U.S. patent application number 10/659195 was filed with the patent office on 2005-08-11 for physiologically active polypeptide conjugate having prolonged in vivo half-life.
Invention is credited to Bae, Sung-Min, Kim, Dae-Jin, Kim, Young-Min, Kwon, Se-Chang, Lee, Gwan-Sun, Lim, Chang-Ki.
Application Number | 20050176108 10/659195 |
Document ID | / |
Family ID | 36096009 |
Filed Date | 2005-08-11 |
United States Patent
Application |
20050176108 |
Kind Code |
A1 |
Kim, Young-Min ; et
al. |
August 11, 2005 |
Physiologically active polypeptide conjugate having prolonged in
vivo half-life
Abstract
A protein conjugate having a prolonged in vivo half-life of a
physiological activity, comprising i) a physiologically active
polypeptide, ii) a biocompatible non-peptidic polymer, and iii) an
immunoglobulin, is useful for the development of a peptide drug due
to the enhanced in vivo stability and prolonged half-life in blood,
while reducing the possibility of inducing an immune response.
Inventors: |
Kim, Young-Min; (Yongin-si,
KR) ; Kim, Dae-Jin; (Seoul, KR) ; Bae,
Sung-Min; (Seoul, KR) ; Lim, Chang-Ki;
(Seongnam-si, KR) ; Kwon, Se-Chang; (Seoul,
KR) ; Lee, Gwan-Sun; (Seoul, KR) |
Correspondence
Address: |
David A. Einhorn, Esq.
Anderson Kill & Olick, P.C.
1251 Avenue of the Americas
New York
NY
10020
US
|
Family ID: |
36096009 |
Appl. No.: |
10/659195 |
Filed: |
September 9, 2003 |
Current U.S.
Class: |
435/70.21 ;
424/178.1; 530/391.1 |
Current CPC
Class: |
A61K 47/60 20170801;
C07K 16/00 20130101; A61K 47/6883 20170801; A61K 47/6811 20170801;
A61K 47/643 20170801 |
Class at
Publication: |
435/070.21 ;
530/391.1; 424/178.1 |
International
Class: |
A61K 039/395; C12P
021/04; C07K 016/46 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 13, 2003 |
KR |
2003-0015744 |
Jun 5, 2003 |
KR |
2003-0036408 |
Claims
1. A protein conjugate comprising i) a physiologically active
polypeptide, ii) a non-peptidic polymer, and iii) an
immunoglobulin, which are covalently linked to one another, and
having a prolonged in vivo half-life of the physiologically active
polypeptide.
2. The protein conjugate according to claim 1, wherein the
non-peptidic polymer has two reactive groups at both ends, through
which the polymer is covalently linked to the physiologically
active polypeptide and the immunoglobulin.
3. The protein conjugate according to claim 2, wherein the
immunoglobulin is covalently linked to at least two complexes of
the physiologically active polypeptide and the non-peptidic
polymer.
4. The protein conjugate according to claim 1, wherein the
immunoglobulin is selected from the group consisting of IgG, IgA,
IgD, IgE, IgM and a mixture thereof.
5. The protein conjugate according to claim 4, wherein the
immunoglobulin is selected from the group consisting of IgG1, IgG2,
IgG3, IgG4 and a mixture thereof.
6. The protein conjugate according to claim 4, wherein the
immunoglobulin is a human immunoglobulin.
7. The protein conjugate according to claim 2, wherein the reactive
group of the non-peptidic polymer is selected from the group
consisting of aldehyde, propion aldehyde, maleimide and succinamide
derivative.
8. The protein conjugate according to claim 7, wherein the
succinamide derivative is succinimidyl propionate, succinimidyl
carboxymethyl, hydroxy succinimidyl or succinimidyl carbonate.
9. The protein conjugate according to claim 7, wherein the
non-peptidic polymer has aldehyde groups at both ends.
10. The protein conjugate according to claim 1, wherein the
non-peptidic polymer is covalently linked at the ends thereof to
the amino terminal, lysine residue, histidine residue or cysteine
residue of the immunoglobulin and the amino terminal, lysine
residue, hisitidine residue or cysteine residue of the
physiologically active polypeptide, respectively.
11. The protein conjugate according to claim 1, wherein the
non-peptidic polymer is selected form the group consisting of
poly(ethylene glycol), poly(propylene glycol), ethylene
glycol-propylene glycol copolymer, polyoxyethylated polyol,
polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether,
poly(lactic-glycolic acid), biodegradable polymer, lipid polymer,
chitin, hyaluronic acids, and a mixture thereof.
12. The protein conjugate according to claim 11, wherein the
non-peptidic polymer is poly(ethylene glycol).
13. The protein conjugate according to claim 1, wherein the
physiologically active polypeptide is selected from the group
consisting of hormone, cytokine, enzyme, antibody, growth hormone,
transcription regulatory factor, blood factor, vaccine, structure
protein, ligand protein and receptor.
14. The protein conjugate according to claim 13, wherein the
physiologically active polypeptide is selected from the group
consisting of human growth hormone, growth hormone releasing
hormone, growth hormone releasing peptide, interferons, colony
stimulating factor, interleukins, glucocerebrosidae, macrophage
activating factor, macrophage peptide, B cell factor, T cell
factor, protein A, suppressive factor of allergy, cell necrosis
glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor,
tumor inhibitory factor, transforming growth factor, alpha-1
antitrypsin, albumin, apolipoprotein-E, erythropoietin,
hyper-glycosylated erythropoietin, factor VII, factor VIII, factor
IX, plaminogen activator, urokinase, streptokinase, protein C,
C-reactive protein, renin inhibitor, collagenase inhibitor,
superoxide dismutase, platelet derived growth factor, epidermal
growth factor, osteogenic growth factor, osteogenesis stimulating
protein, calcitonin, insulin, atriopeptin, cartilage inducing
factor, connective tissue activator protein, follicle stimulating
hormone, leutinizing hormone, FSH releasing hormone, nerve growth
factor, parathyroid hormone, relaxin, secretin, somatomedin,
insulin-like growth factor, adrenocorticotrophic hormone, glucagon,
cholecystokinin, pancreatic polypeptide, gastrin releasing peptide,
corticotrophin releasing, thyroid stimulating hormone, monoclonal
antibody, polyclonal antibody, antibody derivatives including
[Fab]', [Fab]'2 and scFv, and virus-derived vaccine antigen.
15. The protein conjugate according to claim 14, wherein the
physiologically active polypeptide is human growth hormone,
interferon-alpha, granulocyte colony stimulating factor or
erythropoietin.
16. A method for preparing the protein conjugate of claim 1,
comprising (a) covalently linking at least one physiologically
active polypeptide, at least one immunoglobulin with at least one
non-peptidic polymer having reactive groups at both ends; and (b)
isolating a protein conjugate comprising essentially the active
polypeptide, the immunoglobulin and the non-peptidic polymer, which
are linked covalently.
17. The method according to claim 16, wherein step (a) further
comprises: (a1) covalently coupling one end of the non-peptidic
polymer with either an immunoglobulin or a physiologically active
polypeptide; (a2) isolating from the resulting reaction mixture a
complex comprising the non-peptidic polymer coupled with the
immunoglobulin or the physiologically active polypeptide; and (a3)
covalently coupling the free end of the non-peptidic polymer of the
complex with the immunoglobulin or physiologically active
polypeptide, to produce a protein conjugate comprising the
physiologically active polypeptide, the non-peptidic polymer and
the immunoglobulin, which are covalently interlinked.
18. The method according to claim 17, wherein the molar ratio of
the physiologically active polypeptide to the non-peptidic polymer
in step (a1) ranges from 1:2.5 to 1:5.
19. The method according to claim 17, wherein the molar ratio of
the immunoglobulin to the non-peptidic polymer in step (a1) ranges
from 1:5 to 1:10.
20. The method according to claim 17, wherein the molar ratio of
the complex obtained in step (a2) to physiologically active
polypeptide or immunoglobulin in step(a3) ranges from 1:1 to
1:3.
21. The method according to claim 17, wherein steps (a1) and (a3)
are performed in the presence of a reducing agent.
22. The method according to claim 21, wherein the reducing agent is
sodium cyanoborohydride, sodium borohydride, dimethylamine borate
or pyridine borate.
23. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 1 and a pharmaceutically acceptable carrier.
24. A method for prolonging the in vivo half-life of a
physiologically active polypeptide, which comprises the step of
covalently linking a non-peptidic polymer having reactive groups at
both ends with a physiologically active polypeptide and an
immunoglobulin.
25. The method according to claim 24, wherein the immunoglobulin is
covalently linked to at least two complexes of the physiologically
active polypeptide and the non-peptidic polymer.
26. The method according to claim 24, wherein the immunoglobulin is
selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA,
IgD, IgE, IgM and a mixture thereof.
27. The method according to claim 26, wherein the immunoglobulin is
a human immunoglobulin.
28. The method according to claim 24, wherein the reactive group of
the non-peptidic polymer is selected from the group consisting of
aldehyde, propion aldehyde, maleimide and succinamide
derivative.
29. The method according to claim 24, wherein the non-peptidic
polymer is selected from the group consisting of poly(ethylene
glycol), poly(propylene glycol), ethylene glycol-propylene glycol
copolymer, polyoxyethylated polol, polyvinyl alcohol,
polysaccharide, dextran, polyvinyl ethyl ether,
poly(lactic-glycolic acid), biodegradable polymer, lipid polymer,
chitin, hyaluronic acids, and a mixture thereof.
30. The method according to claim 29, wherein the non-peptidic
polymer is poly(ethylene glycol).
31. The method according to claim 24, wherein the physiologically
active polypeptide is selected from the group consisting of
hormone, cytokine, enzyme, antibody, growth hormone, transcription
regulatory factor, blood factor, vaccine, structural protein,
ligand protein and receptor.
32. The method according to claim 31, wherein the physiologically
active polypeptide is selected from the group consisting of human
growth hormone, growth hormone releasing hormone, growth hormone
releasing peptide, interferons, colony stimulating factor,
interleukins, glucocerebrosidae, macrophage activating factor,
macrophage peptide, B cell factor, T cell factor, protein A,
suppressive factor of allergy, cell necrosis glycoprotein,
immunotoxin, lymphotoxin, tumor necrosis factor, tumor inhibitory
factor, transforming growth factor, alpha-1 antitrypsin, albumin,
apolipoprotein-E, erythropoietin, hyper-glycosylated
erythropoietin, factor VII, factor VIII, factor IX, plasminogen
activator, urokinase, streptokinase, protein C, C-reactive protein,
renin inhibitor, collagenase inhibitor, superoxide dismutase,
platelet derived growth factor, epidermal growth factor, osteogenic
growth factor, osteogenesis stimulating protein, calcitonin,
insulin, atriopeptin, cartilage inducing factor, connective tissue
activator protein, follicle stimulating hormone, leutinizing
hormone, FSH releasing hormone, nerve growth factor, parathyroid
hormone, relaxin, secretin, somatomedin, insulin-like growth
factor, adrenocorticotrophic hormone, glucagon, cholecystokinin,
pancreatic polypeptide, gastrin releasing peptide, corticotropin
releasing factor, thyroid stimulating hormone, monoclonal antibody,
polyclonal antibody, antibody derivatives including [Fab]',
[Fab]'2, and scFv, and virus-derived vaccine antigen.
33. The method according to claim 32, wherein the physiologically
active polypeptide is human growth hormone, interferon-alpha,
granulocyte colony stimulating factor or erythropoietin.
34. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 2 and a pharmaceutically acceptable carrier.
35. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 3 and a pharmaceutically acceptable carrier.
36. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 4 and a pharmaceutically acceptable carrier.
37. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 5 and a pharmaceutically acceptable carrier.
38. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 6 and a pharmaceutically acceptable carrier.
39. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 7 and a pharmaceutically acceptable carrier.
40. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 8 and a pharmaceutically acceptable carrier.
41. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 9 and a pharmaceutically acceptable carrier.
42. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 10 and a pharmaceutically acceptable
carrier.
43. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 11 and a pharmaceutically acceptable
carrier.
44. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 12 and a pharmaceutically acceptable
carrier.
45. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 13 and a pharmaceutically acceptable
carrier.
46. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 14 and a pharmaceutically acceptable
carrier.
47. A pharmaceutical composition having a prolonged half-life of a
physiologically active polypeptide, which comprises a protein
conjugate of claim 15 and a pharmaceutically acceptable carrier.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a long acting protein
having a prolonged in vivo half-life and a preparation method
thereof.
BACKGROUND OF THE INVENTION
[0002] Polypeptides are susceptible to denaturation or enzymatic
degradation in the blood, liver or kidney. Because of the low
stability of polypeptides, it has been required to administer a
peptide drug in a sustained frequency to a subject in order to
maintain an effective plasma concentration of the active substance.
Moreover, since peptide drugs are usually administrated by
infusion, frequent injection of peptide drugs causes considerable
discomfort to a subject. Thus, there have been many studies to
develop a peptide drug which has an increased circulating half-life
in the blood, while maintaining a high pharmacological efficacy
thereof. Such desirous peptide drugs should also meet the
requirements of enhanced serum stability, high activity,
applicability to various polypeptides and a low probability of
inducing an undesired immune response when injected into a
subject.
[0003] One of the most widely used methods for improving the
stability of proteins is the chemical modification of a polypeptide
with highly soluble macromolecules such as polyethylene glycol
("PEG") which prevents the polypeptides from contacting with
proteases. It is also well known that, when linked to a peptide
drug specifically or non-specifically, PEG increases the solubility
of the peptide drug and prevents the hydrolysis thereof, thereby
increasing the serum stability of the peptide drug without
incurring any immune response due to its low antigenecity (Sada et
al., J. Fermentation Bioengineering, 1991, 71: 137-139). However,
such pegylated polypeptides have the disadvantages of lowering both
the activity and production yield of an active substance as the
molecular weight of PEG increases. An interferon conjugated with
two activated PEGs as well as a PEG spacer which is linked to two
polypeptides having different activities are disclosed in U.S. Pat.
No. 5,738,846 and International Patent Publication No. WO92/16221,
respectively; however, they do not show any distinctive effect in
terms of sustained activity of the physiologically active
polypeptides in vivo.
[0004] It is also reported that the circulating half-life of a
recombinant human granulocyte-colony stimulating factor ("G-CSF")
can be prolonged by covalently linking it to albumin through a
hetero-bifunctional PEG (Kinstler et al., Pharmaceutical Research,
1995, 12(12): 1883-1888). However, the stability of the modified
G-CSF-PEG-albumin is merely 4 times higher than that of authentic
G-CSF and, thus, it has not yet been put to practical use.
[0005] As another approach for enhancing the in vivo stability of
physiologically active polypeptides, an active polypeptide fused
with a stable protein is produced in a transformant by using
recombinant technologies. For example, albumin is known as one of
the most effective proteins for enhancing the stability of
polypeptides fused thereto and there are many such fusion proteins
reported (International Patent Publication Nos WO93/15199 and
93/15200, and European Patent Publication No. 413,622). However, a
fusion protein coupled with albumin still has the problem of
reduced activity.
[0006] U.S. Pat. No. 5,045,312 discloses a method for conjugating
growth hormone to bovine serum albumin or mouse immunoglobulin
using a cross-linking agent such as carbodiimide, glutaraldehyde,
acid chloride, etc. in order to enhance the activity of the growth
hormone. However, this method is solely aimed at enhancing the
activity of a target growth hormone. In addition, the use of
chemical compounds such as carbodiimide, glutaraldehyde, acid
chloride, etc. as a cross-linking agent is disadvantageous due to
their potent toxicity and non-specificity of reaction.
[0007] Although there have been many attempts to combine a
physiologically active polypeptide with various macromolecules, all
have failed to simultaneously increase the stability and the
activity.
[0008] As an improved method for enhancing the stability of an
active polypeptide and simultaneously maintaining the in vivo
activity thereof, the present invention provides a protein
conjugate comprising a physiologically active polypeptide,
non-peptidic polymer and immunoglobulin, which are covalently
interlinked to one another.
SUMMARY OF THE INVENTION
[0009] Accordingly, a primary object of the present invention is to
provide a protein conjugate having a prolonged in vivo half-life of
a physiologically active polypeptide without inducing an immune
response in a subject, while minimizing the reduction in the
polypeptide's activity.
[0010] Another object of the present invention is to provide a
method for preparing a protein conjugate comprising a
physiologically active polypeptide, a biocompatible non-peptidic
polymer and an immunoglobulin, which are covalently
interlinked.
[0011] A further object of the present invention is to provide a
pharmaceutical composition comprising said physiologically active
polypeptides having a prolonged in vivo half-life.
[0012] A still further object of the present invention is to
provide a method for enhancing the in vivo stability and prolonging
the circulating half-life of a physiologically active polypeptide,
without sacrificing the activity thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The above and other objects and features of the present
invention will become apparent from the following description of
the invention, when taken in conjunction with the accompanying
drawings, which respectively show:
[0014] FIG. 1: a chromatogram of hGH-PEG-IgG conjugates;
[0015] FIG. 2: SDS-PAGE results of hGH-PEG-IgG conjugates;
[0016] FIG. 3: SDS-PAGE results of Interferon-PEG-IgG
conjugates;
[0017] FIG. 4: SDS-PAGE results of hGH-PEG-IgG conjugates before
and after the treatment of DDT;
[0018] FIG. 5: a mass spectrum of hGH-PEG-IgG conjugates;
[0019] FIG. 6: a pharmacokinetic graph showing that hGH-PEG-IgG
conjugates have serum stability superior to PEG-hGH complex;
[0020] FIG. 7: a pharmacokinetic graph showing that
erythropoietin-PEG-IgG conjugates have an enhanced circulating
half-life as compared with the free erythropoietin or
erythropoietin stabilized by hyper-glycosylation;
[0021] FIG. 8: in vivo activity of hGH-PEG-IgG conjugates based on
the time-dependent weight change of rats after the injection of
solvent only (30 .mu.g/day; group 1), natural hGH (30 .mu.g/day;
group 2), hGH-PEG (30 .mu.g/day; group 3), hGH-PEG-IgG conjugate
(30 .mu.g/day; group 4), and hGH-PEG-IgG conjugate (10 .mu.g/day;
group 5); and
[0022] FIG. 9: in vivo activity of G-CSF-PEG-IgG conjugates based
on the time-dependent change in the number of neutrophils: no
treatment (group 1); solvent injection only (group 2); natural
G-CSF (group 3); 20 kDa PEG-G-CSF (group 4); and
.sup.17S-G-CSF-PEG-IgG conjugate treatment (group 5).
DETAILED DESCRIPTION OF THE INVENTION
[0023] The term "physiologically active polypeptide" as used herein
refers to any polypeptide or protein having a useful biological
activity when administered to a mammal including a human, which is
interchangeable with the term "physiologically active protein",
"active protein", "active polypeptide" or "peptide drug".
[0024] The term "protein conjugate" or "conjugate" refers to a
compound comprising a physiologically active polypeptide, a
non-peptidic polymer and an immunoglobulin which are covalently
interlinked to one another in accordance with the present
invention.
[0025] The term "complex", as distinguished from the term
"conjugate", is used herein to mean those compounds comprising only
two components selected from a physiologically active polypeptide,
an immunoglobulin and a non-peptidic polymer.
[0026] The term "non-peptidic polymer" refers to a biocompatible
polymer comprising at least two monomers, in which the monomers are
linked together via any covalent bond other than a peptide
bond.
[0027] In accordance with one aspect of the present invention,
there is provided a protein conjugate comprising i) a
physiologically active polypeptide, ii) a non-peptidic polymer, and
iii) an immunoglobulin, which are covalently linked to one another,
and having a prolonged in vivo half-life of the physiologically
active polypeptide.
[0028] For example, the protein conjugate of the invention may
comprise at least one unit structure of [active
polypeptide/non-peptidic polymer/immunoglobulin], in which all of
the components are covalently interlinked in a linear form. The
non-peptidic polymer may have two reactive groups at both ends,
through which the polymer is covalently linked to the
physiologically active polypeptide and the immunoglobulin,
respectively. In a preferred embodiment, two complexes of the
physiologically active polypeptide and the non-peptidic polymer may
be covalently linked to an immnunoglobulin.
[0029] The molar ratio of the physiologically active polypeptide to
the immunoglobulin may range from 1:1 to 10:1, preferably 1:1 to
4:1.
[0030] One kind of polymer as well as a combination of different
kinds of polymers may be used as the non-peptidic polymer.
[0031] In the protein conjugate of the present invention, the
suitable binding sites of the immunoglobulin may include a free
amino group in the variable region or the constant region of the
immunoglobulin. Suitable sites of the immunoglobulin for covalent
bonding with the non-peptidic polymer or active polypeptide may
include an amino-terminal group within the variable region,
amine-group of lysine residue or histidine residue, and free --SH
group of cysteine, and the suitable site of the non-peptidic
polymer is a terminal reactive group.
[0032] The immunoglobulin may be selected from the group consisting
of IgG, IgA, IgD, IgE, IgM, a combination thereof and all the
subtypes of IgG such as IgG1, IgG2, IgG3 and IgG4. In order not to
induce an immune response in a patient, the immunoglobulin is
preferably a human immunoglobulin.
[0033] As a component constituting the protein conjugate of the
invention, the immunoglobulin may be either a natural one isolated
from the blood or a recombinant prepared by genetic engineering.
Any immunoglobulin modified with substitution, deletion or addition
of amino acid residues in various sites therein as well as any
hyper-glycosylated derivative thereof also may be used for the
present invention, as long as such immunoglobulin or derivative is
substantially equivalent to a wild-type in terms of the function,
structure and stability thereof. Amino acid residue Nos. 214 to
238, 297 to 299, 318 to 322, and 327 to 331 of an immunoglobulin G,
which have been known as important sites for binding, may be used
as a suitable site for the modification.
[0034] A suitable non-peptidic polymer has a reactive group
selected from the group consisting of aldehyde, propionic aldehyde,
maleimide and succinamide derivative. The succinamide derivative
may be selected from the group consisting of succinimidyl
propionate, succinimidyl carboxymethyl, hydroxy succinimidyl and
succinimidyl carbonate. A non-peptidic polymer having aldehyde
groups at both ends is effective in minimizing non-specific
coupling, thereby linking the non-peptidic polymer with a
physiologically active polypeptide and an immunoglobulin at each
end of the polymer, respectively. A protein conjugate produced by
reductive alkylation of an aldehyde group is more stable than that
coupled via an amide bond.
[0035] The reactive groups at the both ends of the non-peptidic
polymer may be identical to or different from each other. For
example, a non-peptidic polymer may have a maleimide group at one
end, and a maleimide group, an aldehyde group or a propionic
aldehyde group at the other. When poly(ethylene glycol) is used as
the non-peptidic polymer, a commercially available product may be
used for preparing the protein conjugate of the invention, or the
termial hydroxy groups of the commercial PEG may be further
converted to other reactive groups before the coupling
reaction.
[0036] The non-peptidic polymer may serve as a spacer which
covalently links the amino terminal, lysine residue, histidine
residue or cysteine residue of the immonoglobulin and one of the
reactive groups of the physiologically active polypeptide,
respectively.
[0037] The non-peptidic polymer is preferably selected from the
group consisting of poly(ethylene glycol), poly(propylene glycol),
ethylene glycol-propylene glycol copolymer, polyoxyethylated
polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl
ether, poly(lactic-glycolic acid), biodegradable polymer, lipid
polymer, chitin, hyaluronic acids, and a combination thereof.
Derivatives of the above known in the art may be used for the same
purpose. More preferable non-peptidic polymer is poly(ethylene
glycol).
[0038] Previously reported cross-linking agents for combining two
polypeptides by gene cloning, such as oligopeptides, increase the
possibility of undesired immune response and limit the binding site
to N-terminal or C-terminal of the polypeptides. Accordingly, one
advantage of the use of a non-peptidic polymer over the
oligopeptides lies in the reduction of toxicity and immunogenecity.
Another advantage is its broad applicability due to the diversity
of the sites to be bound.
[0039] When used as a cross-linking agent, small chemical compounds
such as carbodiimide and glutaraldehyde may result in denaturation
of polypeptides to be linked therethrough, or may obstruct a
controlled binding and purification of the resultant. Contrary to
such chemicals, the protein conjugate of the invention, which
comprises a non-peptidic polymer, is advantageous in terms of
easiness of controlling the binding, purifying the resulting
conjugates and minimizing non-specific coupling reaction.
[0040] The protein conjugate of the present invention shows a
prolonged in vivo half-life and activity remarkably superior to a
polypeptide-PEG complex or a polypeptide-PEG-albumin complex.
According to pharmacokinetic analyses, the half-life of an
hGH-PEG-IgG conjugate of the present invention was about thirteen
times longer than that of wild-type hGH, while an hGH-PEG complex
and an hGH-PEG-albumin complex show half-lives seven times and five
times longer than the wild-type protein, respectively (see Test
Example 2, Table 2). Similar results were obtained from tests using
G-CSF, .sup.17S-G-CSF, interferons or EPO instead of hGH. Compared
with active polypeptide complexes modified with PEG only or a
PEG-albumin complex, the protein conjugate of the present invention
shows considerable increases in both mean residence time ("MRT")
and serum half-life, which are higher by a factor of 2.about.70
than those of conventional complexes (see Test Example 2, Tables 3
to 6).
[0041] The results of pharmacokinetic analyses show that the
protein conjugates applied to various polypeptides including hGH,
interferon, EPO, G-CSF and its derivative exhibit excellent
performance characteristics in terms of in-blood half-life and MRT,
and, thus, can be advantageously employed in preparing a peptide
drug formulation having a prolonged in vivo half-life.
[0042] Exemplary classes of the physiologically active polypeptides
include the following polypeptides, and muteins and other analogs
thereof: hormone, cytokine, enzyme, antibody, growth hormone,
transcription regulatory factor, blood factor, vaccine, structural
protein, ligand protein and receptor.
[0043] Specific examples of the physiologically active polypeptides
suitable for preparing the protein conjugate of the invention
include human growth hormone, growth hormone releasing hormone,
growth hormone releasing peptide, interferons, colony stimulating
factor, interleukins, glucocerebrosidae, macrophage activating
factor, macrophage peptide, B cell factor, T cell factor, protein
A, suppressive factor of allergy, cell necrosis glycoprotein,
immunotoxin, lymphotoxin, tumor necrosis factor, tumor inhibitory
factor, transforming growth factor, alpha-1 antitrypsin, albumin,
apolipoprotein-E, erythropoietin, hyper-glycosylated
erythropoietin, factor VII, factor VIII, factor IX, plasminogen
activator, urokinase, streptokinase, protein C, C-reactive protein,
renin inhibitor, collagenase inhibitor, superoxide dismutase,
platelet derived growth factor, epidermal growth factor, osteogenic
growth factor, osteogenesis stimulating protein, calcitonin,
insulin, atriopeptin, cartilage inducing factor, connective tissue
activator protein, follicle stimulating hormone, leutinizing
hormone, FSH releasing hormone, nerve growth factor, parathyroid
hormone, relaxin, secretin, somatomedin, insulin-like growth
factor, adrenocorticotrophic hormone, glucagon, cholecystokinin,
pancreatic polypeptide, gastrin releasing peptide, corticotropin
releasing factor, thyroid stimulating hormone, monoclonal antibody,
polyclonal antibody, antibody derivatives including [Fab]', [Fab]'2
and scFv, and virus-derived vaccine antigen.
[0044] A particularly preferred polypeptide is the one selected
from the group consisting of human growth hormone, interferons,
granulocyte colony stimulating factor and erythropoietin in light
of the fact that these polypeptides need more frequent
administration than others for the purpose of treating or
preventing relevant diseases.
[0045] List of the physiologically active polypeptides, to which
the present invention can be applied, is not limited to those
recited in the above but includes any muteins or derivatives
thereof inasmuch as the function, structure, activity and stability
of the mutein or derivative can be recognized as an equivalent or
superior to those of the wild-type polypeptides.
[0046] Another aspect of the present invention is to provide a
method for preparing said protein conjugate, which comprises the
steps of:
[0047] (a) covalently linking at least one physiologically active
polypeptide, at least one immunoglobulin with at least one
non-peptidic polymer having reactive groups at both ends; and
[0048] (b) isolating a protein conjugate comprising essentially the
active polypeptide, the immunoglobulin and the non-peptidic
polymer, which are interlinked covalently.
[0049] In step (a) of the above method, polypeptides,
immunoglobulins and non-peptidic polymers may be covalently linked
by a two-step reaction or a simultaneous reaction. The two-step
reaction (e.g., a non-peptidic polymer is covalently linked to an
active polypeptide or an immunoglobulin and, then, the resulting
complex is covalently linked to an active polypeptide or an
immunoglobulin to give a conjugate thereof, in which the active
polypeptide and the immunoglobulin are linked to each other via the
non-peptidic polymer) is advantageous in reducing the production of
undesirable by-products.
[0050] Accordingly, the step (a) of the above method may
comprise:
[0051] (a1) covalently coupling one end of the non-peptidic polymer
with either an immunoglobulin or a physiologically active
polypeptide;
[0052] (a2) isolating from the reaction mixture a complex
comprising the non-peptidic polymer coupled with the immunoglobulin
or the physiologically active polypeptide; and
[0053] (a3) covalently coupling the free end of the non-peptidic
polymer of the complex with the immunoglobulin or physiologically
active polypeptide, to produce a protein conjugate in which the
non-peptidic polymer covalently interlinks the physiologically
active polypeptide and immunoglobulin.
[0054] The molar ratio of the physiologically active polypeptide to
the non-peptidic polymer in step (a1) may preferably range from
1:2.5 to 1:5 and the molar ratio of the immunoglobulin to the
non-peptidic polymer in step (a1), preferably from 1:5 to 1:10. The
molar ratio of the complex obtained in step (a2) to the
physiologically active polypeptide or immunoglobulin in step (a3)
may range from 1:1 to 1:3.
[0055] Steps (a1) and (a3) may be preferably performed in the
presence of a reducing agent, which may be selected from the group
consisting of sodium cyanoborohydride, sodium borohydride,
dimethylamine borate and pyridine borate.
[0056] The procedures for conducting steps (a2) and (b) may be
based on conventional methods used for purifying proteins, such as
size exclusion chromatography, ion exchange chromatography, etc.
and a combination thereof, in accordance with the extent of
required purity and the properties of the resulting conjugate
including molecular weight and electricity.
[0057] Still another aspect of the present invention is to provide
a pharmaceutical composition of a physiologically active
polypeptide having a prolonged in vivo half-life in comparison with
unmodified polypeptides, which comprises the protein conjugate of
the invention and a pharmaceutically acceptable carrier.
[0058] The pharmaceutical composition of the present invention can
be administered via various routes including oral, transdermal,
subcutaneous, intravenous and intramuscular introduction, and
injection is more preferred. The composition of the invention may
be formulated so as to provide a quick, sustained or delayed
release of the active ingredient after it is administered to a
patient, by employing any one of the procedures well known in the
art. The formulation may be in the form of a tablet, pill, powder,
sachet, elixir, suspension, emulsion, solution, syrup, aerosol,
soft and hard gelatin capsule, sterile injectable solution, sterile
packaged powder and the like. Examples of suitable carriers,
excipients or diluents are lactose, dextrose, sucrose, sorbitol,
mannitol, starches, gum acacia, alginates, gelatin, calcium
phosphate, calcium silicate, polyvinylpyrrolidone, cellulose,
methylcellulose, microcrystalline cellulose, water,
methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium
stearate and mineral oil. The formulation may additionally include
fillers, anti-agglutinating agents, lubricating agents, wetting
agents, flavoring agents, emulsifiers, preservatives and the
like.
[0059] The amount of the active ingredient actually administered
ought to be determined in light of various relevant factors
including the condition to be treated, the chosen route of
administration, the age, sex and body weight of the individual
patient, and the severity of the patient's symptom, especially, the
kind of active ingredient. Owing to the enhanced stability of a
protein conjugate of the invention, the total number and frequency
of the administration of the peptide drug formulation comprising
the protein conjugate can be considerably reduced.
[0060] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only and are not intended to limit the scope of the
invention.
EXAMPLE 1
Preparation of hGH-PEG-IgG conjugate I
[0061] (Step 1) Preparation of hGH-PEG Complex
[0062] Human growth hormone (hGH, M.W. 22 kDa) was dissolved in 100
mM phosphate buffer solution to a concentration of 5 mg/ml, and
polyethylene glycol containing aldehyde groups at both ends
(ALD-PEG-ALD, Shearwater Inc, USA) which has a molecular weight of
3.4 kDa was added to the resulting buffer solution in an amount
corresponding to an hGH:PEG molar ratio of 1:1, 1:2.5, 1:5, 1:10 or
1:20. Sodium cyanoborohydride (NaCNBH.sub.3, Sigma) was added
thereto to a final concentration of 20 mM as a reducing agent, and
the reaction mixture was stirred at 4.degree. C. for 3 hours. To
separate an hGH-PEG complex in which PEG is selectively linked to
the terminal amino residue of hGH in a molar ratio of 1:1, the
reaction mixture was subjected to Superdex.RTM. (Pharmacia, USA)
size exclusion chromatography. The hGH-PEG complex was eluted and
purified from the column with 10 mM potassium phosphate buffer (pH
6.0) to remove contaminants such as unmodified hGH, unreacted PEG
and dimmeric by-products having two molecules of hGH linked at both
ends of PEG. The purified hGH-PEG complex was concentrated to 5
mg/ml. It has been found that an optimal hGH:PEG molar ratio for
obtaining the best result was in the range of 1:2.5 to 1:5.
[0063] (Step 2) Formation of Conjugate Between hGH-PEG Complex and
IgG
[0064] Immunoglobulin G (IgG, Green Cross, Korea) having a
molecular weight of 150 kDa was dissolved in 100 mM phosphate
buffer solution. To conjugate IgG to the aldehyde group of the
PEG-hGH complex purified in Example 1, the PEG-hGH complex was
added to an IgG-containing buffer solution in an amount
corresponding to a hGH-PEG complex:IgG molar ratio of 1:1, 1:2, 1:4
or 1:8. NaCNBH.sub.3 was added thereto to a final concentration of
20 mM as a reducing agent, and the reaction mixture was gradually
stirred at 4.degree. C. for 20 hours. To purify the hGH-PEG-IgG
conjugate from contaminants after the conjugation reaction, the
reaction mixture was subjected to anion exchange chromatography
using a DEAE column (Pharmacia, USA) equilibrated with 20 mM Tris
buffer solution (pH 7.5). The mobile phase was changed from Buffer
A (20 mM Tris buffer, pH 7.5) to Buffer B (20 mM Tris buffer
containing 1.0 M NaCl, pH 7.5) in a linear fashion (NaCl
concentration: 0 M.fwdarw.0.5 M). To remove small quantities of
unreacted IgG and unmodified hGH from the eluted hGH-PEG-IgG
conjugate, the eluting solution was subjected to cation exchange
chromatography using an SP5PW column (Waters, USA) equilibrated
with 10 mM sodium acetate (pH 4.5). The mobile phase was changed
from Buffer A (10 mM sodium acetate, pH 4.5) to Buffer B (10 mM
sodium acetate containing 1.0 M NaCl, pH 7.5) in a linear fashion
(NaCl concentration: 0 M.fwdarw.0.5 M), which results in purifying
the hGH-PEG-IgG conjugate (FIG. 1).
[0065] It has been found that the optimal hGH-PEG complex:IgG molar
ratio for obtaining the best result was 1:2.
EXAMPLE 2
Preparation of hGH-PEG-IgG Conjugate II
[0066] (Step 1) Preparation of I2G-PEG Complex
[0067] IgG (Green Cross, Korea) was dissolved in 100 mM phosphate
buffer to a concentration of 15 mg/ml, and 3.4 kDa of ALD-PEG-ALD
(Shearwater Inc, USA) was added to the resulting buffer solution in
an amount corresponding to an IgG:PEG molar ratio of 1:1, 1:2.5,
1:5, 1:10 or 1:20. NaCNBH.sub.3 was added thereto to a final
concentration of 20 mM as a reducing agent, and the reaction
mixture was stirred at 4.degree. C. for 3 hours. To separate
IgG-PEG complex in which PEG is selectively linked to the terminal
amino residue of IgG in a molar ratio of 1:1, the reaction mixture
was subjected to Superdex.RTM. (Pharmacia, USA) size exclusion
chromatography. The IgG-PEG complex was eluted and purified from
the column with 10 mM potassium phosphate buffer (pH 6.0) to remove
contaminants such as unmodified IgG, unreacted PEG and dimmeric
by-products having two molecules of IgG linked at both ends of PEG.
The purified IgG-PEG complex was concentrated to 15 mg/ml. It has
been found that an optimal IgG:PEG molar ratio for obtaining the
best result was in the range of 1:5 to 1:10.
[0068] (Step 2) Formation of Conjugate Between IgG-PEG Complex and
hGH
[0069] To conjugate hGH (M.W. 22 kDa) to the IgG-PEG complex
purified in Example 1, hGH dissolved in 100 mM phosphate buffer was
reacted with the IgG-PEG complex in a molar ratio of 1:1, 1:1.5,
1:3 or 1:6. NaCNBH.sub.3 was added thereto to a final concentration
of 20 mM as a reducing agent, and the reaction mixture was stirred
at 4.degree. C. for 20 hours. The reaction mixture was subjected to
purification according to the same method described in step 2 of
Example 1 to remove unreacted substances and by-products, and the
IgG-PEG-hGH conjugate was purified therefrom.
EXAMPLE 3
Preparation of IFN .alpha.-PEG-IgG Conjugate
[0070] An IFN .alpha.-PEG-IgG conjugate was prepared and purified
according to the same method described in Example 1, except that
interferon alpha 2b (IFN .alpha. 2b, M. W. 20 kDa) was employed
instead of hGH and the IFN .alpha. 2b:ALD-PEG-ALD (M.W. 3.4 kDa)
molar ratio was 1:5.
EXAMPLE 4
Preparation of Human G-CSF-PEG-IgG Conjugate
[0071] A G-CSF-PEG-IgG conjugate was prepared and purified
according to the same method described in Example 1, except that
human granulocyte colony stimulating factor (G-CSF, M. W. 18.7 kDa)
was employed instead of hGH and the G-SCF:ALD-PEG-ALD (M.W. 3.4
kDa) molar ratio was 1:5.
[0072] Further, G-SCF derivative-PEG-IgG conjugate was prepared and
purified according to the same method described above using G-SCF
derivative (.sup.17S-G-CSF) which was attained by replacing the
17.sup.th amino acid of wild-type G-CSF with serine.
EXAMPLE 5
Preparation of EPO-PEG-IgG Conjugate
[0073] An EPO-PEG-IgG conjugate was prepared and purified according
to the same method described in Example 1, except that human
erythropoietin (EPO, M. W. 35 kDa) was employed instead of hGH and
the EPO:ALD-PEG-ALD (M.W. 3.4 kDa) molar ratio was 1:5.
EXAMPLE 6
Preparation of Protein Conjugate using PEG having a Different
Functional Group
[0074] hGH-PEG-IgG conjugates were prepared using PEG having
different functional groups other than aldehyde groups at both ends
as following. 10 mg of hGH dissolved in 100 mM phosphate buffer was
reacted with PEG containing succinimidyl propionate (SPA) groups at
both ends (SPA-PEG-SPA, Shearwater Inc, USA, M. W. 3.4 kDa) in an
amount corresponding to an hGH:PEG molar ratio of 1:1, 1:2.5, 1:5,
1:10 or 1:20. The reaction mixture was stirred at room temperature
for 2 hours. To obtain an hGH-PEG complex in which PEG is
selectively linked to the lysine residue of hGH in a molar ratio of
1:1, the reaction mixture was subjected to Superdex.RTM.
(Pharmacia, USA) size exclusion chromatography. The PEG-hGH complex
was eluted and purified from the column with 10 mM potassium
phosphate buffer (pH 6.0) to remove contaminants such as unmodified
hGH, unreacted PEG and dimmeric by-products having two molecules of
hGH linked at both ends of PEG. The purified IgG-PEG complex was
concentrated to 5 mg/ml. The hGH-PEG-IgG conjugate was prepared
using the hGH-PEG complex concentrated and purified according to
the same method described in Example 1. It has been found that an
optimal hGH:PEG molar ratio for obtaining the best result was in
the range of 1:2.5 to 1:5.
[0075] Another hGH-PEG-IgG conjugate was prepared and purified
according to the same method described above, except that PEG
containing N-hydroxysuccinimidyl (NHS) groups at both ends
(NHS-PEG-NHS, Shearwater Inc, USA) was employed instead of
SPA-PEG-SPA.
EXAMPLE 7
Preparation of Protein Conjugate using PEG having a Different
Molecular Weight
[0076] An hGH-PEG-IgG conjugate was prepared and purified according
to the same method described in Example 1, except that PEG
containing aldehyde groups at both ends and having a molecular
weight of 10,000 daltons (ALD-PEG-ALD, Shearwater Inc, USA) was
employed. At this time, it has been found that an optimal hGH:PEG
molar ratio for obtaining the best result was in the range of 1:2.5
to 1:5. The purified hGH-PEG complex was concentrated to 5 mg/ml.
An hGH-PEG-IgG conjugate was prepared using the hGH-PEG complex
concentrated and purified according to the same method described in
step 2 of Example 1.
COMPARATIVE EXAMPLE 1
Preparation of PEG-hGH Complex
[0077] 5 mg of hGH was dissolved in 100 mM potassium phosphate
buffer (pH 6.0) to obtain 5 ml of a solution, and an activated
methoxy-PEG-ALD having 40 kDa of PEG was added to the solution in
an amount corresponding to an hGH:PEG molar ratio of 1:4.
NaCNBH.sub.3 was added thereto to a final concentration of 20 mM as
a reducing agent, and the reaction mixture was gradually stirred at
4.degree. C. for 18 hours. Then, ethanolamine was added thereto to
a final concentration of 50 mM to inactivate unreacted PEG.
[0078] To further remove unreacted PEG, the reaction mixture was
subjected to Sephadex.RTM. G-25 column (Pharmacia, USA)
chromatography. The column was equilibrated with 2 column volume
(CV) of 10 mM Tris-HCl (pH 7.5) buffer before loading the reaction
mixture. Elution fractions were analyzed for the absorbance at 260
nm using a UV spectrophotometer. The PEG modified hGH which has a
large molecular weight was eluted first before unreacted PEG.
[0079] The PEG-modified hGH was further purified from the elution
fraction as following. A column packed with 3 ml of PolyWAX LP
(Polywax Inc, USA) was equilibrated with 10 mM Tris-HCl (pH 7.5)
buffer. The elution fraction containing the PEG modified hGH was
loaded to the column at a flow rate of 1 ml/min, and the column was
washed with 15 ml of the equilibration buffer. Tri-, di- and
mono-PEG linked hGHs were fractionated in order by a salt
concentration gradient method (NaCl concentration: 0% .fwdarw.70%)
using 1 M NaCl buffer for 30 min.
[0080] To further purify the mono-PEG linked hGH complex from the
mixture, the column effluent was subjected to size exclusion
chromatography. The concentrated effluent was loaded onto a
Superdex 200 (Pharmacia, USA) column equilibrated with 10 mM sodium
phosphate buffer and eluted with the same buffer solution at a flow
rate of 1 ml/min. The tri- and di-PEG linked hGH complexes which
eluted earlier than the mono-PEG linked hGH complex were removed to
obtain purified mono-PEG linked hGH complex.
[0081] PEG-IFN, PEG-.sup.17S-G-CSF derivative and PEG-G-CSF in
which 40 kDa PEG is linked to the terminal amino residues of IFN
.alpha. and G-CSF, respectively, were prepared and purified
according to the same method described above.
COMPARATIVE EXAMPLE 2
Preparation of Albumin-hGH Complex
[0082] To conjugate albumin with the hGH-PEG complex obtained in
Example 1, human serum albumin (HSA, M.W. about 67 kDa,) (Green
Cross, Korea) dissolved in 10 mM phosphate buffer solution was
reacted with the hGH-PEG complex in an amount corresponding to an
hGH-PEG complex:HSA molar ratio is 1:1, 1:2, 1:4 or 1:8. The
reaction mixture was concentrated to 100 mM phosphate buffer, and
NaCNBH.sub.3 was added thereto to a final concentration of 20 mM as
a reducing agent. The reaction mixture was stirred at 4.degree. C.
for 20 hours. It has been found that an optimal hGH-PEG
complex:albumin molar ratio for obtaining the best result was
1:2.
[0083] After the conjugation reaction, the reaction mixture was
subjected to Superdex size exclusion chromatography to remove
unreacted starting materials and by-products. The reaction mixture
was concentrated and loaded onto the column at a flow rate of 2.5
ml/min using 10 mM sodium acetate (pH 4.5) to obtain purified
hGH-PEG-albumin conjugate. Since the purified hGH-PEG- albumin
conjugate was still contaminated by small quantities of unreacted
albumin and hGH dimmer, anion exchange chromatography was further
performed to remove these contaminants. The hGH-PEG-albumin
conjugate effluent was loaded onto a SP5PW (Waters, USA) column
equilibrated with 10 mM sodium acetate (pH 4.5), and fractionated
with 10 mM sodium acetate (pH 4.5) containing 1.0 M NaCl in a
linear fashion (NaCl concentration: 0 M.fwdarw.0.5 M), to recover
pure hGH-PEG-albumin.
[0084] IFN .alpha.-PEG-albumin, G-CSF-PEG-albumin and
.sup.17S-G-CSF derivative-PEG-albumin in which albumin is linked to
IFN .alpha., .sup.17S-G-CSF and G-CSF, respectively, were prepared
and purified according to the same method described above.
TEST EXAMPLE 1
Confirmation and Quantification of Protein Conjugates
[0085] (1) Confirmation of Protein Conjugates
[0086] Protein conjugates prepared in above Examples were analyzed
for their modification state by SDS-PAGE using a gel having a
concentration gradient of 4 to 20% and ELISA (R&D System,
USA).
[0087] hGH, hGH-PEG, IFN and IFN-PEG were each developed on
SDS-PAGE and a mixture with 50 mM DTT (dithiothreitol), while IgG,
hGH-PEG-IgG and IFN-PEG-IgG without DTT.
[0088] FIGS. 2 and 3 show the SDS-PAGE results obtained for the
hGH-PEG-IgG and IFN-PEG-IgG conjugates, respectively. Numbers
listed on left margin are molecular weight markers (kDa).
[0089] As shown in FIGS. 2 and 3, the appearance molecular weight
of hGH-PEG-IgG conjugate is about 170 kDa. However, since it is
difficult to discriminate the molecular weight difference between
the IgG protein conjugates and wild-type IgG in SDS-PAGE, the
hGH-PEG-IgG conjugate and IgG were reduced by DTT treatment,
separated into heavy- and light-chains, and confirmed its
conjugated state by SDS-PAGE, respectively (FIG. 4).
[0090] When IgG was treated with DTT, the light chain of IgG was
separated first, and the heavy chain of IgG, later according to
their molecular weight. Bands of hGH-PEG-IgG conjugate treated with
DTT appeared at positions which corresponding to molecular weights
calculated by adding the molecular weight of hGH-PEG (3.4 kDa) to
the molecular weight of light- and heavy chain fragments,
respectively. The light chain of hGH-PEG-IgG conjugate formed a
band at a lower position (smaller molecular weight) than the heavy
chain of hGH-PEG-IgG conjugate whose band was found at a position
corresponding to about 80 kDa. From the above results, it has been
found that hGH coupled with light and heavy chains with equal
probability, and that IgG reacts with hGH in a molar ratio of
1:1.
[0091] (2) Quantitative Analysis of Protein Conjugates
[0092] The amount of each protein conjugate prepared in the above
Examples was determined by calculating its peak area observed in
size exclusion chromatography (column: Superdex, elution solution:
10 mM potassium phosphate buffer solution (pH 6.0)) and comparing
with that of control. After conducting size exclusion
chromatography using pre-quantified hGH, IFN, G-CSF,
.sup.17S-G-CSF, EPO and IgG, respectively, relative response
factors of the peak areas were determined. The size exclusion
chromatography was performed using a constant amount of each
protein conjugate with a same condition, and the quantitative value
of biologically active protein existed in each protein conjugate
was determined by subtracting the peak area corresponding to IgG
from the peak area of each protein conjugate obtained above.
[0093] ELISA (R&D System, USA) analysis was also carried out
besides chromatography. If a portion of IgG is conjugated to a
biologically active site of a polypeptide, the value obtained by
ELISA using an antibody specific for the biologically active sire
would be lower than the value calculated by chromatography. In case
of the hGH-PEG-IgG conjugate, it has been found that the value
measured by ELISA was only about 30% of the value determined by
chromatography.
[0094] (3) Confirmation of Purity and Mass of Protein
Conjugates
[0095] The protein conjugate obtained in each Example was analyzed
for its absorbance value at 280 nm during size exclusion
chromatography, and found that hGH-PEG-IgG, IFN-PEG-IgG, G-CSF and
.sup.17S-G-CSF-PEG-IgG each showed a single peak corresponding to a
molecular weight of from 170,000 to 180,000 daltons. The peak of
EPO-PEG-IgG was observed at a position corresponding to a molecular
weight of 200,000 daltons.
[0096] To determine the exact molecular weight of each protein
conjugate, the purified samples were analyzed using MALDI-TOF
(Voyager DE-STR, Applied Biosystems, USA) superspeed mass
spectrometry. Sinapinic acid was employed as a matrix. 0.5 .mu.l of
each sample was spread on a slide glass and dried in the air. After
an equal volume of the matrix was dropped on the slide glass, the
slide glass was dried in the air and installed in an ion source.
Detection was performed by a linear mode TOF equipment using a
positive method, and ions were accelerated by a total potential
difference of about 2.5 kV in a divided extraction supply source
using a delayed ion extractor at a delayed extraction time of 750
nsec/1500 nsec. The results of mass spectrometry analyses of
hGH-PEG-IgG conjugate are shown in Table 1 and FIG. 5.
1TABLE 1 Mass spectrometry analysis of IgG-protein conjugates
Theoretical value Measured value (kDa) (kDa) hGH-PEG-IgG (Exp. 1)
175.4 175.4 IFN .alpha.-PEG-IgG (Exp. 3) 172.6 172.6 G-CSF-PEG-IgG
(Exp. 4) 172.1 173.0 .sup.17S-G-CSF derivative- 171.9 172.2 PEG-IgG
(Exp. 4) EPO-PEG-IgG (Exp. 5) 185.4 183.0
[0097] The results showed that the purity of hGH-PEG-IgG conjugate
was 90% or more, and that the measured molecular weight was nearly
equal to the theoretical value. Further, the hGH-PEG-IgG conjugate
was in the form of IgG bound to the hGH-PEG complex in a molar
ratio of 1:1.
TEST EXAMPLE 2
Pharmacokinetics Analysis
[0098] In vivo stabilities and pharmacokinetic coefficients of the
IgG-protein conjugates, PEG-protein and albumin-protein complexes
(test group) prepared in Examples and Comparative Examples were
compared with those of biologically active wild-type protein
(control group). 5 Sprague-Dawley (SD) rats were used for each
group in the following experiments. Mice received subcutaneous
injections of 100 .mu.g/kg of the control, PEG-complex,
albumin-protein conjugate and IgG-protein conjugate, respectively.
Blood samples were taken from the control group at 0.5, 1, 2, 4, 6,
12, 24, 30, 71 and 96 hour after the injection, and the samples of
the test groups, at 1, 6, 12, 24, 30, 48, 72, 96, 120, 240 and 320
hour after the injection. Blood samples were collected in an
eppendorf tube coated with heparin to prevent blood coagulation,
and subjected to high-speed micro centrifugation at 4.degree. C.,
3,000.times.g for 30 min to remove cells. The protein concentration
in sera was measured by ELISA method using the respective antibody
specific for each biologically active protein.
[0099] Pharmacokinetic values of the wild-type hGH, IFN, G-CSF and
EPO, and protein conjugates, complexes thereof are shown in Tables
2 to 6, in which T.sub.max means the time to reach the maximum drug
concentration, T.sub.1/2, half-life of a drug in blood, and MRT
(mean residence time), average retention time in a body.
2TABLE 2 Pharmacokinetic values of hGH hGH-PEG- hGH-PEG- Wild-type
hGH-40K PEG albumin IgG hGH (Com. Exp. 1) (Com. Exp. 2) (Exp. 1)
T.sub.max (hr) 1.0 12 12 12 T.sub.1/2 (hr) 1.1 7.7 5.9 13.9 MRT
(hr) 2.1 18.2 13.0 19.0
[0100]
3TABLE 3 Pharmacokinetic values of IFN .alpha. IFN .alpha.-PEG- IFN
.alpha.-PEG- Wild-type IFN .alpha.-40K PEG albumin IgG IFN .alpha.
(Com. Exp. 1) (Com. Exp. 2) (Exp. 3) T.sub.max (hr) 1.0 30 12 30
T.sub.1/2 (hr) 1.7 35.8 17.1 76.7 MRT (hr) 2.1 71.5 32.5 121.0
[0101]
4TABLE 4 Pharmacokinetic values of G-CSF G-CSF-40K G-CSF-PEG-
G-CSF-PEG- Wild-type PEG albumin IgG G-CSF (Com. Exp. 1) (Com. Exp.
2) (Exp. 4) T.sub.max (hr) 2.0 12 12 12 T.sub.1/2 (hr) 2.8 4.8 5.2
8.4 MRT 5.2 24.5 25.0 35.7 (hr)
[0102]
5TABLE 5 Pharmacokinetic values of .sup.17S-G-CSF Wild-type
.sup.17S-G-CSF .sup.17S-G-CSF .sup.17S-G-CSF .sup.17S-G-
derivative-40K derivative-PEG- derivative- CSF PEG albumin PEG-IgG
derivative (Com. Exp. 1) (Com. Exp. 2) (Exp. 4) T.sub.max (hr) 2.0
24 24 48 T.sub.1/2 (hr) 2.9 4.3 6.4 7.2 MRT (hr) 5.8 24.4 25.1
42.6
[0103]
6TABLE 6 Pharmacokinetic values of EPO Highly Wild-type
glycosylated EPO EPO-PEG-IgG EPO (Darbepoetin-.alpha.) (Exp. 5)
T.sub.max (hr) 6.0 12 48 T.sub.1/2 (hr) 9.4 18.4 5.2 MRT (hr) 21.7
36.7 95.6
[0104] As can be seen in Tables 2 to 6, the half-life of the
hGH-PEG-IgG conjugate was 13.9 hr, which is about 13-fold higher
than that of wild-type hGH and about 2-fold higher than that of the
hGH-40K PEG complex (7.7. hr) prepared in Comparative Example. The
half-life of the hGH-PEG-albumin conjugate in which albumin is
linked to the one end of PEG, not directly to hGH, was 5.9 hr. This
result confirms that the inventive protein conjugate shows far
superior durability in vivo.
[0105] Further, in Table 3, the results for IFN .alpha. were
similar to those of hGH, but the effect of increasing the blood
half-life observed in the inventive protein conjugate was far
higher. While the half-life of wild-type IFN .alpha. was 1.7 hr,
the half-life of 40 kDa PEG-IFN .alpha. complex increased to 35.8
hr and the half-life of IFN .alpha.-PEG-albumin conjugate, to 17.1
hr. As compared with these, the half-life of IFN .alpha.-PEG-IgG
conjugate remarkably increased to 76.7 hr.
[0106] As shown in Tables 4 and 5, the in vivo durability of G-CSF
and its derivatives showed a tendency similar to that of hGH and
IFN. The half-life of 40 kDa PEG modified protein complexes and
albumin conjugates were longer than those of wild-type G-CSF and
its derivative. However, the inventive IgG protein conjugate showed
a much longer half-life. Such an ability of the conjugated IgG to
increase the drug stability in blood was also observed for amino
acid modified derivatives. From these results, it can be
anticipated that the inventive protein conjugate applied to other
proteins would also exert the desired effect described above.
[0107] FIG. 7 and Table 6 show that the effect of increasing the
in-blood half-life of the inventive protein conjugate is evident
for EPO having a glycosylated moiety. Namely, the in-blood
half-life of wild-type EPO was 9.4 hr and that of highly
glycosylated EPO having high blood stability, i.e,
Darbepoetin-.alpha. (Aranesp, Amgen, USA), was 18.4 hr. In case of
EPO-PEG-IgG conjugate, the blood half-life remarkably increased to
52.2 hr.
[0108] As can be seen above results, the inventive protein
conjugate covalently bonded IgG with a non-peptide polymer has an
in-blood half-life which is dozen times higher than the wild-type
protein.
[0109] Especially, as compared with 40 kDa PEG modified protein
complex which has the highest in-blood durability among the
previously reported PEG formulations, the inventive IgG protein
conjugate exhibits far better durability. Further, relative to the
protein conjugate coupled with albumin instead of IgG, the
inventive protein conjugate showed markedly higher durability.
These results suggest that the inventive protein conjugate can be
effectively used for preparing a sustained formulation of a protein
drug. The present findings, that the inventive protein conjugates
exhibit markedly higher in-blood stability and longer MRT than
previously reported PEG binding protein or albumin protein
conjugate for a wide range of proteins including the G-CSF
derivative having a point mutation, strongly suggests that such
effect of increasing the in-blood stability and durability observed
for the inventive protein conjugate would also be realized for any
other biologically active peptides.
[0110] The half-life of hGH-PEG-IgG conjugate (Example 7) prepared
using 10 kDa PEG as a non-peptide polymer was measured by the same
method described above to be 14.7 hr, which is slightly higher than
that of hGH-PEG-IgG conjugate using 3.4 kDa PEG (13.9 hr). The
appearance molecular weight and in-blood half-lives observed for
those prepared using PEG having different functional groups, e.g.,
succinimidyl propionate or N-hydroxysuccinimidyl groups, were
similar to those prepared using PEG having aldehyde groups.
TEST EXAMPLE 3
Measurement of in vivo Activity
[0111] (1) Comparison of in vivo Activity of hGH Protein
Conjugates
[0112] In vivo activities of the hGH-PEG-IgG conjugate, 40 kD
PEG-hGH complex and hGH-PEG-albumin conjugate were measured by
using rat node lymphoma cell line Nb2 (European Collection of Cell
Cultures, ECCC #97041101) that undergo hGH dependent mitosis as
follows.
[0113] Nb2 cells were cultivated in Fisher's medium supplemented
with 10% fetal bovine serum (FBS), 0.075% NaCO.sub.3, 0.05 mM
2-mercaptoethanol and 2 mM glutamine. The cells were incubated for
additional 24 hours in the same medium without 10% FBS. After about
2.times.10.sup.5 cells per well were added to a 96-well plate,
various dilutions of hGH-PEG-IgG, 40 kDa PEG-hGH, hGH-PEG-albumin
and a control (National Institute for Biological Standards and
Control, NIBSC) were added to each well and the plates were
incubated for 48 hours at 37.degree. C. in a CO.sub.2 incubator. To
measure the extent of cell growth (the number of cells existed in
each well), 25 .mu.l of cell titer 96 Aqueous One Solution
(Promega, USA) was added to each well and incubated for 4 hours at
37.degree. C. Absorbance at 490 nm was measured to calculate the
titer of each sample, and the calculated titers as shown in Table
7.
7TABLE 7 In vitro activity analysis of hGH Relative activity
Specific activity* to wild-type hGH Conc. (ng/ml) (U/mg) (%)
Wild-type hGH 100 2.71E+0.6 100 Control (NIBSC) 100 2.58E+0.6 95.2
HGH-40K PEG 100 0.206E+0.6 7.6 HGH-PEG- 100 0.141E+0.6 5.2 albumin
hGH-PEG-IgG 100 0.86E+0.6 31.7 *specific activity = 1/ED.sub.50
.times. 10.sup.6 (ED.sub.50: the amount of protein representing 50%
of the maximum cell growth)
[0114] As can be seen from Table 7, all samples used in the
experiments have in vitro activity. In addition, the in vitro
activity of PEG modified hGH complex was lower than that of the
unmodified hGH. In case of interferon, it was reported that 12 kDa
PEG and 40 kDa PEG conjugates with IFNs showed activities which
were only about 25% and 7% of the wild-type, respectively (P.
Bailon et al., Bioconjugate Chem. 12:196-202, 2001). The larger the
molecular weight of PEG increases, the lower the in vitro activity
of PEG complex decreases. The in vitro activity of 40 kDa PEG
modified hGH complex was only about 7% of wild-type hGH, and the
hGH-PEG-albumin conjugate also showed a very low in vitro activity
of about 5.3% of the wild-type. However, in case of conjugating IgG
with the hGH-PEG complex, its relative activity was significantly
enhanced to 30% or more of the wild-type. These results suggest
that the inventive protein conjugates have both higher in vivo
activity as well as prolonged in-blood half-life. In case of the
IgG protein conjugates of the present invention, the increased
protein activity is believed to be due to the increased in-blood
stability caused by conjugation with IgG which plays the role of
preserving the binding affinity to a receptor, and the non-peptidic
polymer providing a spatial room. Such effect is expected to occur
for IgG protein conjugates of any other biologically active
proteins.
[0115] (2) Comparison of in vivo Activity of IFN .alpha. Protein
Conjugates
[0116] To compare the in vivo activity of IFN .alpha. protein
conjugates, anti-viral activity of IFN .alpha.-PEG-IgG complex
(Example 3), 40 kDa PEG-IFN .alpha. conjugate (Comparative Example
1) and IFN .alpha.-PEG-albumin conjugate (Comparative Example 2)
were measured by a cell culture biopsy method using Madin-Darby
bovine kidney cells (MDBK cells; ATCC CCL-22) saturated with
vesicular stomatitis virus (VSV). IFN .alpha. 2b having no PEG
modification (NIBSC IFN) was employed as a control.
[0117] MDBK cells were cultured in MEM (minimum essential medium,
JBI) supplemented with 10% FBS and 1% penicillin-streptomycin at
37.degree. C. in a 5% CO.sub.2 incubator. Samples and a control
(NIBSC IFN) were diluted with the same culture medium to a constant
concentration, and 100 .mu.l of each dilution was added to 96-well
plate. 100 .mu.l of the cultured cell solution was added to each
well, and the microplate was incubated at 37.degree. C. for about 1
hr in a 5% CO.sub.2 incubator. After an hour, 50 .mu.l of VSV
having a viral concentration of 5.about.7.times.10.sup.3 PFU was
added to each well, and further incubated for 16 to 20 hours at
37.degree. C. under 5% CO.sub.2. Wells containing only cells and
virus without samples or the control were employed as a negative
control, and wells containing only cells without added viruses, as
a positive control.
[0118] To remove the culture medium and to stain living cells, 100
.mu.l of a neutral red solution was added to each well and further
incubated at 37.degree. C. for 2 hours in a 5% CO.sub.2 incubator.
After removing the supernatant by aspirating, the extraction
solution (100 .mu.l g of a mixture of 100% ethanol and 1% acetate
(1:1)) was added to each well. The stained cells were resuspended
in the extraction solution with shaking and the absorbance at 540
nm was measured. ED.sub.50 representing 50% of the maximum cell
growth was calculated by regarding the cell growth of the positive
control as 100% relative to the cell growth of the negative
control.
8TABLE 8 In vitro activity analysis of IFN .alpha. Relative
activity Concentration to wild-type IFN (ng/ml) ED50 (IU/mg) (%)
Wild-type IFN .alpha. 100 4.24E+0.8 100 IFN .alpha.-40K PEG 100
2.04E+0.7 4.8 IFN .alpha.-PEG- 100 2.21E+0.7 5.2 albumin IFN
.alpha.-PEG-IgG 100 4.75E+0.7 11.2
[0119] As shown in Table 8, the activity of PEG modified IFN
complex was lower than that of unmodified IFN. Especially, the
in-blood stability increased as the molecular weight of PEG moiety
increased, but the relative activity gradually decreased. A 40 kDa
PEG modified IFN complex showed a very low in vitro activity
corresponding to about 4.8% of the wild-type activity. As mentioned
above, there was a previous report that 12 kDa PEG and 40 kDa PEG
conjugated IFNs showed about 25% and 7% in vitro activity of the
wild-type, respectively (P. Bailon et al., Bioconjugate Chem.
12:196-202, 2001). Namely, since if the molecular weight of PEG
increases, the blood half-life increases but its pharmaceutical
effect suddenly decreases, there has been a need to develop a
substance having improved pharmaceutical activity and prolonged
half-life. The IFN .alpha.-PEG-albumin conjugate also showed a very
low in vitro activity corresponding to only about 5.2% of the
wild-type. However, in case of modifying IFN .alpha. with IgG (IFN
.alpha.-PEG-IgG conjugate), the relative activity increased to
11.2% of the wild-type. These results show that the inventive IgG
protein conjugate exhibits high in vivo activity together with
prolonged half-life.
[0120] (3) Comparison of in vivo Activity of G-CSF Protein
Conjugates
[0121] The in vivo activities of wild-type G-CSF (Filgrastim),
.sup.17Ser-G-CSF derivative, 20 kDa PEG-G-CSF complex (Neulasta,
USA), 40 kDa PEG-.sup.17S-G-CSF derivative complex,
.sup.17Ser-G-CSF derivative-PEG-albumin conjugate and
.sup.17S-G-CSF derivative-PEG-IgG conjugate were measured.
[0122] First, human myelogenous originated cells, HL-60 (ATCC
CCL-240, Promyelocytic leukemia patient/36 yr old Caucasian female)
cells, were cultivated in RPMI1640 medium supplemented with 10%
FBS, and the number of cells were adjusted to about
2.2.times.10.sup.5 cells/ml. DMSO (dimethylsulfoxide, culture
grade/SIGMA) was added to the cells to a concentration of 1.25%
(v/v). 90 .mu.l of the DMSO treated culture solution having about
2.times.10.sup.4 suspended cells per well was added to 96-well
plate (Coming/low evaporation 96 well plate) and incubated at
37.degree. C. for 72 hours in a 5% CO.sub.2 incubator.
[0123] The concentration of each sample was determined by using a
G-CSF ELISA kit (R & D Systems, USA), and each sample was
diluted with RPMI1640 medium at a proper ratio to a concentration
of 10 .mu.g/ml. The resulting solution was subjected to 19 cycles
of sequential half dilution with RPMI1640 medium.
[0124] 10 .mu.l of each sample prepared above was added to each
well having HL-60 cells on cultivation, and the concentration was
reduced by half from 1000 ng/ml. The microplates treated with
protein samples were further incubated at 37.degree. C. for 72
hour.
[0125] To examine the extent of cell growth after the incubation,
the number of cells were determined by measuring absorbance at 670
nm using CellTiter96.TM. (Promega, USA).
9TABLE 9 In vitro activity analysis of G-CSF derivative Relative
activity ED50 (IU/ml) to G-CSF (%) Wild-type G-CSF 0.30 100
(Filgrastim) .sup.17Ser-G-CSF derivative 0.26 115 20K-PEG-G-CSF
1.20 25 (Neulasta) .sup.17Ser-G-CSF derivative- 10.0 <10.0 40K
PEG .sup.17Ser-G-CSF derivative- 1.30 23.0 PEG-albumin
.sup.17Ser-G-CSF derivative- 0.43 69.0 PEG-IgG
[0126] As can be seen from Table 9, the IgG protein conjugate of
.sup.17Ser-G-CSF derivative having an amino acid modification
showed an effect similar to that observed for the protein conjugate
of the wild-type. It has been already confirmed that the
.sup.17Ser-G-CSF derivative modified with PEG shows a longer
half-life but a lower activity than the unmodified (Korean Patent
Application No. 2003-17867). Specially, while the in-blood
stability of PEG modified .sup.17Ser-G-CSF derivative increased as
the molecular weight of the PEG moiety increased, its relative
activity gradually decreased. 40 kDa PEG modified .sup.17Ser-G-CSF
derivative complex showed a very low in vitro activity
corresponding to about 10% of the wild-type. Namely, as the
molecular weight of PEG increases, the in-blood half-life increases
but its pharmaceutical effect suddenly decreases, there has been a
need to develop a substance having improved pharmaceutical activity
and prolonged half-life. Meanwhile, the .sup.17Ser-G-CSF derivative
modified with albumin showed a relatively low in vitro activity
corresponding to only about 23% of the wild-type. However, in case
of modifying .sup.17Ser-G-CSF derivative with IgG
(.sup.17Ser-G-CSF-PEG-IgG conjugate), its relative activity
increased in a level which is 69% or more of the wild-type. These
results show that the inventive IgG protein conjugate exhibits high
in vivo activity together with prolonged half-life.
TEST EXAMPLE 4
Measurement of in vivo Activity in Animal Model
[0127] (1) Comparison of in vivo Activity of hGH Protein
Conjugates
[0128] 10 hypsectomized Sprague Dawley rats (5-week old, SLC, USA)
were employed for each group in a body weight gaining test to
measure the in vivo activities of hGH-PEG-IgG conjugate, hGH-40K
PEG complex and wild-type hGH. A solvent control, wild-type hGH,
hGH-PEG-IgG conjugate and hGH-40K PEG complex were subcutaneously
injected into the rat's back of the shoulder using a 26 G syringe
(1 ml, Korea Vaccine Co., Ltd.) according to the administration
schedule and dose described in Table 10. Rats' weights were
measured before the injection and 16 hours after the injection.
Rats were sacrificed with ether 24 hours after the final injection,
and the presence of pituitary gland was examined with the naked eye
to exclude the rats having observable residual pituitary gland from
the result.
10TABLE 10 Condition for in vivo activity test of hGH in animal
model Average daily Total dose amount of Administration Group Drug
(day) administration schedule 1 Solvent -- PRS (0.5 ml) Once/day,
control Daily administration for 12 days 2 Wild-type 30 .mu.g 360
mIU Once/day, hGH (30 .mu.g/time) Daily administration for 12 days
3 hGH-40K 30 .mu.g 360 mIU Once/6 days, PEG (180 .mu.g/time) Twice
administration 4 hGH-PEG- 30 .mu.g 360 mIU Once/6 days, IgG (180
.mu.g/time) Twice administration 5 hGH-PEG- 10 .mu.g 120 mIU Once/6
days, IgG (60 .mu.g/time) Twice administration
[0129] The change in the weight after the administration of each
sample was showed in FIG. 8. Since the wild-type hGH used as a
standard (control) must be administered everyday to maintain its in
vivo activity, it was administered once a day for 12 days, and
accordingly, rats in Group 1 gained in weight during the
administration. In rats in Group 2 administered with the hGH-40 kDa
PEG complex once a week, gained weight continuously till 3 days
after the administration, and the rate of increase slowed down
thereafter. Theses results coincided with the expectation based on
the results of Test Examples 1 and 2 that the hGH-PEG complex
showed far longer half-life and higher in vivo activity than the
wild-type hGH. Especially, the effect generated by administering
hGH-PEG-IgG conjugate once a week in an amount corresponding to a
third of the wild-type dose equal or better than daily
administration of the wild-type. This means that the in vivo
activity of hGH-PEG-IgG conjugate is more than 3-fold higher than
that of the wild-type.
[0130] (2) Comparison of in vivo Activity of G-CSF Derivative
Protein Conjugates
[0131] In order to examine the effect of the inventive protein
conjugates with .sup.17Ser-G-CSF having a substitution of 17.sup.th
amino acid by serine, the in vivo activities of wild-type G-CSF, a
commercially available 20 kDa PEG-G-CSF complex and
.sup.17Ser-G-CSF-PEG-IgG conjugate were compared. The
.sup.17Ser-G-CSF-PEG-IgG conjugate of the present invention was
dissolved in a solvent comprising 20 mM sodium phosphate, 1%
glycine and 0.25% mannitol (pH 7.0). Wild-type methionyl G-CSF
complex (Filgrastim, Amgen, USA) and 20 kDa PEG modified G-CSF
(Neulasta, Amgen, USA) dissolved in the same solvent were employed
as a comparative group. Female 7-week-old ICR mice were purchased
from Samtaco Bio (Korea) and subjected to an acclimation period for
a week before the experiment. At the beginning of the experiment,
the weight of ICR mice were in the range of 30.about.35 g. They
were allowed to freely ingest formula feed (Samyang Corporation,
Korea) and water during the acclimation and experiment, and kept in
a cage under the condition of 22.+-.3.degree. C., 55.+-.5% of
relative humidity, 1.about.12 times/hr ventilation, 150.about.200
lux of illumination intensity and a daily lighting cycle of 12 hrs
light/12 hrs dark. Each experimental group consisted of 5 mice, and
a complex anticancer agent and each sample were administered to the
mice according to the administration schedule and dose described in
Table 11. Neutropenia animal model was prepared by injecting once a
mixture of 130 mg/kg of cyclohexamide (CPA; Sigma, USA), 4.5 mg/kg
of doxorubicin (DXR; Sigma, USA) and 1 mg/kg of vincristin (VCR,
Sigma, USA) into the abdominal cavity of ICR mice. No treatment
group did not receive the anticancer agent administration and show
no reduction of neutrophil. The solvent control is the group which
was administered with anticancer agent to reduce the number of
neutrophil and with adjuvant only instead of a drug sample. The
wild-type G-CSF was subcutaneously injected at a dose of 100
.mu.g/kg/day around 10 a.m. everyday from the first day till the
fifth day after the anticancer agent administration. The
.sup.17S-G-CSF-IgG and 20 kDa PEG-G-CSF complexes (Neulasta, Amgen,
USA) were injected once at the first day after the anticancer agent
administration at a dose of 1,000 .mu.g/kg that corresponds to a
dose for five days when a twofold amount of the wild-type dose was
regarded as a daily dose (200 .mu.g/kg/day). 0.3.about.0.5 ml of
blood was taken from mice's orvital vein at day 1, 2, 3, 4, 5, 6
and 8 after the anticancer agent administration. Blood collection
was performed around 4 p.m., 6 hours after the injection of a drug
sample. The numbers of white blood cells (WBC), red blood cells
(RBC) and platelet were measured using an automatic hematocyte
counter. In addition, a blood spread specimen was prepared and
subjected to Giemsa staining. Each hematocyte was differentially
calculated to obtain the ratio of neutrophil, and then, the number
of neutrophil was calculated by formula 2 based on the ratio of
neutrophil.
the number of neutrophil (cells/mm.sup.3)=total number of WBC
(cells/mm.sup.3).times.the ratio of neutrophil (%).times.1/100
<Formula 2>
[0132] To examine the statistical significance of the values
observed for the no treatment group, solvent control group and
.sup.17S-G-CSF derivative PEG-IgG group, statistical analysis was
performed about the number of hematocyte and weight of each group
using Student's t-test.
11TABLE 11 Condition of in vivo activity test of increasing the
number of neutrophil in animal model Average Total amount daily
dose of Administration Group Drug (kg/day) administration schedule
1 No treatment -- PRS (0.5 ml) Once/day, Daily administration for 5
days 2 Solvent -- PRS (0.5 ml) Once/day, control Daily
administration for 5 days 3 Wild-type G- 100 .mu.g 500 .mu.g/kg/5
Once/day, CSF times Daily administration (Filgrastim) for 5 days 4
20K PEG-G- 200 .mu.g 1,000 .mu.g/kg/ Once administration CSF time
(Neulasta) 5 .sup.17S-G-CSF 200 .mu.g 1,000 .mu.g/kg/ Once
administration derivative time PEG-IgG
[0133] The recovery of neutrophil after the administration of each
sample is shown in FIG. 9. When the wild-type G-CSF used as a
standard was injected everyday for 5 days, the number of neutrophil
gradually increased during the administration and finally reached a
maximum at day 5. While the 20 kDa PEG-G-CSF complex administered
once at twofold amount of the daily dose showed only two-thirds of
the in vivo activity observed for the daily administration of
wild-type G-CSF, the .sup.17S-G-CSF derivative-PEG-IgG conjugate
exhibited an activity which was 3-fold higher than the in vivo
activity of 20 kDa PEG-G-CSF complex. Further, the inventive
protein conjugate generated two-fold higher effect for recovering
neutrophil than daily administration of G-CSF, which coincided with
the expectation based on the result that the .sup.17S-G-CSF
derivative-PEG-IgG conjugate had significantly longer in-blood
half-life and higher in vivo activity than the wild-type. These
results show that the same effect of the inventive protein
conjugate caused by covalently binding IgG to PEG can be expected
of a protein derivative having an amino acid modification as well
as the wild-type.
* * * * *