gln His Gln His Gln His Gln Gln 1 5 10 15 6
45 DNA Artificial Sequence misc_feature DNA encoding the tag of SEQ
ID NO5 6 atgaaacacc aacaccaaca tcaacatcaa catcaacatc aacag 45 7 30
PRT Homo sapiens SIGNAL (1)..(30) 7 Met Ala Gly Pro Ala Thr Gln Ser
Pro Met Lys Leu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp His Ser
Ala Leu Trp Thr Val Gln Glu Ala 20 25 30 8 615 DNA Artificial
Sequence misc_feature Sequence encoding hG-CSF with signal peptide
of SEQ ID NO7 8 atggccggcc ctgccacaca gtcccccatg aagctgatgg
ccctgcagct gctgctgtgg 60 cactccgccc tgtggacagt gcaggaggcc
acccctctgg gccccgccag ctccctgcct 120 cagtccttcc tgctgaagtg
cctggagcag gtgagaaaga tccagggcga cggcgccgcc 180 ctgcaggaga
agctgtgcgc cacatacaag ctgtgccacc ctgaggagct ggtgctgctg 240
ggccacagcc tgggcatccc ctgggcccct ctgtccagct gcccctccca ggccctgcag
300 ctggccggct gcctgtccca gctgcactcc ggcctgttcc tgtaccaggg
cctgctgcag 360 gccctggagg gcatctcccc cgagctgggc cccacactgg
ataccctgca gctggacgtg 420 gccgatttcg ccaccacaat ctggcagcag
atggaggagc tgggcatggc ccctgccctg 480 cagcctaccc agggcgccat
gcctgccttt gcctccgcct ttcagagacg ggccggcggc 540 gtgctggtgg
ccagccacct gcagagcttt ctggaggtgt cctacagagt gctgcggcac 600
ctggcccagc cttga 615 9 6 PRT Artificial Sequence PEPTIDE (1)..(6)
Synthetic tag 9 His His His His His His 1 5 10 8 PRT Artificial
Sequence PEPTIDE (1)..(8) Synthetic tag 10 Met Lys His His His His
His His 1 5 11 10 PRT Artificial Sequence PEPTIDE (1)..(10)
Synthetic tag 11 Met Lys His His Ala His His Gln His His 1 5 10 12
14 PRT Artificial Sequence PEPTIDE (1)..(14) Synthetic tag 12 Met
Lys His Gln His Gln His Gln His Gln His Gln His Gln 1 5 10 13 10
PRT Artificial Sequence PEPTIDE (1)..(10) Synthetic tag 13 Glu Gln
Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 14 8 PRT Artificial Sequence
PEPTIDE (1)..(8) Synthetic tag 14 Asp Tyr Lys Asp Asp Asp Asp Lys 1
5 15 9 PRT Artificial Sequence PEPTIDE (1)..(9) Synthetic tag 15
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5
* * * * *
References