U.S. patent application number 12/631617 was filed with the patent office on 2010-09-02 for mutants of staphylokinase carrying amino and carboxy-terminal extensions for polyethylene glycol conjugation.
Invention is credited to Kanak Lata Dikshit, Satish Singh.
Application Number | 20100221236 12/631617 |
Document ID | / |
Family ID | 42067534 |
Filed Date | 2010-09-02 |
United States Patent
Application |
20100221236 |
Kind Code |
A1 |
Singh; Satish ; et
al. |
September 2, 2010 |
Mutants of Staphylokinase Carrying Amino and Carboxy-Terminal
Extensions for Polyethylene Glycol Conjugation
Abstract
The present invention relates to the development of new
derivatives of a bacterial plasminogen activator, Staphylokinase
(SAK), having one or more amino acid residues with single or
multiple cysteines at the amino and/or carboxy terminal ends and
their conjugation with PEG (Polyethylene Glycol), resulting in new
Staphylokinase derivatives that display altered oligomeric states,
enhanced thermal and protease stability and extended plasma
half-life. Also included is the cloning and expression in a
suitable bacterial host; purification of Staphylokinase derivatives
to homogeneity and their chemical modification by integrating a PEG
molecule to create new biologically active Staphylokinases having
higher protein stability and improved in vivo plasma half life,
that may enhance the clinical potential of Staphylokinase in
thrombolytic therapy for the treatment of cardiovascular
diseases.
Inventors: |
Singh; Satish; (Chandigarh,
IN) ; Dikshit; Kanak Lata; (Chandigarh, IN) |
Correspondence
Address: |
King & Spalding LLP
P.O. Box 889
Belmont
CA
94002-0889
US
|
Family ID: |
42067534 |
Appl. No.: |
12/631617 |
Filed: |
December 4, 2009 |
Current U.S.
Class: |
424/94.63 ;
435/188; 435/212; 435/252.33 |
Current CPC
Class: |
A61K 38/4886 20130101;
A61K 47/60 20170801; A61K 38/00 20130101; C12Y 304/24029 20130101;
A61P 9/04 20180101; C12N 9/52 20130101; A61P 9/10 20180101; C12N
9/48 20130101 |
Class at
Publication: |
424/94.63 ;
435/212; 435/188; 435/252.33 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61P 9/10 20060101 A61P009/10; A61P 9/04 20060101
A61P009/04; C12N 9/48 20060101 C12N009/48; C12N 9/96 20060101
C12N009/96; C12N 1/21 20060101 C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 5, 2008 |
IN |
2757/DEL/2008 |
Claims
1. A Staphylokinase variant, wherein at least one cysteine residue
is added to the amino-terminal region of SEQ ID NO:1, to the
carboxy-terminal region of SEQ ID NO:1 or to the amino-terminal
region and the carboxy-terminal region of SEQ ID NO:1.
2. The Staphylokinase variant of claim 1, wherein the at the least
one cysteine residue is added to the amino-terminal residue of SEQ
ID NO:1.
3. The Staphylokinase variant of claim 1, wherein the at least one
cysteine residue is added to the carboxy-terminal residue of SEQ ID
NO:1.
4. A Staphylokinase variant of claim 1, comprising the sequence
selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5 and SEQ ID NO:6.
5. The Staphylokinase variant of claim 1, wherein the at least one
cysteine residue is modified with a cysteine-reactive moiety.
6. The Staphylokinase variant of claim 1, wherein the at least one
cysteine residue is conjugated to a polyethylene glycol (PEG)
molecule.
7. The variant of claim 6, wherein the PEG molecule is linear or
branched.
8. The variant of claim 7, wherein the molecular size of the PEG
molecule ranges from 5-20 Kilodaltons.
9. The variant of claim 6, wherein the variant has proteolytic
stability which is greater than the proteolytic stability of the
polypeptide identified as SEQ ID NO:1.
10. The variant of claim 6, wherein the variant has in vivo
immunogenicity which is less that the in vivo immunogenicity of the
polypeptide identified as SEQ ID NO:1.
11. The variant of claim 6, wherein the variant has an in vivo half
life which is greater than the in vivo half life of the polypeptide
identified as SEQ ID NO:1.
12. The variant of claim 1, wherein the variant has temperature
stability which is greater than the temperature stability of the
polypeptide identified as SEQ ID NO:1, wherein the temperature
stability is determined at a temperature ranging from about
20.degree. C. to about 80.degree. C.
13. A recombinant E. coli host cell comprising a vector, wherein
the vector comprises a DNA sequence encoding a polypeptide selected
from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5
or SEQ ID NO:6.
14. The host cell of claim 13, wherein the host cell is deposited
in the International Depository Authority (IDA), and wherein the
host cell has the deposit number MTCC 5437, MTCC 5438, MTCC 5439,
or MTCC 5440.
15. A pharmaceutical composition comprising the Staphylokinase
variant of claim 6, further comprising a pharmaceutically
acceptable carrier.
16. A method for treating a cardiovascular disorder selected from
the group consisting of myocardial infarction, vascular thromboses,
pulmonary embolism, stroke a vascular event, disease or disorder
selected from a group consisting of myocardial infarction, angina,
pulmonary embolism, transient ischemic attack, deep vein
thrombosis, thrombotic re-occlusion subsequent to a coronary
intervention procedure, heart surgery or vascular surgery,
peripheral vascular thrombosis, Syndrome X, heart failure, and a
disorder in which a narrowing of at least one coronary artery
occurs, comprising administering the pharmaceutical composition of
claim 15.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of India Application No.
2757/DEU2008, filed Dec. 5, 2008, incorporated herein by reference
in its entirety.
REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM
[0002] A Sequence Listing is being submitted electronically via EFS
in the form of a text file, created Dec. 4, 2009 and named
"SAK8001seqlist.txt" (9000 bytes), the contents of which are
incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0003] The present invention relates to cysteine variants of
Staphylokinase (SAK) wherein its amino and carboxy terminal ends
are extended by adding extra amino acids having at least one
cysteine residue where Polyethylene Glycol (PEG) can be
conjugated.
[0004] This invention relates to the field of cardiovascular
therapeutics, particularly to the thrombolytic drugs that are
utilized for the treatment of various cardiovascular diseases.
[0005] These fibrinolytic agents, e.g., Tissue plasminogen
activator (tPA), Urokinase (UK), Streptokinase (SK), are routinely
utilized for the treatment of various circulatory disorders, e.g.,
deep vein thrombosis, myocardial infarction etc. [Castellino, F. J.
(1981) Recent advances in the chemistry of the fibrinolytic system.
Chem. Rev. 81; 431-446; Vorcheimer, D. A. (1999) Current state of
thrombolytic therapy. Curr. Cardiol Rep. 1; 212-220] due to their
ability to activate blood zymogen, plasminogen (PG) into plasmin
(Pm) that degrades fibrin mesh of the blood clot into a soluble
product, thus, clearing the blockage and leading to coronary
recanalization within the body.
[0006] The present invention relates to the development of new
variants of a bacterial plasminogen activator, Staphylokinase, that
has the ability to activate human plasminogen into active plasmin
[Lack, C. H. (1948) Staphylokinase: an activator of plasma
protease. Nature 161; Lijnen, H. R., Collen, D. (1996)
Staphylokinase, a fibrin-specific bacterial plasminogen activator.
Fibrinolysis, 10; 119-126; Matsuo, O., Okada, K., Fukao, H.,
Tomioka, Y., Ueshima, S., Watanuki, M., Sakai, M (1990)
Thrombolytic properties of Staphylokinase. Blood 76; 925-929]
similar to widely utilized thrombolytic agent, Streptokinase but
unlike SK, SAK exerts its plasminogen activation activity in a
fibrin specific manner due to its unique ability to display
plasminogen activator activity around thrombi as during blood
circulation SAK:PG complex is disrupted by the activity of alpha-2
antiplasmin [Lijnen H. R., Van Hoef B., De Cock F., Okada K.,
Ueshima S., Matsuo O., Callen D. (1991) On the mechanism of
fibrin-specific plasminogen activation by staphylokinase. J. Biol.
Chem. 266; 11826-11832; Collen, D. (1996) Fibrin-selective
thrombolytic therapy for acute myocardial infarction. Circulation
93; 857-865); Collen, D. (1998) Staphylokinase: a potent, uniquely
fibrin-selective thrombolytic agent. Nat. Med. 4; 279-284] whereas
in a clot environment, binding of SAK: plasmin complex on the
fibrin surface become stronger [Sakharov, D. V., Lijnen, H, R. and
Rijken, D. C. (1996) Interaction between plasmin(ogen) and fibrin.
J. Biol. Chem. 271; 27912-27918]. However, being a bacterial
product, clinical administration of SAK creates an allergic
reaction and due to its short plasma half-life (3 min) relatively
large therapeutic dose may be required. The invention disclosed
herein pertains to the development of SAK variants that display
higher temperature stability, low protease sensitivity and extended
in vivo plasma half-life.
[0007] Thus, the present invention relates to the integration of
one or more amino acid residues at the amino- and/or carboxy-
terminal regions of Staphylokinase to engineer sites for PEG
linkage to create new variants of staphylokinase. More
specifically, the extended terminal regions of SAK carry one or
more cysteine residues that allow SAK to make subunit association
resulting in dimeric or multimeric state of SAK that display
enhanced thermal stability and full biological activity comparable
to its unmodified form.
[0008] The present invention also relates to covalent linkage of
Polyethylene Glycol (PEG), ranging from 5 to 20 kDa of molecular
weight, within the extended terminal region of SAK resulting in
derivatives that display low protease sensitivity, higher thermal
stability and extended plasma half life in vivo.
[0009] The present invention also relates to engineering of one or
more cysteine residues within the extended region, specifically at
the carboxy-terminal region, for PEG conjugation that can mask one
of the antigenic sites (Lys135-Lys136) of SAK near the C-terminus
and, thus, can reduce the antigenicity of protein resulting in a
SAK derivative having increased stability, longer in vivo half life
and reduced antigenicity.
[0010] The present invention, therefore, relates to engineered
forms of SAK carrying amino and carboxy-terminal extension and
their PEG conjugated variants that acquire new functions such as
higher temperature stability, low protease sensitivity, reduced
antigenicity and extended in vivo half life and, therefore, can
have enhanced benefits in pharmaceutical composition for the
treatment of various cardiovascular complications.
BACKGROUND
[0011] Staphylokinase is a profibrinolytic protein secreted by
certain strains of Staphylococcus aureus that forms a
stoichiometric complex with human plasminogen and displays
localized plasminogen activation activity in a fibrin specific
manner [Lack, C. H. (1948) Staphylokinase : an activator of plasma
protease. Nature 161; Collen, D., De Cock, F., Vanlinthout, I.,
Declerck, P. J., Lijnen, H. R. and Stasen, J. M. (1992) Comparative
thomboytic and immunogenic properties of staphylokinase and
streptokinase. Fibrinolysis 6; 232-242; Collen D., Lijnen, H.R.
(1993) On the future of thrombolytic therapy for acute myocardial
infarction. Am J Cardiol. 72; 46-50]. This is due to its ability to
bind plasmin at the clot surface with nearly 150-fold higher
affinity than the circulating plasminogen [Sakharov, D. V., Lijnen,
H. R. and Rijken, D. C. (1996) Interaction between plasmin(ogen)
and fibrin. J. Biol. Chem. 271; 27912-27918] where
staphylokinase:plaminogen complex is rapidly inhibited by the blood
component alpha 2-antiplasmin. In a clot environment plasminogen is
partially degraded which results in conformational changes whereby
binding with staphylokinase becomes stronger, therefore, resulting
in a highly localized plasminogen activation activity around
thrombi [Collen, D., De Cock, F., Vanlinthout, I., Declerck, P. J.,
Lijnen, H. R. and Stasen, J. M. (1992) Comparative thomboytic and
immunogenic properties of staphylokinase and streptokinase.
Fibrinolysis 6; 232-242; Callen D., Lijnen, H.R. (1993) On the
future of thrombolytic therapy for acute myocardial infarction. Am
J Cardiol. 72; 46-50]. Since staphylokinase has a weak affinity for
circulating but a high affinity for fibrin-bound plasminogen
[Sakharov, D. V., Lijnen, H. R. and Rijken, D. C. (1996)
Interaction between plasmin(ogen) and fibrin. J. Biol. Chem. 271;
27912-27918] it offers an advantage as a potential clot-dissolving
agent with greater fibrin-specificity, considerably reduced
antigenicity, and an efficacy at least as good as t-PA in terms of
arterial patency [Vanderschueren S, Stockx L, Wilms G, Lacroix H,
Verhaeghe R, Vermylen J, Collen D. (1995) Thrombolytic therapy of
peripheral arterial occlusion with recombinant Staphylokinase.
Circulation 92; 2050-2057; Vanderschueren, S., Van Vlaenderen, I.
and Collen, D. (1997) Intravenous thrombolysis with recombinant
staphylokinase versus tissue type plasminogen activator in a rabbit
embolic stroke model. Stroke 28; 1783-1788].
[0012] Staphylokinase is a single chain 16 kDa protein, consisting
of 136 amino acid residues. It forms a bimolecular complex with the
blood proteins, such as plasminogen (PG) and plasmin (Pm) and
exerts its fibrinolytic effects through conversion of an active
non-specific serine protease, plasmin (Pm) to a highly specific
proteolytic enzyme that can recognize blood zymogen, PG, as a
substrate and convert it into plasmin that is capable of degrading
blood clots. In a plasma milieu, SAK is able to dissolve fibrin
clots without any associated fibrinogen degradation [Lijnen H. R.,
Van Hoef B., De Cock F., Okada K., Ueshima S., Matsuo O., Collen D.
(1991) On the mechanism of fibrin-specific plasminogen activation
by staphylokinase. J Biol. Chem. 266; 11826-11832.; Collen D.,
Lijnen, H. R. (1993) On the future of thrombolytic therapy for
acute myocardial infarction. Am J Cardiol. 72; 46-50]. Clinical
trials have shown that Staphylokinase is as effective as t-PA at
achieving early perfusion in myocardial infarction patients and its
utility in thrombolytic treatment has now been established by
several limited clinical trials [Collen D., Lijnen, H. R. (1993) On
the future of thrombolytic therapy for acute myocardial infarction.
Am J Cardiol. 72; 46-50.; Lijnen, H. R., Collen, D.(1996)
Staphylokinase, a fibrin-specific bacterial plasminogen activator.
Fibrinolysis, 10; 119-126].
[0013] Staphylokinase is produced in very low amounts by its
natural host, Staphylococcus aureus [Lack, C. H. (1948)
Staphylokinase: an activator of plasma protease. Nature 161].
Considering its therapeutic applicability and clinical implications
in thrombolytic therapy, several alternative sources of SAK
production have been developed through recombinant routes. The
staphylokinase gene has been cloned from the bacteriophages sakC
[Sako, T., Sawaki, S., Sakurai, T, Ito, S., Yoshizawa, Y., Kondo,
I. (1983) Cloning and expression of the staphylokinase gene of
Staphylococcus aureus in Escherichia coli. Mol. Gen. Genet. 190;
271-277) and sak42D (Schlott, B., Hartmann, M., Guhrs, K. H.,
Birch-Hirschfeild, E., Pohl, H. D., Vanderschueren, S., van de
Werf, F., Michoel, A., Collen, D. and Behnke, D. (1994) High yield
production and purification of recombinant staphylokinase for
thrombolytic therapy. Biotechnology 12; 185-189] as well as from
the genomic DNA of a lysogenic Staphylococcus aureus strain
[Behnke, D., Gerlach, D. (1984) Cloning and expression in
Escherichia coli, Bacillus subtilis and Streptococcus sanguis of a
gene for staphylokinase, a bacterial plasminogen activator. Mol.
Gen. Genet. 210; 528-534]. The staphylokinase gene encodes a
protein of 163 amino acids, with amino acid 28 corresponding to the
NH2-terminal residue of full-length mature staphylokinase. The gene
encoding for SAK has been overexpressed into various heterologous
hosts, e.g., E. coli, Bacillus and Yeast [Sako, T., Sawaki, S.,
Sakurai, T, Ito, S., Yoshizawa, Y., Kondo, I. (1983) Cloning and
expression of the staphylokinase gene of Staphylococcus aureus in
Escherichia coli. Mol. Gen. Genet. 190; 271-277; Behnke, D.,
Gerlach, D. (1984) Cloning and expression in Escherichia coli,
Bacillus subtilis and Streptococcus sanguis of a gene for
staphylokinase, a bacterial plasminogen activator. Mol. Gen. Genet.
210; 528-534; Schlott, B., Hartmann, M., Guhrs, K. H.,
Birch-Hirschfeild, E., Pohl, H. D., Vanderschueren, S., van de
Werf, F., Michoel, A., Collen, D. and Behnke, D.(1994) High yield
production and purification of recombinant staphylokinase for
thrombolytic therapy. Biotechnology 12; 185-189] to produce SAK in
large quantity in purified form for testing its clinical
applicability.
[0014] Currently, attempts are being made to commercialize
Staphylokinase for clinical use after several successful clinical
and animal trial studies [Vanderschueren, S., Barrios, L.,
Kerdsinchai, P., Van den Heuvel, P., Hermans, L., Vrolix, M., De
Man F., Benit, E, Muyldermans, L., Collen, D., Van de Werf, F.,
(2001) A randomized trial of recombinant staphylokinase versus
alteplase for coronary artery patency in acute myocardial
infarction. Circulation 92; 2044-2049; Armstrong, P. W., Burton,
J., Pakola, S., Molhoek, P. G., Betriu, A. Tendera, M., Bode, C.,
Adgey, A. A., Bar, F., Van de Werf, F. (2003) Collaborative
Angiographic Patency Trial of Recombinant Staphylokinase (CAPTORS
II). Am Heart J 146; 484-488]. However, being a product of
bacterial origin, Staphylokinase elicits considerable allergic
response during drug administration [Collen, D., De Cock, F.,
Vanlinthout, I., Declerck, P. J., Lijnen, H. R. and Stasen, J. M.
(1992) Comparative thomboytic and immunogenic properties of
staphylokinase and streptokinase. Fibrinolysis 6; 232-242].
Attempts have been made to reduce its anitigenicity through the
development of various mutant forms of Staphylokinase [Collen, D.
(1996) Fibrin-selective thrombolytic therapy for acute myocardial
infarction. Circulation 93; 857-865] where distinct mutations were
created within its antigenic epitopes. Another limiting factor of
Staphylokinase, that can hamper its use in thrombolytic therapy, is
its relatively short plasma half-life (3-4 min) due to that
repeated dose of this drug might be required to get effective
recanalization during thrombolytic therapy and that in turn might
exert higher allergic response in the patients. Therefore,
development of second-generation SAK derivatives, where these
shortcomings of native SAK are eliminated, would prove more
advantageous. To overcome these problems, derivatives of SAK
carrying PEG attachment within the protein at various sites have
been generated [Vanwetswinkel, S., Plaisance, S., Zhi-Yong,
Vanlinthout, I., Brepoels, K., Lasters, I., Collen, D., and
Jespers, L. (2000) Pharmacokinetic and thrombolytic properties of
cysteine-linked polyethylene glycol derivatives of staphylokinase.
Blood. 95; 936-942; Verhamme, P., Goossens, G., Maleux, G., Collen,
D. and Stas, M. (2007) A dose-finding clinical trial of
staphylokinase SY162 in patients with long-term venous access
catheter thrombotic occlusion. J Thromb Thrombolysis. 24; 1-5],
however, derivatives carrying PEG at internal sites displayed
significantly lower specific activity [Vanwetswinkel, S.,
Plaisance, S., Zhi-Yong, Vanlinthout, I., Brepoels, K., Lasters,
I., Collen, D., and Jespers, L. (2000) Pharmacokinetic and
thrombolytic properties of cysteine-linked polyethylene glycol
derivatives of staphylokinase. Blood. 95; 936-942] suggesting
internal sites within the core region of SAK may not be suitable
for the chemical modification of SAK.
[0015] Cysteine derivatives of Staphylokinase have been described
in the prior art [Vanwetswinkel, S., Plaisance, S., Zhi-Yong,
Vanlinthout, I., Brepoels, K., Lasters, I., Collen, D., and
Jespers, L. (2000) Pharmacokinetic and thrombolytic properties of
cysteine-linked polyethylene glycol derivatives of staphylokinase.
Blood. 95; 936-942; U.S. Pat. No. 6,383,483 "Staphylokinase
derivatives with cysteine substitutions"; U.S. Pat. No. 6,902,733
"Staphylokinase derivatives with polyethylene glycol"] where
cysteine residue has been substituted within the core region and
amino-terminal part of Staphylokinase. Derivatization of cysteine
substituted SAK mutants with PEG within the core region resulted in
substantial loss of its plasminogen activation ability. Therefore,
ideal site for the PEG conjugation within the core region has not
been found and the approach to conjugate PEG with SAK has not been
successful as these SAK derivatives display significantly lower
plasminogen activation ability than the native form of SAK.
Recombinant Staphylokinase variants obtained by site-directed
substitution with cysteine, within the NH.sub.2-terminal region of
SAK (serine 2 [Ser2] and/or Ser3), that is released from the core
of the protein during plasminogen activation process, were
derivatized with thiol-specific (ortho-pyridyl-disulfide or
maleimide) polyethylene glycol (PEG) molecules, resulting in a SAK
derivative that displayed a plasma half-life 4-5 fold higher
(.about.13 min) than the unmodified form (>3 min). The specific
activity and thrombolytic potency of this SAK derivative in human
plasma was found comparable to that of native SAK and currently
this SAK variant is under clinical trial [Verhamme, P., Goossens,
G., Maleux, G., Collen, D. and Stas, M. (2007) A dose-finding
clinical trial of Staphylokinase SY162 in patients with long-term
venous access catheter thrombotic occlusion. J Thromb Thrombolysis.
24; 1-5]. Although circulating half-life of PEG-linked SAK
derivatives, described in the known literature or disclosed in
known patents [Johnson, C., Royal, M., Moreadith, R., Bedu-Addo,
F., Advant, S. , Wan, M., and Conn, G. (2003) Monitoring
manufacturing process yields, purity and stability of structural
variants of PEGylated staphylokinase mutant SY161 by quantitative
reverse-phase chromatography. Biomed Chromatogr. 17; 335-344] have
been claimed to increase to certain extent, their use as a single
bolus injection for clinical intervention, might require further
improvement in the stability and half life of SAK molecule. The
engineering of SAK for further improvement has been limited due to
its smaller size and difficulty in targeting specific regions of
protein without compromising functional properties of SAK as most
of the regions of either involved in the interaction with the
partner plasmin(ogen) or substrate plasminogen [Parry, M. A.,
Fernandez-Catalan, C., Bergner, A., Huber, R., Hopfner, K. P.,
Scholott, B., Guhrs, K. H. Bode, W (1998). The ternary
microplasmin-staphylokinase-microplasmin complex is a
protease-cofactor-substrate complex in action. Nat. Struct. Biol.
10; 917-923].
[0016] The present invention, therefore, unravels a novel strategy
for engineering a SAK molecule to improve its thrombolytic
properties by enhancing its plasma half life and stability and a
low immune reactivity. The said properties are achieved although
their clot dissolving ability is maintained similar to that of the
wild type molecule. Here, design and development of new SAK
derivatives has been discussed where amino and/or carboxy terminal
regions of SAK have been extended by introducing new amino acid
sequences, particularly one or more cysteine residues, to create
dimeric/multimeric forms of SAK, and their modification by
attaching a PEG molecule of different sizes within the extended
region so that the integrated PEG with SAK remains away from the
core functional region and does not interfere with biological
function of SAK but simultaneously can increase overall stability
and shelf life of the protein. Moreover, the Staphylokinase
derivatives, thus generated, can have extended in vivo plasma
half-life, thus, creating new SAK mutants that can be more
advantageous in thrombolytic therapy. In principal, the present
invention, disclosed herein, relates to new derivatives of
Staphylokinase displaying higher thermal stability and increased
in-vivo half-life than the unmodified Staphylokinase.
[0017] Therefore, details disclosed in the present invention,
provide new strategy and design for the modification of SAK for
engineering and chemical modification of SAK for enhancing its
thrombolytic potential. SAK derivatives, thus generated, having
multimeric forms and/or conjugated with PEG, disclosed in the
present invention, display significant improvement in their
functional properties over an unmodified SAK form and other known
derivatives with respect to stability and circulating half-life and
can be more useful for clinical purposes for the treatment of
cardiovascular complications providing the advantage of higher
temperature stability that can increase the shelf life of the
protein and extended half life that can reduce the requirement of
repeated dose during thrombolytic therapy.
[0018] The prime objective of the present invention is to develop a
cysteine variant of Staphylokinase wherein at least one cysteine
residue is added at amino and carboxy-terminal extension of SEQ ID
NO: 1.
[0019] Another object of the present invention is to develop
biologically functional derivatives of SAK that can display higher
thermal and protease stability so that the shelf life of protein is
increased and its plasma half-life in vivo is extended so that it
can be more beneficial for the thrombolytic therapy. Integration of
these two attributes in a SAK molecule can tremendously increase
therapeutic potential of Staphylokinase for the treatment of
circulatory disorders.
[0020] The other objective of the present invention is to extend
amino and/or carboxy terminal regions of SAK carrying one or more
cysteine residues to alter subunit association properties of SAK
and to conjugate different lengths of PEG molecule away from the
main functional regions of protein so that biological activity of
protein is not compromised after PEG attachment but simultaneously
can provide protection to the molecule from the protease attack so
that its circulating half life in vivo is extended.
[0021] Yet another objective of the invention is to prepare a piece
of DNA carrying complete genetic information for the production of
SAK derivatives in a suitable host such as E. coli, Bacillus, Yeast
or any microbial system using known recombinant DNA techniques for
high level intracellular production of various SAK derivatives so
that large amount of SAK mutant proteins can be obtained in high
yield.
[0022] Yet another objective of the invention is to prepare SAK
derivatives in purified form using known protein purification
techniques and then conjugate one or more PEG molecule within the
extended region of SAK to prepare mono or di PEGylated forms of
SAK.
[0023] Overall objective of the present invention, therefore, is to
develop new derivatives of SAK that can display better stability,
enhanced shelf life and extended plasma half-life in vivo. These
attributes in the staphylokinase derivatives, disclosed herein,
will significantly improve thrombolytic potential of staphylokinase
for the treatment of cardiovascular disorders.
BRIEF SUMMARY
[0024] Accordingly, the present invention provides a cysteine
variant of Staphylokinase (a Staphylokinase variant) wherein at
least one cysteine residue is added at amino and carboxy-terminal
extension of SEQ ID NO:1. In one embodiment, the at least one
cysteine residue is added to the N-terminal region of SEQ ID NO:1
by substituting at least one amino acid residue in the N-terminal
region of SEQ ID NO:1 with the at least one cysteine residue. In
another embodiment, the at least one cysteine residue is added to
the N-terminal region of SEQ ID NO:1 by adding the at least one
cysteine residue to the N-terminal amino acid of SEQ ID NO:1. In
another embodiment, the at least one cysteine residue is added to
the C-terminal region of SEQ ID NO:1 by adding the at least one
cysteine residue to the C-terminal amino acid of SEQ ID NO:1.
[0025] In an embodiment of the present invention, a cysteine
variant of Staphylokinase is provided, wherein the cysteine variant
of Staphylokinase comprises a sequence selected from the group
consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID
NO:6. In another embodiment, the cysteine variant of Staphylokinase
consists of a sequence selected from the group consisting of SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6
[0026] In an embodiment of the present invention, a cysteine
variant of Staphylokinase wherein one or more cysteine residues are
added to the regions comprising of before N-terminal amino-acid of
Staphylokinase polypeptide, after the C-terminal amino-acid of
Staphylokinase polypeptide. In one embodiment, one or more cysteine
residues are added to the N-terminal residue of SEQ ID NO:1. In
another embodiment, the one or more cysteine residues is added to
the C-terminal residue of SEQ ID NO:1.
[0027] In another embodiment of the present invention, a cysteine
variant of Staphylokinase, wherein cysteine variant has N and/or
C-terminus extension of amino-acids.
[0028] In another embodiment of the present invention, a cysteine
variant of Staphylokinase wherein the substituted cysteine residue
is modified with a cysteine-reactive moiety.
[0029] In yet another embodiment of the present invention, a
cysteine variant of staphylokinase is provided, wherein at least
one or more substituted or added cysteine residue is modified with
or conjugated to a polyethylene glycol (PEG) molecule.
[0030] In yet another embodiment of the present invention, the PEG
molecule attached to the PEGylated cysteine variant is a linear or
branch polymer of molecular size ranging from 5-20 Kilodaltons.
[0031] In still another embodiment of the present invention, the
PEGylated cysteine variant has increased proteolytic stability as
compared to their original unmodified counterparts. In yet another
embodiment, the Staphylokinase variant has proteolytic stability
greater than the proteolytic stability of SEQ ID NO:1.
[0032] In still another embodiment of the present invention, the
PEGylated cysteine variant, wherein said variant has decreased
antigenicity and in vivo immunogenicity when compared to their
original unmodified counterparts. In yet another embodiment, the
Staphylokinase variant has in vivo immunogenicity which is less
than the in vivo immunogenicity of SEQ ID NO:1.
[0033] In still another embodiment of the present invention, the
PEGylated cysteine variant, wherein said variant has slow renal
clearance hence increased in vivo half life as compared to their
original unmodified counterparts. In yet another embodiment, the
Staphylokinase variant has an in vivo half life which is greater
than the in vivo half life of SEQ ID NO:1.
[0034] In still another embodiment of the present invention, a
cysteine variant of staphylokinase wherein SAK derivatives and
their PEG conjugated forms display higher temperature stability
ranging from 20.degree. C. to 80.degree. C. than their unmodified
form and wild type SAK.
[0035] In still another embodiment of the present invention, a
cysteine variant of staphylokinase wherein the extended
carboxy-terminal end of staphylokinase carries a single cysteine
residue and the plasmid DNA encoding this SAK variant (SAK 1C-CT),
has been transformed into an E. coli host to express this SAK
derivative.
[0036] In still another embodiment of the present invention, the
recombinant E. coli strain has been deposited in International
Depository (IDA) section of Microbial type culture collection under
designation of MTCC 5437.
[0037] In still another embodiment of the present invention, a
cysteine variant of staphylokinase wherein the carboxy terminal
region of staphylokinase derivative carry two cysteine residues and
a plasmid DNA encoding this SAK variant (SAK 2C-CT) has been
transformed into an E. coli host to express this SAK
derivative.
[0038] In still another embodiment of the present invention, the
recombinant E. coli strain has been deposited in International
Depository (IDA) section of Microbial type culture collection under
designation of MTCC 5438.
[0039] In still another embodiment of the present invention, a
cysteine variant of staphylokinase wherein the extended
amino-terminal end of staphylokinase carries a single cysteine
residue and the plasmid DNA encoding this SAK variant (SAK 1C-NT)
has been transformed into an E. coli host to express this SAK
derivative.
[0040] In still another embodiment of the present invention, the
recombinant E. coli strain has been deposited in International
Depository (IDA) section of Microbial type culture collection under
designation of MTCC 5439.
[0041] In further embodiment of the present invention, a cysteine
variant of staphylokinase wherein the extended amino-terminal
region of staphylokinase derivative carries two cysteine residues
and a plasmid DNA encoding this SAK variant (SAK 2C-NT) has been
transformed into an E. coli host to express this SAK
derivative.
[0042] In further embodiment of the present invention, the
recombinant E. coli strain has been deposited in International
Depository (IDA) section of Microbial type culture collection under
designation of MTCC 5440.
[0043] In one embodiment of the present invention, a vector
comprising a DNA molecule encoding SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5 or SEQ ID NO:6 is provided.
[0044] In one embodiment, a recombinant E. coil host cell having a
vector comprising a DNA molecule encoding SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5 or SEQ ID NO:6 is provided.
[0045] In yet further embodiment of the present invention, a
pharmaceutical composition comprising at least one of the pegylated
derivatives together with, or without, excipient(s).
[0046] In yet further embodiment of the present invention, a
pharmaceutical composition, for treating circulatory disorder
selected from the group consisting of myocardial infarction,
vascular thromboses, pulmonary embolism, stroke a vascular event,
disease or disorder selected from a group consisting of myocardial
infarction, angina, pulmonary embolism, transient ischemic attack,
deep vein thrombosis, thrombotic re-occlusion subsequent to a
coronary intervention procedure, heart surgery or vascular surgery,
peripheral vascular thrombosis, Syndrome X, heart failure, and a
disorder in which a narrowing of at least one coronary artery
occurs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1. Restriction map of recombinant plasmid pRM1 showing
site of integration of a SAK encoding gene.
[0048] FIG. 2. Pegylation of SAK mutants carrying amino and carboxy
terminal extension.
[0049] FIG. 3. Stability of various SAK derivatives at 65.degree.
C.
[0050] FIG. 4. Trypsin digestion profile of SAK and PEGylated
derivatives of SAK.
[0051] FIG. 5. Half life of wild type and PEG conjugated SAK
mutants.
DETAILED DESCRIPTION
[0052] The various aspects and embodiments will now be fully
described herein. These aspects and embodiments may, however, be
embodied in many different forms and should not be construed as
limiting; rather, these embodiments are provided so the disclosure
will be thorough and complete, and will fully convey the scope of
the present subject matter to those skilled in the art.
[0053] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0054] The present invention provides a cysteine variant of
Staphylokinase wherein at least one cysteine residue is added at
amino and carboxy-terminal extension of SEQ ID NO: 1.
[0055] The invention also pertains to new derivatives of
stapylokinase carrying extended amino and carboxy terminal ends
where one or more cysteine residues were introduced to modify
oligomeric state of protein, resulting in staphylokinase mutants
displaying enhanced thermal stability. The variants, carrying
cysteine residue(s) at one or both ends of polypeptide, were
further modified by conjugation of PEG molecule, ranging from 5 to
20 kDa, to enhance proteolytic resistance and plasma half life of
Staphylokinase.
[0056] The present invention also relates to peptide sequence of
Staphylokinase having SEQ ID NO:1, its corresponding gene with
nucleotide sequence given in SEQ ID NO:2, or mutants or derivatives
thereof developed using known procedures, their expression in a
heterologous host, e.g., E. coil, Bacillus, Yeast, their production
and purification in large amount using general methods of
recombinant DNA and protein purification, covalent modification of
mutant polypeptides by conjugating PEG molecule of different sizes.
SAK derivatives, thus created, display enhanced thermal and
protease stability and extended in vivo retention.
[0057] The present invention also pertains to covalent modification
of SAK with PEG attachment to mask one of the antigenic sites
without affecting biological activity so that resulting molecule
can have reduced antigenicity.
[0058] The present invention also pertains to the pharmaceutical
use of Staphylokinase derivatives and their PEG conjugated forms
along with suitable composition of carriers and stabilizers for
clinical administration in the body for the treatment of
cardiovascular diseases. The invention also relates to modified
polypeptides carrying amino acid sequence of SEQ ID NOs. 3, 4, 5
and 6 and their encoding gene sequences for modified Staphylokinase
derivatives, as well as their respective expression plasmids
ligated with these mutant SAK-encoding nucleic acid sequences. E.
coli host cells transformed with the plasmids encoding the
Staphylokinase derivative proteins were deposited with the
International Deposition Authority (IDA) section of Microbial type
culture collection and were assigned deposit numbers MTCC 5437 (SAK
1C-CT, SEQ ID NO:5), MTCC 5438 (SAK 2C-CT, SEQ ID NO:6), MTCC 5439
(SAK 1C-NT, SEQ ID NO:3), and MTCC 5440 ((SAK 2C-NT, SEQ ID NO:4),
and are capable of producing large amounts of mutant SAK proteins
in the intracellular compartment of the recombinant E. coli cells.
This invention further relates to a process for the purification of
Staphylokinase derivatives and their further modification by
covalent linkage of PEG outside the functional region of protein,
particularly within the extended amino or carboxy terminal regions
of SAK, and lastly, a method of dissolving blood clot in a subject
in need thereof.
[0059] The invention discloses the integration of one or more amino
acid residues, carrying one or multiple cysteines at amino and/or
carboxy terminal regions of SAK using known genetic engineering
techniques, creating SAK derivatives that are able to attain
dimeric and multimeric forms and display higher stability than the
unmodified form.
[0060] In a preferred embodiment, single and/or double cysteine
residues are added at amino and/or carboxy-terminal region of SAK
resulting in SAK derivatives showing higher protein stability than
the unmodified form.
[0061] In another embodiment, SAK derivatives having one or more
cysteine residue(s) within the extended region of SAK are produced
into a suitable host such as E. coli, Bacillus, Yeast etc. using
any recombinant DNA techniques known in the prior art and purified
from the cell extract through various known protein purification
techniques such as Ion Exchange Chromatography, Hydrophobic
Interaction Chromatography, Gel filtration techniques etc.
[0062] In another preferred embodiment, purified protein
preparation of SAK derivatives or their mutants are chemically
modified to conjugate PEG molecule of 5, 10 or 20 kDa using known
procedures.
[0063] The following examples are given by way of illustration of
the present invention and therefore should not be construed to
limit the scope of the present invention
GENERAL METHODS UTILIZED IN THE EXAMPLE
[0064] Recombinant DNA techniques: Conventional and well-known
techniques of recombinant DNA and molecular biology were utilized.
Details of these techniques are available in various standard text
books or manuals related to this field for example, Sambrook et.
al., Molecular Cloning: A Laboratory Manual (2.sup.nd edition, Cold
spring Harbor Press, N.Y., 1989. The source of SAK gene for the
purpose of modification is plasmid, pRM1 (as detailed in EP Patent
No. 1608677 "A method for oxygen regulated production of
recombinant Staphylokinase"). The SAK gene was engineered to extend
amino and/or carboxy terminal ends following polymerase chain
reaction (PCR) following standard protocols of genetic engineering
techniques [Sambrook J., Fritsch E. F., Maniatis T. (1989)
Molecular cloning a laboratory manual. 2nd Ed. Cold Spring Harbor,
N.Y. Cold Spring Harbor Laboratory Press] and mutant SAK genes were
cloned on a T7 RNA polymerase based expression vector, pET9b and
was transformed in the Escherichia coli BL21 DE3 strain obtained
from New England Biolabs.
[0065] Electrophoretic analysis of proteins: Purified preparation
of SAK or its presence in vivo was analysed through SDS-PAGE,
essentially according to Laemmli procedure.
Casein-Plasminogen overlay assay for detection of SAK activity:
Bacterial colonies producing Staphylokinase were detected by
overlay of casein and human plasminogen in soft agar as mentioned
by Behnke et al., 1984. Briefly, 10 ml of soft agarose mixture
carrying 0.8% agarose, 10% skim milk, 100 mU of human plasminogen,
150 mM NaCl and 50 mM Tris-CI (pH.8.0) is poured on to top of the
plates carrying bacterial colonies expressing staphylokinase. These
plates were incubated at 37.degree. C. for 4-5 h and SAK carrying
colonies were identified by the presence of a clearing zone around
the colonies. Biological activity of SAK protein can also be tested
on plate overlayered with casein and human plasminogen after
putting a spot of purified protein on the plate.
[0066] Assay for Staphylokinase mediated plasminogen activation
using chromogenic peptide substrate. Plasminogen activation ability
of Staphylokinase and its modified forms were checked through known
procedures (Jackson, K. W., Esmon, N., Tang, J. (1981)
Streptokinase and Staphylokinase. Methods enzymol. 80; 387-394).
Briefly, 10 .mu.l of appropriately diluted sample of SAK was mixed
with 25 .mu.l of sample buffer (50 mM Tris.Cl, pH 7.5) and 15 .mu.l
of human plasminogen (0.5 mg/ml) and incubated at 37.degree. C. for
15 minutes and then 18 .mu.l of NaCl (1.77 M in 50 mM Tris. Cl, pH
7.5) is added. The amount of plasmin thus generated was measured
after addition of 12 .mu.l of chromogenic substrate, Chromozyme PL
(5 mg/ml in water, Boehringer Mannheim), and tubes were further
incubated at 37.degree. C. for 10 minutes. SAK activity was
measured at 405 nm due to the release of yellow colored
p-4-nitroaniline.
[0067] Clot lysis assay: Clot lysis ability of SAK or its modified
forms was tested following the standard methods (British
Pharmacopia, 1980 edition). Fibrin clot lysis was carried out in
the presence of citrated human plasma or human fibrinogen
containing different concentrations of SAK or its analogs. Briefly,
fibrin clot was labeled by radioiodination (I-125) and mixed with
appropriate concentrations of SAK and incubated at 37.degree. C.
and rotated slowly. 0.1 ml aliquot was removed at regular intervals
and release of soluble fibrin was measured by the amount of
radioactivity released using gamma counter.
[0068] Temperature stability. Stability of SAK derivatives or their
PEG conjugated forms was determined by incubating the purified
preparation of each derivative at 4.degree. C., 37.degree. C.,
42.degree. C. and 65.degree. C. for different time periods and then
testing their ability for the plasminogen activation using known
standard technique mentioned above.
[0069] Proteolytic stability. Each SAK derivative or their PEG
conjugated form was incubated with trypsin in the ratio of 1:100
and 1:200 at 37.degree. C. and its degradation was analyzed on
SDS-PAGE and also after checking their biological activity by
casein-plasminogen assay after spotting on LB-Agar plate.
[0070] Determination of plasma half life of SAK derivatives: SAK
derivatives and their PEG conjugated forms were labeled with
I.sup.125 (Perkin Elmer) using lodogen (1,3, 4, 6-Tetrachloro-3a-6
alpha-diphenylgiucoluril) method following standard procedure
[Fraker, P. J., Speck, J. C. (1978) Protein and cell membrane
iodination with a sparingly soluble choramide 1, 3, 4,
6-tetrachloro-3a,6a- diphenylgylycoril. Biochem. Biophys. res.
commun. 80; 849-857]. Iodinated SAK derivatives were injected into
blood plasma as well as in live mice and samples were withdrawn at
different time intervals to check the stability of SAK derivatives
in blood plasma.
[0071] Immunogenicity of PEGylated cysteine derivatives of SAK:
Immunological properties of various SAK cysteine derivatives was
checked using SAK specific polyclonal antisera raised against WT
SAK. Immunoreactivity of various PEGylated SAK derivatives and WT
SAK was checked against this antibody by ELISA plate method. The
microtitre plate was incubated at 4.degree. C. overnight with SAK
or PEGylated cysteine mutants containing 1 .mu.g of protein in
coating buffer i.e. 0.2 M bicarbonate buffer pH 9.2. The plate was
washed with 200 .mu.l PBS-T three times. 100 .mu.l of diluted
(1:40000 in PBS) primary antibody against SAK was added to each
well and incubated at 37.degree. C. for 4 hrs. After three
washings, 100 .mu.l of HRP conjugated antibody was added in 1:5000
dilution and kept for 1 hr at 37.degree. C. The plate was washed
three times with PBS. 100 .mu.l of TMB substrate was added to each
well and incubated for 20-30 min at 37.degree. C. Finally 50 .mu.l
of H.sub.2SO.sub.4 was added to stop the reaction and the plate was
read at 450 nm. The immmunoreactivity of SAK and different
PEGylated derivatives was evaluated by comparing the absorbance
values at 405 nm.
EXAMPLES
[0072] The following examples illustrate certain aspects and
advantages of the present invention, however, the present invention
is in no way considered to be limited to the particular embodiments
described below.
Example 1
[0073] Construction of SAK mutants carrying extended amino and
carboxy terminal regions: SAK is the smallest known protein that
has an efficient plasminogen activator activity. Major portion of
SAK polypeptide is important for either forming a bimolecular
complex with plasminogen or interaction with a substrate molecule.
Therefore, addition or a deletion within the core region of SAK
often results [Schlott, B., Gurhs, H. K., Hartmann, M., Rocker, A.,
Collen, D. (1997) Staphylokinase requires NH2-terminal proteolysis
for plasminogen activation. J. Biol. Chem. 273; 22346-22350;
Rajamohan, G. and Dikshit, K. L.(2000) Role of the N-terminal
region of Staphylokinase (SAK) evidence for the participation of
the N-terminal region of SAK in the enzyme-substrate complex
formation. FEBS Lett. 474; 151-158] in a non-functional form of
protein. Therefore, to generate derivatives of SAK, extension of
one or more amino acid residues carrying one or multiple cysteine
residues at terminal positions, was done using recombinant DNA
techniques so that this region can be targeted for engineering new
attributes to the protein. Amino or carboxy-terminal extension of
SAK was done by polymerase chain reaction using oligonucleotide
primers and extension of SAK was done at both the ends either
individually or in combination. Table 1 shows the amino acid
sequences of first or last seven residues of SAK derivatives. PCR
amplified product of each SAK derivative was cloned on an
expression vector pET 9b. Primary recombinant plasmid, pRM1 has
been described earlier (EP Patent No.1608677 "A method for oxygen
regulated production of recombinant Staphylokinase"; US patent
pending). Map of this recombinant plasmid is given in FIG. 1. The
nucleotide sequence of each SAK derivative was confirmed through
automated DNA sequencing (Applied Biosystems). Details of amino
acid and nucleotide sequence of wild-type SAK are given in SEQ ID
NO:1 and SEQ ID NO:2, respectively.
[0074] Amino acid sequence of SAK derivatives is shown in the
sequence listing.
TABLE-US-00001 TABLE 1 Staphylokinase Derivatives Carrying Extended
Amino and/or Carboxy Terminals Sequence of First or Last 7 Amino
SAK Derivatives Extended Region Acid Residues SAK WT None SSSFDKG
(SEQ ID NO: 1) (N-terminus) KVVIEKK (C-terminus) S3C None SSCFDKG
(SEQ ID NO: 7) (cysteine residue substituted with serine at
3.sup.rd position of amino terminal region) SAK 2CNT Two cysteine
residues added CCSSSFD (SEQ ID NO: 4) at the N-terminal region of
SAK SAK 1CNT One cysteine residue added CSSSFDK (SEQ ID NO: 3) at
the N-terminal region of SAK SAK 2CCT Two cysteine residues added
VIEKKCC (SEQ ID NO: 6) at the carboxy-terminal region of SAK SAK
1CCT One cysteine residue added VIEKKAC (SEQ ID NO: 5) at the
carboxy-terminal region of SAK
Example 2
[0075] Intracellular production of SAK derivatives and their
recovery from recombinant E. coli: E. coli cells transformed with
recombinant plasmid carrying mutant SAK gene were streaked on a
Luria Bertani (LB) agar plate containing 50 .mu.g/ml Kanamycin and
kept in an incubator set at 37.degree. C. for overnight. Individual
colonies appearing on the plate were used to raise seed culture in
a 10 ml liquid LB medium supplemented with 50 .mu.g/ml Kanamycin
and grown at 37.degree. C., 200 rpm on a gyratory shaker for 8-10
h. This primary seed culture (1% v/v) was used to inoculate 1 liter
of LB medium containing 50 .mu.g/ml Kanamycin and allowed to grow
at 37.degree. C. at 200 rpm till its optical density (OD.sub.600
nm) reaches to 0.4-0.5. The culture was then induced for SAK
production by adding 0.1 mM IPTG and further grown at 37.degree. C.
for another 6-8 hours. Cells were then harvested by spinning them
down by centrifugation at 6000.times.g in a GS-3 rotor (Sorvall)
for 30 min at 4.degree. C. The supernatants were discarded and the
cell pellet was resuspended in 15 ml of 10 mM Tris.CI buffer and
lysed either by sonication or chemical lysis using 6M-guanidium
hydrochloride and 20 mM sodium phosphate buffer, pH 7.2. The cell
lysate was centrifuged at 6000.times.g at 4.degree. C. for 15 min
and clear lysates were diluted two fold with 10mM Tris.Cl buffer
and thereafter applied at room temperature to a 10 times 32 cm
column of SP-sepharose at a flow rate of 1 liter per hour. The
column was washed with 10 mM Tris.Cl buffer, pH 6.2 and eluted with
a gradient of 0.1 to 0.5 M NaCl. The SAK containing fraction was
checked by spot test by mixing 1 .mu.l fraction with 1 mU of human
plasminogen (0.5 mg/ml) and 1 mU of chromozyme PL. The SAK
containing fractions exhibited development of yellow color. These
fractions were pooled and adjusted to 2.5 M with solid sodium
chloride and subjected to hydrophobic interaction chromatography on
a 10.times.20 cm column of phenyl-sepharose at room temperature and
flow rate of 1 liter/hour. The column was washed with 0.1 M
phosphate buffer and SAK was eluted with 0.01 M phosphate buffer
(pH 6.2). Aliquots from each fraction were analyzed on 15% SDS-PAGE
to examine the relative purity of the eluted protein. On SDS-PAGE,
it showed a single band of 16 kDa. Table 2 shows the specific
activity of SAK and its derivatives by standard procedure of
plasminogen activation assay.
TABLE-US-00002 TABLE 2 Functional Properties of SAK Derivatives SAK
Derivatives Specific Activity (U/mg) SAK WT* 67 U/mg SAK
(Recombinant) 66 .+-. 3.5 U/mg SAK S3C 60 .+-. 4.1 U/mg SAK 1CNT 40
.+-. 4.3 U/mg SAK 2CNT 57 .+-. 3.8 U/mg SAK 1CCT 64 .+-. 4.5 U/mg
SAK 2CCT 65 .+-. 3.8 U/mg *WT SAK with 67 U/mg was obtained from
World Health Organization and used as a standard to compare
specific activity of SAK derivatives.
Example 3
[0076] Determination of sub-unit association properties of SAK
derivatives: In order to check how the extension of amino terminal
regions of SAK and placement of cysteine residues within this
region has affected the oligomeric state of protein, purified
preparation of each SAK derivative was run on a native 10%
polyacrylamide gel without adding any SDS and mercaptoethanol. Gel
was stained with coomassie blue to check change in the oligomeric
state of the protein. Checking the molecular mass of each SAK
derivative following standard method of size exclusion
chromatography on a G75 Sephadex column further substantiated
change in the oligomeric state of protein.
Example 4
[0077] Conjugation of Polyethylene Glycol with SAK derivatives
extended at amino and carboxy terminal regions: Maleimide activated
methoxy Polyethylene Glycol of different sizes 5, 10, 20 kDa was
used to conjugate PEG molecule with SAK derivatives carrying single
or multiple cysteine residues within their extended amino or
carboxyl ends. Initially, different molar ratio of SAK derivative
and PEG (ranging from 1:2 to 1:5) were mixed in a pegylation
reaction buffer (comprising of 50 mM Tris.Cl (pH 8.0), 100 mM NaCl)
and kept at room temperature for different time periods to find out
the ideal condition for PEG linkage with individual SAK derivative.
Particularly in the case of SAK derivative carrying more than one
cysteine, condition for the PEG linkage has been optimized by
standardizing the ratio of protein and PEG as well as time of
incubation so that all cysteine residues can be pegylated
maximally. Passing through the Amicon column having 15 kDa cut off
then separated unlinked PEG molecule from the protein. Mono and
dipegylated form of protein were separated through size exclusion
chromatography on a Superdex G 75 column through protein
purification system linked with online data recording (Acta Prime,
Amersham Biosciences).
[0078] Integration of PEG molecule with SAK was checked by
analyzing the Pegylated forms of SAK derivatives on 10% SDS-PAGE.
SAK derivative carrying cysteine residue at one end displayed only
monopegylated form along with some unpegylated form as shown in
FIG. 2. efficiency of SAK mutants to form mono and dipegylated
forms were different in each case (FIG. 2).
Example 5
[0079] Temperature stability of SAK derivatives and their PEG
conjugated forms: PEG conjugated forms of different SAK derivatives
were purified after Gel-filtration column using standard
biochemical procedure and concentrated. 200 .mu.l of purified
protein preparation of SAK derivatives and their PEG linked forms
were kept at 4.degree. C., 37.degree. C., 42.degree. C. and
65.degree. C. at concentration 1mg/ml in 50 mM tris buffer (pH 7.5)
and aliquot of 10 .mu.l was removed at different time intervals and
their residual functional activity was checked by testing their
plasminogen activator activity. Stability profile of different SAK
variants at 65.degree. C. is shown in FIG. 3.
Example 6
[0080] Protease susceptibility of SAK derivatives and their PEG
conjugated forms: In order to check the response of SAK derivatives
and their PEG linked forms against protease susceptibility, native
SAK and its PEG derivatives and their unmodified forms were tested
against a general protease, i.e., Trypsin. 100 .mu.g of each SAK
derivative and the native SAK was incubated with Trypsin in 100:1
and 200:1 ratio (w/w) in 10 mM Tris.Cl (pH 8.0) and 50 mM NaCl for
1 to 2 hr at 37.degree. C. and an aliquot was taken out at
different intervals of time and analyzed on SDS-PAGE. PEG
conjugated derivatives of SAK displayed significant increase in
resistance against trypsin attack as compared to unmodified as well
as wild type SAK (FIG. 4).
Example 7
[0081] Testing of in vivo plasma half life of SAK derivatives and
their PEG conjugated forms: Since native SAK and SAK derivatives
were expressed in a bacterial host, E. coli, presence of some
endotoxins in the purified protein can be expected. To remove
endotoxins from the protein preparation, each SAK derivatives as
well as wild type SAK protein was passed twice through a Polymyxin
B Agarose column (Sigma Aldrich) and then used for animal studies.
Endotoxin-free preparation of SAK derivatives as well as wild type
SAK was radiolabelled with I.sup.125 following known procedures
[Fraker, P. J., Speck, J. C. (1978) Protein and cell membrane
iodination with a sparingly soluble choramide 1, 3, 4,
6-tetrachloro-3a,6a-diphenylgylycoril. Biochem. Biophys. Res.
Commun. 80; 849-857] and unincorporated free iodine was removed by
dialysis against 50 mM phosphate buffer. The radiolabelled
preparation of SAK derivative and wild type SAK was then used for
studying their pharmacological characteristics by injecting into
mice to test their in vivo stability. Mice (Swiss) were used for
injecting various radiolabelled SAK derivatives. First, mice were
treated with 3% iso-fluorane to anaesthesize and then vasodilation
was induced in the tail to inject nearly 7-8 .mu.g of iodinated
protein via the tail vein. About 30-50 .mu.l of blood sample was
collected immediately after injection and after the different
intervals of time from tail vein or from ear vein. In vivo plasma
half-life of SAK derivative was determined by checking the level of
residual level of radioactivity left in the plasma processed from
the collected sample at different time points. Simultaneously,
withdrawn samples were TCA-precipitated after adding equal volume
of 20% TCA into each sample and incubating on ice for 20 min. The
samples were then centrifuged at 5000 rpm for 10 min and
supernatant was removed to separate free radioactivity and
precipitate was analyzed for the radioactivity associated with the
plasma protein by checking radioactive counts left in the
precipitate (FIG. 5). The results were further confirmed by
autoradiography after running the samples on SDS-PAGE. Two
independent sets of experiments were conducted to conclude the
results.
[0082] Results show that different PEG conjugated SAK derivatives
have different degrees of in vivo plasma half-life as shown in
Table 3. Integration of two cysteines in SAK results in 5-8 fold
increase in plasma half life than SAK derivatives carrying only one
cysteine. Also, the presence of cysteine in the extended amino and
carboxy ends of SAK have different effects on in vivo stability of
SAK. Retention of SAK derivatives in animal model was also tested
by taking blood samples at different time points and checking the
retention of protein by autoradiography which indicated that after
5 minutes of injection, wild type SAK gets cleared from the blood
sample, whereas, PEG conjugated SAK derivatives are retained in the
blood for a longer time. Retention time differed in case of
different mutants as shown in Table 3.
TABLE-US-00003 TABLE 3 In vivo plasma half life of SAK derivatives
injected in mice In vivo SAK Derivatives Plasma Half-Life SAK <5
min SAK S3C-PEG kDa ~20 min SAK 1CNT PEG 20 kDa >60 min SAK 2CNT
PEG 20 kDa >3 h SAK 1CCT PEG 20 kDa >90 min SAK 2CCT PEG 20
kDa >3 h SAK 1CCT PEG 5 kDa >10 min SAK 1CCT PEG 10 kDa
>20 min SAK 2CCT PEG 5 kDa >40 min SAK 2CCT PEG 10 kDa >90
min
Example 8
[0083] Immunogenicity of PEGylated cysteine derivatives of SAK: To
check the immunogenicity of various SAK cysteine derivatives,
polyclonal antisera raised against SAK in rabbit was used to check
the reactivity with various PEGylated SAK derivatives and WT SAK by
ELISA plate method. The microtitre plate was incubated at 4.degree.
C. overnight with 100 .mu.l/well of SAK and PEGylated cysteine
mutants containing 1 .mu.g of protein in coating buffer i.e. 0.2 M
bicarbonate buffer pH 9.2. The coated plates were washed with 200
.mu.l PBS-T (PBS, pH 7.5, containing 0.05% v/v Tween 20) three
times for10 min each. The unoccupied sites were blocked with 5%
skim milk in PBS-T and kept for 2 hrs at 37.degree. C. The plate
was washed with 200 .mu.l PBS-T three times. 100 .mu.l of diluted
(1:40000 in PBS) primary antibody against SAK was added to each
well and incubated at 37.degree. C. for 4 hrs. After three
washings, 100 .mu.l of HRP conjugated antibody was added in 1:5000
dilutions and kept for 1 hr at 37.degree. C. The plate was washed
three times with PBS. 100 .mu.l of TMB substrate was added to each
well and incubated for 20-30 min at 37.degree. C. Finally 50 .mu.l
of H2SO4 was added to stop the reaction and the plate was read at
450 nm. The immmunoreactivity of SAK and different PEGylated
derivatives was evaluated by comparing the absorbance values. The
SAK cysteine derivatives showed varied degrees of immunoreactivity
against polyclonal sera of SAK. The percentage reactivity of all
SAK derivatives has been shown in Table 4.
TABLE-US-00004 TABLE 4 Immunogenicity of PEGylated cysteine
derivatives of SAK Percentage SAK Derivatives Immunoreactivity PEG
1CCT 5 kDa 70% PEG 2CCT 5 kDa 65-70% PEG 1CCT 10 kDa 60% PEG 2CCT
10 kDa 50% PEG 1CCT 20 kDa 35-40% PEG 2CCT 20 kDa 30-35%
[0084] Based on the above disclosure, it is evident that the
present invention offers flexibility of designing new amino acid
sequences for a single and a multiple PEG conjugation which can
improve the functional properties of staphylokinase without
interference in its native sequence. This is because the mutations
are not done in the native peptide but rather in the introduced N
and C terminal extensions which therefore does not affect the
inherent properties of the SAK protein.
[0085] The PEG conjugated derivatives of Staphylokinase mentioned
in the present invention display the following advantages: they
have increased plasma half life, increased temperature stability,
low immune reactivity and increased clinical potential of SAK for
the treatment of thrombolytic complications.
Sequence CWU 1
1
71136PRTStaphylococcus aureus 1Ser Ser Ser Phe Asp Lys Gly Lys Tyr
Lys Lys Gly Asp Asp Ala Ser1 5 10 15Tyr Phe Glu Pro Thr Gly Pro Tyr
Leu Met Val Asn Val Thr Gly Val 20 25 30Asp Gly Lys Gly Asn Glu Leu
Leu Ser Pro His Tyr Val Glu Phe Pro 35 40 45Ile Lys Pro Gly Thr Thr
Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val 50 55 60Glu Trp Ala Leu Asp
Ala Thr Ala Tyr Lys Glu Phe Arg Val Val Glu65 70 75 80Leu Asp Pro
Ser Ala Lys Ile Glu Val Thr Tyr Tyr Asp Lys Asn Lys 85 90 95Lys Lys
Glu Glu Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly Phe Val 100 105
110Val Pro Asp Leu Ser Glu His Ile Lys Asn Pro Gly Phe Asn Leu Ile
115 120 125Thr Lys Val Val Ile Glu Lys Lys 130
1352411DNAStaphylococcus aureus 2tcaagttcat tcgacaaagg aaaatataaa
aagggcgatg acgcgagtta ttttgaacca 60acaggcccgt atttgatggt aaatgtgact
ggagttgatg gtaaaggaaa tgaattgcta 120tcccctcatt atgtcgagtt
tcctattaaa cctgggacta cacttacaaa agaaaaaatt 180gaatactatg
tcgaatgggc attagatgcg acagcatata aagagtttag agtagttgaa
240ttagatccaa gcgcaaagat cgaagtcact tattatgata agaataagaa
aaaagaagaa 300acgaagtctt tccctataac agaaaaaggt tttgttgtcc
cagatttatc agagcatatt 360aaaaaccctg gattcaactt aattacaaag
gttgttatag aaaagaaata a 4113138PRTStaphylococcus aureus 3Met Cys
Ser Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp1 5 10 15Ala
Ser Tyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr 20 25
30Gly Val Asp Gly Lys Gly Asn Glu Leu Leu Ser Pro His Tyr Val Glu
35 40 45Phe Pro Ile Lys Pro Gly Thr Thr Leu Thr Lys Glu Lys Ile Glu
Tyr 50 55 60Tyr Val Glu Trp Ala Leu Asp Ala Thr Ala Tyr Lys Glu Phe
Arg Val65 70 75 80Val Glu Leu Asp Pro Ser Ala Lys Ile Glu Val Thr
Tyr Tyr Asp Lys 85 90 95Asn Lys Lys Lys Glu Glu Thr Lys Ser Phe Pro
Ile Thr Glu Lys Gly 100 105 110Phe Val Val Pro Asp Leu Ser Glu His
Ile Lys Asn Pro Gly Phe Asn 115 120 125Leu Ile Thr Lys Val Val Ile
Glu Lys Lys 130 1354139PRTStaphylococcus aureus 4Met Cys Cys Ser
Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp1 5 10 15Asp Ala Ser
Tyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val 20 25 30Thr Gly
Val Asp Gly Lys Gly Asn Glu Leu Leu Ser Pro His Tyr Val 35 40 45Glu
Phe Pro Ile Lys Pro Gly Thr Thr Leu Thr Lys Glu Lys Ile Glu 50 55
60Tyr Tyr Val Glu Trp Ala Leu Asp Ala Thr Ala Tyr Lys Glu Phe Arg65
70 75 80Val Val Glu Leu Asp Pro Ser Ala Lys Ile Glu Val Thr Tyr Tyr
Asp 85 90 95Lys Asn Lys Lys Lys Glu Glu Thr Lys Ser Phe Pro Ile Thr
Glu Lys 100 105 110Gly Phe Val Val Pro Asp Leu Ser Glu His Ile Lys
Asn Pro Gly Phe 115 120 125Asn Leu Ile Thr Lys Val Val Ile Glu Lys
Lys 130 1355138PRTStaphylococcus aureus 5Ser Ser Ser Phe Asp Lys
Gly Lys Tyr Lys Lys Gly Asp Asp Ala Ser1 5 10 15Tyr Phe Glu Pro Thr
Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val 20 25 30Asp Gly Lys Gly
Asn Glu Leu Leu Ser Pro His Tyr Val Glu Phe Pro 35 40 45Ile Lys Pro
Gly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val 50 55 60Glu Trp
Ala Leu Asp Ala Thr Ala Tyr Lys Glu Phe Arg Val Val Glu65 70 75
80Leu Asp Pro Ser Ala Lys Ile Glu Val Thr Tyr Tyr Asp Lys Asn Lys
85 90 95Lys Lys Glu Glu Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly Phe
Val 100 105 110Val Pro Asp Leu Ser Glu His Ile Lys Asn Pro Gly Phe
Asn Leu Ile 115 120 125Thr Lys Val Val Ile Glu Lys Lys Ala Cys 130
1356138PRTStaphylococcus aureus 6Ser Ser Ser Phe Asp Lys Gly Lys
Tyr Lys Lys Gly Asp Asp Ala Ser1 5 10 15Tyr Phe Glu Pro Thr Gly Pro
Tyr Leu Met Val Asn Val Thr Gly Val 20 25 30Asp Gly Lys Gly Asn Glu
Leu Leu Ser Pro His Tyr Val Glu Phe Pro 35 40 45Ile Lys Pro Gly Thr
Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val 50 55 60Glu Trp Ala Leu
Asp Ala Thr Ala Tyr Lys Glu Phe Arg Val Val Glu65 70 75 80Leu Asp
Pro Ser Ala Lys Ile Glu Val Thr Tyr Tyr Asp Lys Asn Lys 85 90 95Lys
Lys Glu Glu Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly Phe Val 100 105
110Val Pro Asp Leu Ser Glu His Ile Lys Asn Pro Gly Phe Asn Leu Ile
115 120 125Thr Lys Val Val Ile Glu Lys Lys Cys Cys 130
1357136PRTStaphylococcus aureus 7Ser Ser Cys Phe Asp Lys Gly Lys
Tyr Lys Lys Gly Asp Asp Ala Ser1 5 10 15Tyr Phe Glu Pro Thr Gly Pro
Tyr Leu Met Val Asn Val Thr Gly Val 20 25 30Asp Gly Lys Gly Asn Glu
Leu Leu Ser Pro His Tyr Val Glu Phe Pro 35 40 45Ile Lys Pro Gly Thr
Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val 50 55 60Glu Trp Ala Leu
Asp Ala Thr Ala Tyr Lys Glu Phe Arg Val Val Glu65 70 75 80Leu Asp
Pro Ser Ala Lys Ile Glu Val Thr Tyr Tyr Asp Lys Asn Lys 85 90 95Lys
Lys Glu Glu Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly Phe Val 100 105
110Val Pro Asp Leu Ser Glu His Ile Lys Asn Pro Gly Phe Asn Leu Ile
115 120 125Thr Lys Val Val Ile Glu Lys Lys 130 135
* * * * *