U.S. patent application number 12/992879 was filed with the patent office on 2011-03-31 for targeted coagulation factors and method of using the same.
This patent application is currently assigned to BAYER HEALTHCARE LLC. Invention is credited to Richard Feldman, Haiyan Jiang, Ji-Yun Kim, Kirk Mclean, Junliang Pan, Glenn Pierce, James Wu, Xiao-Yan Zhao.
Application Number | 20110077202 12/992879 |
Document ID | / |
Family ID | 41319075 |
Filed Date | 2011-03-31 |
United States Patent
Application |
20110077202 |
Kind Code |
A1 |
Feldman; Richard ; et
al. |
March 31, 2011 |
Targeted Coagulation Factors and Method of Using the Same
Abstract
Targeted coagulation factors comprising a coagulation factor
linked with at least one domain that specifically binds to a
membrane protein on a blood cell is provided. The disclosed
targeted coagulation factors increase the efficiency of coagulation
factors and prolong their duration of action and thus, are an
improvement for the treatment of hematological diseases such as
hemophilia A.
Inventors: |
Feldman; Richard; (El
Cerrito, CA) ; Kim; Ji-Yun; (Berkeley, CA) ;
Jiang; Haiyan; (San Francisco, CA) ; Mclean;
Kirk; (Orinda, CA) ; Pan; Junliang; (Moraga,
CA) ; Pierce; Glenn; (Rancho Santa Fe, CA) ;
Wu; James; (El Cerrito, CA) ; Zhao; Xiao-Yan;
(Union City, CA) |
Assignee: |
BAYER HEALTHCARE LLC
Tarrytown
NY
|
Family ID: |
41319075 |
Appl. No.: |
12/992879 |
Filed: |
May 15, 2009 |
PCT Filed: |
May 15, 2009 |
PCT NO: |
PCT/US2009/044148 |
371 Date: |
November 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61053932 |
May 16, 2008 |
|
|
|
Current U.S.
Class: |
514/13.7 ;
530/381; 530/383 |
Current CPC
Class: |
A61P 7/00 20180101; C07K
16/2839 20130101; A61K 38/37 20130101; C07K 2319/30 20130101; A61K
38/4846 20130101; C07K 14/755 20130101; A61K 47/64 20170801; A61K
47/6849 20170801; C07K 2317/622 20130101; A61P 7/04 20180101; A61K
38/00 20130101; C07K 16/2848 20130101; A61K 47/6811 20170801; C07K
2319/33 20130101 |
Class at
Publication: |
514/13.7 ;
530/381; 530/383 |
International
Class: |
A61K 38/36 20060101
A61K038/36; C07K 14/745 20060101 C07K014/745; C07K 14/755 20060101
C07K014/755; A61P 7/00 20060101 A61P007/00 |
Claims
1. A targeted coagulation factor comprising a coagulation factor
linked with at least one domain that specifically binds to a
membrane protein on a blood cell.
2. The targeted coagulation factor of claim 1, wherein the
coagulation factor is a functional FVIII polypeptide or FIX.
3. The targeted coagulation factor of claim 1, wherein the domain
is an antibody fragment, a peptide, a peptide mimetic, or a small
molecule.
4. The targeted coagulation factor of claim 1, wherein the blood
cell is a platelet and the membrane protein is GPIIb/IIIa.
5. The targeted coagulation factor of claim 4, wherein the
coagulation factor is a functional FVIII polypeptide.
6. The targeted coagulation factor of claim 4, wherein the domain
is a RGD peptide or a single chain fragment of an anti-GPIIb/IIIa
antibody.
7. The targeted coagulation factor of claim 1, wherein the
coagulation factor is a functional FVIII polypeptide and the domain
is an antibody fragment, a peptide, a peptide mimetic, or a small
molecule that is linked with the B-domain of the FVIII.
8. The targeted coagulation factor of claim 7, wherein the blood
cell is a platelet and the domain is a RGD peptide or a single
chain fragment of an anti-GPIIb/IIIa antibody.
9. A pharmaceutical composition comprising a therapeutically
effective amount of the targeted coagulation factor of claim 1 and
a pharmaceutically acceptable excipient or carrier.
10. A method for treating hematological diseases comprising
administering an effective amount of the targeted coagulation
factor of claim 1 to a patient in need thereof.
11. A method for targeting a coagulation factor to the surface of a
blood cell comprising linking the coagulation factor with at least
one domain that binds to a membrane protein on a blood cell.
12. The method of claim 11, wherein the coagulation factor is a
functional FVIII polypeptide or FIX.
13. The method of claim 11, wherein the blood cell is a platelet or
a red blood cell.
14. The method of claim 11, wherein the domain is an antibody
fragment, a peptide, a peptide mimetic, or a small molecule.
15. The method of claim 11, wherein the blood cell is a platelet
and the membrane protein is GPIIb/IIIa.
16. The method of claim 15, wherein the coagulation factor is a
functional FVIII polypeptide.
17. The method of claim 15, wherein the domain is a RGD peptide or
a single chain fragment of an anti-GPIIb/IIIa antibody.
18. The method of claim 11, wherein the coagulation factor is FVIII
and the domain is an antibody fragment, a peptide, a peptide
mimetic, or a small molecule that is linked with the B-domain of
the FVIII.
19. The method of claim 18, wherein the blood cell is a platelet
and the domain is a RGD peptide or a single chain fragment of an
anti-GPIIb/IIIa antibody.
20. The method of claim 11, wherein the coagulation factor is
further released from the surface of the blood cell.
Description
[0001] This application claims benefit of U.S. Provisional
application Ser. No. 61/053,932; filed on May 16, 2008, the
contents of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The invention relates to targeted coagulation factors having
increased efficacy. The invention further provides methods of
treating patients suffering from a coagulation factor deficiency
disorder by selectively targeting coagulation factors to their
biological sites of action, such as by targeting Factor VIII
(FVIII) to red blood cells and platelets. Pharmaceutical
compositions comprising the targeted coagulation factors according
to the invention are also provided.
BACKGROUND OF THE INVENTION
[0003] The effectiveness of biological drugs is often limited by
their duration of action in patients, particularly when the disease
requires constant modulation by the drug. Consequently, enhancement
of pharmacokinetic properties is often more critical to the success
of a therapeutic agent in the clinic than is optimization of the
drug's potency. One approach to protect drugs from various
mechanism of clearance so to prolong the half-life is to add
targeting domains that promote drug binding to long-lived proteins
in circulation such as matrix proteins, or to the surface of cells,
such as blood cells or endothelial cells. For example, localization
of therapeutic peptides or proteins to blood cell surfaces has been
shown to prolong their circulation half-life by preventing normal
clearance mechanisms (Chen, et al., Blood 105(10):3902-3909, 2005).
A wide variety of molecules may be used as the targeting
domain.
[0004] In another instance, when the Kunitz-type protease inhibitor
(KPI) domain of tick anticoagulant protein was linked with an
anionic phospholipid, phosphatidyl-L-serine (PS) binding protein,
annexin V (ANV), the fusion protein (ANV-KPI) was shown to be more
active and possess higher in vivo antithrombotic activities than
the non-fusion counterpart (Chen, et al., 2005). Because ANV has
strong affinities for PS and phosphatidylethanolamine (PE), it is
hypothesized that the fusion protein ANV-KPI can be specifically
targeted to the PS/PE-rich anionic membrane-associated coagulation
enzyme complexes present at sites of thrombogenesis. Similarly,
Dong, et al., reported fusing the fibrin-selective Desmodus
rotundus salivary PA .alpha.1 (dsPA .alpha.1) to a urokinase
(uPA)/anti-P-selectin antibody (HuSZ51) to produce a fusion protein
that is fully functional with similar antithrombotic activities as
the non-fusion counterpart in in vitro assays. Furthermore, the
fusion protein HuSZ51-dsPA .alpha.1 was shown to bind to
thrombin-activated human and dog platelets (Dong, et al., Thromb.
Haemost. 92:956-965, 2004).
[0005] Other efforts have been made in targeting anticoagulants to
prevent clots and to reduce mortality associated with thrombotic
diseases (see, e.g., WO 94/09034). A more recent development is
demonstrated by Stoll, et al., (Arterioscler. Thromb. Vasc. Biol.
27:1206-1212, 2007), in which a Factor Xa (FXa) inhibitor, tick
anticoagulant peptide (TAP), was targeted to ligand-induced binding
sites (LIBS) on GPIIb/IIIa, a glycoprotein abundantly expressed on
the platelet surface, via an anti-LIBS single-chain antibody
(scFv.sub.anti-LIBS). The fusion protein scFv.sub.anti-LIBS-TAP was
shown to possess an effective anticoagulation activity even at low
doses at which the non-targeted counterpart failed.
[0006] The aforementioned targeted anticoagulants were fusion
proteins designed to target specific cells. According to Stoll, et
al., the targeted anticoagulant should be a small molecule with a
highly potent coagulation inhibition activity that is retained
while fused to an antibody. The release of the anticoagulant from
the fusion proteins in its targeted sites was not discussed.
[0007] The present invention focuses on targeting therapeutic
proteins for the treatment of hematological diseases such as
hemophilia. For example, current treatment of hemophilia A patients
with FVIII concentrates or recombinant FVIII is limited by the high
cost of these factors and their relatively short duration of
action. Hemophilia A patients are currently treated by intravenous
administration of FVIII on demand or as a prophylactic therapy
administered several times a week. For prophylactic treatment,
FVIII is administered three times a week. Unfortunately, this
frequency is cost prohibitive for many patients. Because of its
short half-life in man, FVIII must be administered frequently.
Despite its large size of greater than 300 kD for the full-length
protein, FVIII has a half-life in humans of only about 11-18
(average 14) hours (Gruppo, et al., Haemophila 9:251-260, 2003).
For those who can afford the frequent dosaging recommended, it is
nevertheless very inconvenient to frequently intravenously inject
the protein. It would be more convenient for the patients if a
FVIII product could be developed that had a longer half-life and
therefore required less frequent administration. Furthermore, the
cost of treatment could be reduced if the half-life were increased
because fewer dosages may then be required. It is therefore
desirable to have more efficient forms of FVIII that can lower the
effective dose or have a prolonged duration of action to
significantly improve treatment options for hemophiliacs.
[0008] Also, a sustained plasma concentration of targeted FVIII may
reduce the extent of adverse side effects by reducing the trough to
peak levels of FVIII, thus eliminating the need to introduce
super-physiological levels of protein at early time-points.
Therefore, it is desirable to have forms of FVIII that have
sustained duration and a longer half-life than current marketed
forms.
[0009] An additional disadvantage to the current therapy is that
about 25-30% of patients develop antibodies that inhibit FVIII
activity (Saenko, et al., Haemophilia 8:1-11, 2002). Antibody
development prevents the use of FVIII as a replacement therapy,
forcing this group of patients to seek an even more expensive
treatment with high-dose recombinant Factor VIIa (FVIIa) and immune
tolerance therapy. A less immunogenic FVIII replacement product is
therefore desirable.
[0010] One approach in improving the treatment for hemophiliacs
involves gene therapy. Ectopically targeting FVIII to platelets by
directing FVIII expression in platelets can have therapeutic
effects in the treatment of hemophilia A (Shi, et al., J. Clin.
Invest. 116(7):1974-1982, 2006).
[0011] It is an object of the invention to provide targeted
coagulation factors that have prolonged duration of action, greater
efficacy, fewer side effects, and less immunogenicity compared to
the untargeted protein.
[0012] Another object of the invention is to reduce side effects
associated with therapeutic protein administration by having the
protein targeted to the specific site of desired action and thereby
reducing the exposure of the protein to other potential
biologically active sites that may result in undesired side
effects.
[0013] A further object of the present invention is to obtain
further advantages by designing targeted therapeutic coagulation
factors in which the therapeutic protein is released from the
targeting domain in the immediate vicinity of its site of action in
vivo. A high local concentration of the non-fusion, activated
proteins may be achieved. Thus, the therapeutic efficacy of the
proteins is enhanced.
SUMMARY OF THE INVENTION
[0014] The targeted coagulation factors according to the present
invention comprise a coagulation factor linked with at least one
domain that specifically binds to a membrane protein on a blood
cell. A pharmaceutical composition comprising the newly disclosed
targeted coagulation factors and a method for treating
hematological diseases using the targeted coagulation factors is
also provided. The present invention further provides a method for
targeting a coagulation factor to the surface of a blood cell by
using the newly disclosed targeted coagulation factors to increase
the efficiency of treating hematological disease with coagulation
factors.
DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1: Schematic drawings of full-length FVIII ("Full
Length FVIII) and B-domain deleted FVIII ("FVIII-BDD-TD") in which
a targeting domain ("TD") is inserted into the B-domain and most of
the B-domain is removed.
[0016] FIG. 2: Structures of modified cyclic peptide integrilin,
"BHRF-1" (A) and "BHRF-3" (B), for linking to FVIII through the
B-domain cysteine.
[0017] FIG. 3: Binding affinity of BHRF-1 and BFRH-3 to immobilized
GPIIa/IIIb.
[0018] FIG. 4: BHRF-1-FVIII binding assay to immobilized
GPIIa/IIIb.
[0019] FIG. 5: In vitro clotting activity of BHRF-1-FVIII as
compared with FVIII.
[0020] FIG. 6: In vitro binding of BHRF-1-FVIII to human
platelets.
[0021] FIG. 7: In vitro binding of BHRF-1-FVIII to mouse
platelets.
DESCRIPTION OF THE INVENTION
[0022] The present invention is directed to targeting a coagulation
factor to its site or sites of action, such as to blood cells. In
one embodiment, a targeted coagulation factor is provided that is
specifically targeted to a blood cell through linking the factor to
at least one domain that binds to a membrane protein on the blood
cell. The domain for targeting the coagulation factor to the blood
cell may be without limitation an antibody fragment, an antibody, a
peptide, a receptor ligand, a carbohydrate, or a small molecule
that has a high affinity to a membrane protein on the surface of
the blood cell. The blood cell for example is a red blood cell or a
platelet.
[0023] As used herein, "coagulation factor" refers to a protein
that is involved in the coagulation cascade and has predominantly
procoagulant activity. Coagulation factors are well known in the
art and include without limitation coagulation factors I, II, V,
VI, VII, VIII, IX, X, XI, XII, and XIII, and protein S. The
coagulation factors may be concentrated from plasma or may be
recombinantly produced. If recombinantly produced, the coagulation
factors may have an amino acid structure that varies from the
natural structure as long as sufficient procoagulant activity is
maintained such that the variant is therapeutically useful. In one
embodiment, the coagulation factor is a functional FVIII
polypeptide, such as without limitation a FVIII concentrate from
plasma or recombinantly produced FVIII, or Factor IX (FIX).
[0024] "Functional FVIII polypeptide" as used herein denotes a
functional polypeptide or combination of polypeptides that are
capable, in vivo or in vitro, of correcting human FVIII
deficiencies, characterized, for example, by hemophilia A. FVIII
has multiple degradation or processed forms in the natural state.
These are proteolytically derived from a precursor, one chain
protein. A functional FVIII polypeptide includes such single chain
protein and also provides for these various degradation products
that have the biological activity of correcting human FVIII
deficiencies. Allelic variations likely exist. The functional FVIII
polypeptides include all such allelic variations, glycosylated
versions, modifications and fragments resulting in derivatives of
FVIII so long as they contain the functional segment of human FVIII
and the essential, characteristic human FVIII functional activity.
Those derivatives of FVIII possessing the requisite functional
activity can readily be identified by straightforward in vitro
tests described herein. Furthermore, functional FVIII polypeptide
is capable of catalyzing the conversion of Factor X (FX) to FXa in
the presence of Factor IXa (FIXa), calcium, and phospholipid, as
well as correcting the coagulation defect in plasma derived from
hemophilia A affected individuals. From the published sequence of
the human FVIII amino acid sequence and the published information
on its functional regions, the fragments that can be derived via
restriction enzyme cutting of the DNA or proteolytic or other
degradation of human FVIII protein will be apparent to those
skilled in the art. Specifically included within functional FVIII
polypeptides without limitation is full-length human FVIII (e.g.,
SEQ ID NO: 1 and SEQ ID NO: 2) and B-domain deleted factor VIII
(e.g., SEQ ID NO: 3 and SEQ ID NO: 4) and having the amino acid
sequences as disclosed in WO 2006/053299.
[0025] "Procoagulant activity" of FVIII refers to the activity of
FVIII in the coagulation cascade. FVIII itself does not cause
coagulation, but plays an essential role in the coagulation
cascade. The role of FVIII in coagulation is to be activated to
FVIIIa, which is a catalytic cofactor for intrinsic FX activation
(Thompson, Semin. Thromb. Hemost. 29 :11-22, 2003). FVIII is
proteolytically activated by thrombin or FXa, which dissociates it
from von Willebrand factor (vWf) and activates its procoagulant
function in the cascade. In its active form, FVIIIa functions as a
cofactor for the FX activation enzyme complex in the intrinsic
pathway of blood coagulation, and it is decreased or nonfunctional
in patients with hemophilia A.
[0026] "FIX" means coagulation factor IX, which is also known as
human clotting factor IX, or plasma thromboplastin component.
[0027] As used herein, the term "targeted coagulation factor"
refers to a coagulation factor that is coupled with at least one
domain that specifically binds to a membrane protein on a blood
cell. The targeted coagulation factor should bind potently to the
blood cells, for example, with a half maximal binding <10 nM.
Binding should be specific to the targeted blood cells, for
example, through binding to membrane proteins selectively expressed
on the targeted cell. "Domain" or "targeting domain" as used herein
refers to a moiety that has a high affinity for membrane proteins
on target cells. Domains suitable for the present invention
include, but are not limited to, antibodies, antibody fragments,
such as single chain antibodies (svFv) or FAB fragments, antibody
mimetics, and peptides or small molecules with high affinity for
membrane proteins on the surface of the blood cells. In one aspect,
a single chain antibody fragment or a peptide is used because its
coding sequence can be linked with the FVIII coding sequence and a
fusion protein can be produced using recombinant technology.
[0028] The coagulation factor can be coupled with the domain either
chemically or by recombinant expression of a fusion protein.
Chemical linkage can be achieved by linking together chemical
moieties present on the coagulation factor and the targeting
domain, including chemical linkages using moieties such as amino,
carboxyl, sulfydryl, hydroxyl groups, and carbohydrate groups. A
variety of homo- and hetero-bifunctional linkers can be used that
have groups that are activated, or can be activated to link to
attach these moieties. Some useful reactive groups on linker
molecules include maleimides, N-hydroxy-succinamic esters and
hyrazides. Many different spacers of different chemical composition
and length can be used for separating these reactive groups
including, for example, polyethylene glycol (PEG), aliphatic
groups, alkylene groups, cycloalkylene groups, fused or linked aryl
groups, peptides and/or peptidyl mimetics of one to 20 amino acids
or amino acid analogs in length. For example, the domain may be
linked with the coagulation factor in such a way that in vivo a
functional form of the coagulation factor would be released from
its targeted domain or the release occurs at or near the site of
biological activity of the coagulation factor in the body.
[0029] Accordingly, in one embodiment of the invention, a targeted
coagulation factor is provided wherein the linkage attaching the
coagulation factor to the domain for targeting the coagulation
factor to the blood cell can be cleaved or degraded thereby
releasing the coagulation factor from the conjugate.
[0030] The release of the coagulation factors from their conjugate
form (i.e., from the targeted coagulation factor) can be achieved
by linking the targeting domain to a site on the coagulation factor
that is removed during its activation process, or by using a linker
that degrades in a controlled manner by enzymes in the blood. For
example, sugar polymers or peptides can be used that are
susceptible to general blood proteases or hydrolases. A variety of
such technologies is known in the art and has been used to make
pro-drugs. The linker could be further engineered to be cleaved
specifically at sites where the coagulation factors are most
needed, such as sites of inflammation or blood coagulation
triggered through trauma. For example, the linker may be
susceptible to specific proteases produced only at the desired site
of action, such as proteases released by the inflammation process
or generated by the blood coagulation cascade. This selective
release of the therapeutic protein may lower the potential for side
effects and increase the efficiency of the protein at its site of
action.
[0031] A variety of membrane proteins on blood cells can be
targeted according to the present invention. To specifically and
efficiently target a coagulation factor to a blood cell, however,
it is preferable that the targeted membrane protein is present
abundantly on the blood cell surface. For example, the glycoprotein
GPIIb/IIIa is found to be one of the most abundantly expressed
molecules on the platelet surface.
[0032] Accordingly, in one embodiment, the coagulation factor is
targeted to a platelet through a domain that binds specifically to
a platelet membrane protein such as the glycoprotein GPIIb/IIIa.
Examples of such domains to target the coagulation factor to
GPIIb/IIIa include, but are not limited to, RGD containing peptides
and mimetics (linear peptides, snake venom peptides, and cyclic
peptides) such as integrilin 9 containing the RGD mimetic sequence,
homo-arginine, glycine aspartic acid), non-peptide RGD mimetics,
and anti-GPIIb/IIIa antibodies. If an antibody is used as the
targeting domain, a single chain fragment of the antibody, such as
svFv or FAB fragment, can be used.
Targeting FVIII and FIX
[0033] Targeting FVIII and FIX to the surface of blood cells, such
as platelets or red blood cells, may serve to slow the clearance of
these coagulation factors. Targeting FVIII to the surface of
platelet cells is of particular interest. FVIII is a critical
cofactor in the FIX-mediated activation of FX, which takes place
predominantly on the surface of activated platelet cells that
accumulate at clot sites. Activation of platelets triggers binding
of these coagulation factors to its surface to form a complex that
facilitates FXa generation. Platelets have an average lifespan in
circulation of about 9 days. In contrast, FVIII in plasma (largely
bound to von Willebrand's factor) displays a half-life of about 14
hours. Thus, binding of FVIII to platelets has the potential to
greatly extend the circulation time of the molecule. Targeting
FVIII to the surface of platelet cells via a targeting domain
according to the present invention increases the efficiency of
FVIII action and is anticipated to prolong the half-life of
FVIII.
[0034] In addition to GPIIb/IIIa, other proteins on platelets could
serve as receptors for targeted FVIII, such as GPla and Anexin V.
The glycoprotein GPIIb/IIIa is preferred because it is one of the
most abundantly expressed molecules on the platelet surface. The
concentration of GPIIb/IIIa in blood is estimated to be about 75 nM
based on its surface density on platelets. This represents a
100-fold excess over the maximum concentration of FVIII achieved
after therapeutic application of the FVIII (C.sub.max about 0.7
nM). Therefore, targeting of FVIII to platelets would occupy
roughly 1% or less of available GPIIb/IIIa sites on platelets. This
low level of occupancy would not be expected to alter platelet
function, which requires a much large fraction (i.e., >50-60%)
of GPIIb/IIIa molecules to be blocked. The high concentration of
GPIIb/IIIa would also drive the equilibrium binding of targeted
FVIII to the platelet surface.
[0035] Without restricting the invention in any way, it is believed
that targeting FVIII to GPIIb/IIIa may also have the benefit that
some of the coagulation factors may be internalized through
endocytosis and recycling of GPIIb/IIIa through the open
intracanicular system of platelets. This FVIII can end up in alpha
granules and be re-released upon platelet activation, providing a
source of FVIII when it is needed for coagulation. Bound or
internalized FVIII targeted to platelets may be protected from
inhibitors (e.g., FVIII antibodies) that are present in many
patients. Thus, targeted FVIII may offer a treatment option for
this important group of patients.
[0036] For targeted FVIII to promote coagulation, the molecule must
be capable of being processed to a functional form (FVIIIa), and be
released from its GPIIb/IIIa binding site. In one embodiment, this
is achieved by linking the GPIIb/IIIa targeting domain to the
B-domain of FVIII. The B-domain is removed in a pro-coagulant
environment by thrombin or FXa mediated proteolysis, producing the
mature FVIIIa molecule. Thus, upon activation, FVIIIa will be
released from GPIIb/IIIa and be available for formation of the FX
activation complex.
[0037] The linkage between FVIII and the targeting domain can be
achieved by covalently binding the targeting domain to reactive
groups on FVIII, including amino, sulfhydryl, carboxyl groups and
carbonyl groups using cross-linking approaches described herein.
Targeting domains can also be coupled to carbohydrate present
mostly on the B-domain of the FVIII molecule. For example, mild
oxidation of FVIII with periodate produces aldehydes on
carbohydrate chains, which can then be reacted with amines or
hyrazides, followed optionally by reduction to form more stable
linkages.
[0038] Free cysteine can be selectively generated on the B-domain
of recombinant FVIII through mild reduction with
Tris(2-carboxyethyl)phosphine (TCEP), allowing specific linking of
the B-domain with a targeting domain that reacts with a free
cysteine, such as a domain containing a thiol, triflate, tresylate,
aziridine, oxirane, S-pyridyl, or maleimide moiety. Furthermore,
FVIII can be modified to replace an amino acid residue with
cysteine to provide a specific location for attachment to a
targeting domain. If a B-domain deleted FVIII is used, a variety of
cysteine muteins of B-domain deleted FVIII, such as those disclosed
in WO 2006/053299, can be used to link FVIII with a targeting
domain through chemical binding at a surface cysteine residue.
Examples of amino acid residues that may be modified to replace an
amino acid residue with cysteine include, but not limited to, 81,
129, 377, 378, 468, 487, 491, 504, 556, 570, 1648, 1795, 1796,
1803, 1804, 1808, 1810, 1864, 1911, 2091, 2118, and 2284 (the amino
acid residue is designated by its position in the sequence of
full-length FVIII).
[0039] The coagulation factor may also be coupled to the targeting
domain using recombinant technology. Host cells may be transfected
with a vector comprising a fusion protein of FVIII and the
targeting domain. In one embodiment, the targeting domain may be
inserted into the B-domain of FVIII and most of the B-domain is
deleted with only portions of the B-domain left at the carboxy and
amino terminals to allow for the biological processing of the
B-domain to delete it from the full-length molecule. As illustrated
in FIG. 1, the remaining portions of the B-domain are specified
that allow for biological processing and removal of the B-domain
under physiological conditions.
[0040] The host cell line may be any cell known to those skilled in
the art as useful for producing a coagulation factor such as
without limitation for FVIII CHO cells, HEK cells, BHK cells, and
HKB11 cells (a hybrid of a human embryonic kidney cell line, HEK293
and a human Burkitt B cell lymphoma line, 2B8).
[0041] A number of domains can be linked chemically to FVIII, or
recombinantly expressed with FVIII, to target FVIII to GPIIb/IIIa
on the surface of platelets. Examples of such domains include, but
are not limited to, antibodies against GPIIb/IIIa, RGD peptides,
peptide mimetics, or small molecule mimetics targeting GPIIb/IIIa.
Antibodies, such as single chain antibodies (svFv) or FAB fragments
targeting GPIIb/IIIa, are particularly useful as targeting
domains.
[0042] It has been shown that the B-domain of FVIII can be removed
without loss of FVIII function. Additionally, it has been also
shown that various B-domain truncated forms of FVIII and B-domain
fusions with other protein domains can yield functionally active
FVIII. In one aspect, the invention involves targeting domains that
can be engineered to insert into, replace, or partially replace the
B-domain of FVIII without blocking the normal processing of the
molecule to yield active FVIII. For example, using recombinant DNA
technology, a FVIII molecule can be produced in which single chain
antibody fragments are fused to the C-terminus of the B-domain of
FVIII. Alternatively, svFv fragments can also be used to replace
the whole or a part of the B-domain of FVIII.
[0043] This can be achieved through insertion of the DNA sequence
encoding the svFv fragments, in frame, after the B-domain coding
sequence, or replacing some or all of the B-domain coding sequence.
This strategy will preserve thrombin cleavage sites required for
normal proteolyic activation of FVIII. A variety of antibodies
against GPIIb/IIIa which localize efficiently to platelets are
known (see, e.g., Schwarz, et al., Circ. Res. 99(1):25-33, 2006;
Jacobin, et al., Clin. Immunol. 108(3):199-210, 2003;
Christopoulos, et al., Blood Coagul. Fibrinolysis 4(5):729-37,
1993; and Chung, et al., FASEB J. 18(2):361-363, 2004).
[0044] Likewise, RGD or RGD mimetic containing peptides are also
useful ligands for targeting FVIII since many of such peptides have
been described to have high binding affinity to GPIIb/IIIa. These
include linear peptides, snake venom peptides, and cyclic peptides.
Non-peptide RGD mimetics could also be used. Similar to the
antibody fragments, RGD peptides can be chemically coupled to
FVIII. Alternatively, RGD sequences can be inserted into the
B-domain coding sequence or used to replace, in whole or in part,
the B-domain coding sequence of FVIII and expressed using
recombinant DNA technology.
[0045] A targeted FIX can be prepared using a similar procedure.
For example, targeting domains can be linked to an activation
domain of a FIX molecule (amino acid residues 191-226 or 145-180,
depending on preferences, that is, +/- signal sequence), which is
proteolytically removed in the activation of FIX to FIXa. The
domain can be linked chemically using cross-linkers reactive with
amino acid side chain groups such as sulfhydryls, amines, and
carboxyl groups in the activation domain, or linked through
carbohydrate chains, as was discussed above for FVIII. A fusion
molecule can also be made using recombinant technology where an
amino acid sequence of a targeting domain is inserted into the FIX
activation peptide, or replacing parts of the activation peptide
sequence. The inserted targeting domain sequences can code for a
single chain antibody, or other platelet binding peptide sequence,
such as an RGD binding peptide.
Pharmaceutical Compositions and Uses
[0046] The invention also concerns pharmaceutical compositions
comprising therapeutically effective amounts of the targeted
coagulation factors of the invention and a pharmaceutically
acceptable excipient or carrier. "Pharmaceutically acceptable
excipient or carrier" is a substance that may be added to the
active ingredient to help formulate or stabilize the preparation
and causes no significant adverse toxicological effects to the
patient. Examples of such excipients or carriers are well known to
those skilled in the art and include water, sugars such as maltose
or sucrose, albumin, salts, etc. Other excipients or carriers are
described, for example, in Remington's Pharmaceutical Sciences
(Mack Publishing Co., Easton, Pa., 20.sup.th edition, 2000). Such
compositions will contain an effective amount of the targeted
coagulation factors together with a suitable amount of excipients
or carriers to prepare pharmaceutically acceptable compositions
suitable for effective administration to a patient in need
thereof.
[0047] For example, the conjugate may be parenterally administered
to subjects suffering from hemophilia A at a dosage that may vary
with the severity of the bleeding episode. The average doses
administered intraveneously is in the range of 40 units per
kilogram for pre-operative indications, 15 to 20 units per kilogram
for minor hemorrhaging, and 20 to 40 units per kilogram
administered over an 8-hours period for a maintenance dose.
[0048] In one embodiment, the present invention concerns a method
for treating hematological diseases comprising administering an
therapeutically effective amount of the aforementioned targeted
coagulation factor to a patient in need thereof.
[0049] As used herein, "therapeutically effective amount" means an
amount of a targeted coagulation factor that is need to provide a
desired level of the targeted factor (or corresponding unconjugated
factor released from the targeted form) in the bloodstream or in
the target tissue. The precise amount will depend upon numerous
factors, including, but not limited to the components and physical
characteristics of the therapeutic composition, intended patient
population, individual patient considerations, and the like, and
can readily be determined by one skilled in the art.
[0050] As used herein, "patient" refers to human or animal
individuals receiving medical care and/or treatment.
[0051] The polypeptides, materials, compositions, and methods
described herein are intended to be representative examples of the
invention, and it will be understood that the scope of the
invention is not limited by the scope of the examples. Those
skilled in the art will recognize that the invention may be
practiced with variations on the disclosed polypeptides, materials,
compositions and methods, and such variations are regarded as
within the ambit of the invention.
[0052] The following examples are presented to illustrate the
invention described herein, but should not be construed as limiting
the scope of the invention in any way.
EXAMPLES
[0053] In order that this invention may be better understood, the
following examples are set forth. These examples are for the
purpose of illustration only, and are not to be construed as
limiting the scope of the invention in any manner. All publications
mentioned herein are incorporated by reference in their
entirety.
Example 1
[0054] Modified RGD Peptides with High Affinity for GPIIb/IIIa
Binding
[0055] Cyclic peptides have been described to bind potently and
selectively to GPIIb/IIIa. One such peptide, integrilin, was used
as a targeting domain to link with FVIII as it has been shown that
integrilin can selectively bind to GPIIb/IIIa. Integrilin was
modified by adding a short PEG linker ending in a maleimide moiety
that can selectively couple to free cysteine residues in proteins.
The modified integrilin is termed BHRF-1 with the linker only (FIG.
2A), and BHRF-3 with the linker and a fluorescein (FITC) (FIG. 2B).
As shown in FIG. 3, the modified integrilins retain affinity for
GPIIb/IIIa as they potently blocked fibrinogen (Fbn) binding to
immobilized GPIIa/IIIb.
[0056] Peptide binding to GPIIb-IIIa was measured using a solid
phase binding assay in which competition of fibrinogen binding by
testing compounds is measured. The assay was performed as follows.
Purified GPIIb-IIIa (Innovative Research, Novi, Mich.) was coated
onto 96-well Immulon-B plates at 0. mL/well.times.2 .mu.g/mL,
diluted in Buffer A (20 mM Tris pH 7.5, 0.15 M NaCl, and 1 mM each
of MgCl.sub.2, CaCl.sub.2, and MnCl.sub.2). After overnight
incubation at 4.degree. C., the plate was blocked for 1 hour at
30.degree. C., with 3.5% BSA in Buffer B (50 mM Tris pH 7.5, 0.1 M
NaCl, and 1 mM each of MgCl.sub.2, CaCl.sub.2, and MnCl.sub.2).
After washing 3 times with Buffer B, diluted peptide or protein
solutions were combined with 3.5 nM biotinylated fibrinogen in 0.1%
BSA/Buffer B and added to the wells, incubating at 30.degree. C.
for 2 hr. After washing (3 times, Buffer B), 1:4000
streptavidin-horseradish peroxidase (HRP) was added (Pierce
Chemical Co., Rockford, Ill.) for 1 hour at 30.degree. C. After a
final washing step (3 times, Buffer B), the plate was developed
with Ultra TMB (3,3',5,5'-tetramentylbenzidine) (Pierce Chemical
Co., Rockford, Ill.) for 5 minutes, stopping with an equal volume
of 2 M sulfuric acid. Plate absorbances were read at 450 nm, and
IC.sub.50 values determined using a 4-parameter logistic fit.
[0057] The modified integrilin peptide (BHRF1) is then coupled with
FVIII via the cysteine (Cys) residue located in the B-domain of
FVIII.
Example 2
[0058] Coupling GPIIb/IIIa Binding Peptides to FVIII
[0059] The polypeptide sequence of the full-length FVIII is known
in the art (see, e.g., SEQ ID NO: 1, SEQ ID NO: 2, and as disclosed
in WO 2006/053299.
Concentration of FVIII and uncapping of free sulfhydryl groups
[0060] The Cys residue located in the B-domain of recombinant FVIII
can be capped by cysteine present in the media during protein
expression, but it can be readily removed by treatment with
reducing agents, such as TCEP, as follows. FVIII (20 mL) was thawed
and concentrated in two Amicon.RTM.-15 cartridges (Millipore,
Billerica, Mass.), spun at 2000.times.g (about 3153 rpm) for 25
minutes in the cold. The concentration of the 2.8 mL retentate is
about 0.8-0.9 mg/mL by A280 using a NanoDrop.RTM. spectrophotometer
(ThermoFisher Scientific, Waltham, Mass.). The buffer was then
exchanged using a 10 mL Zeba desalting cartridge, pre-equilibrated
with 50 mM Tris, 150 mM NaCl, 2.5 mM CaCl.sub.2 and 100 ppm
Tween.RTM.-80 (polyoxyethylenesorbitan monooleate). A protein
solution of 2.8 mL with a concentration of 0.88 mg/mL was obtained.
TCEP was then added to a final concentration of 0.68 mM and the
mixture was gently turned end-over-end at 4.degree. C. for about 3
hours. TCEP was removed by two successive Zeba cartridge spins, and
the FVIII was allowed to re-oxidize for at least 30 minutes before
addition of the peptide. After the removal of TCEP, the FVIII
concentration was measured at 0.768 mg/mL ("KG-R").
Coupling of the RGD targeting peptide
[0061] To couple the modified integrilin peptide BHRF-1 to FVIII,
0.294 mg of the peptide (M. W. 1225) was added to 48 .mu.L dry
dimethyl sulfoxide (DMSO) to make a 5 mM stock solution. This stock
solution (34.4 .mu.L) was then added to 2.8 mL KG-R. The reaction
was quenched by addition of an equi-molar amount of cysteine after
80 minutes. The reaction mixture was then extensively dialyzed
against starting Tris buffer (2 liters). The final concentration of
BHRF-1-FVIII was 0.74 mg/mL and the yield was 2 mg. A similar
procedure was also used to prepare BHRF-3-FVIII.
[0062] As shown in FIG. 3, the modified integrilin peptides, BHRF-1
and BHRF-3, retain affinity for GPIIb/IIIa as they potently blocked
fibrinogen (Fbn) binding to immobilized GPIIa/IIIb. FVIII coupled
to BHRF-1 (FVIII-BHRF-1) showed high potency for inhibition of
fibrinogen binding to immobilized GPIIb/IIIa
(IC.sub.50=0.043+/-0.05 nM(N=3)). This was even more potent than
the parent BHRF-1 peptide. Results are shown in Table 1.
TABLE-US-00001 TABLE 1 Conjugate Moiety nM (N) Integrelin 1.3 +/-
1.0 4 BHRF-1 (+linker) 1.2 +/- 0.6 2 BHRF-3 1.5 +/- 1.3 3 (+linker
+ FITC)
Coupling of the RGD targeting peptide to B-domain deleted FVIII
[0063] If a B-domain deleted FVIII ("BDD") is used for coupling, a
variety of Cys muteins of B-domain deleted FVIII as disclosed in WO
2006/053299 can be used to couple BDD to a targeting domain such as
the modified RGD peptides as disclosed herein.
Example 3
[0064] BHRF-1-FVIII Binds to Immobilized GPIIb/IIIa
[0065] To test the binding activity of BHRF-1-FVIII to GPIIb/IIIa,
biotinylated GPIIb/IIIa was immobilized on streptavidin plates and
treated with either BHRF-1-FVIII or unmodified FVIII, both in
binding buffer (50 mM Tris, pH 7.5, 100 mM NaCl.sub.2, 1 mM
CaCl.sub.2, 1 mM MgCl.sub.2, 1 mM MnCl.sub.2 and 1 mg/mL BSA). The
unbound protein was removed by washing three times with binding
buffer. Assay buffer (25 .mu.L) was added to the plate, and FVIII
activity was determined using a chromogenic assay kit (Coatest.RTM.
SP4, Chromogenix, Lexington, Mass.). As shown in FIG. 4, there was
binding of BHRF-1-FVIII, while only little binding of unmodified
FVIII was detected. The increased binding of BHRF-1-FVIII was
completely eliminated by addition of a cyclic RGD peptide
(GpenGRGDSPCA; SEQ ID NO: 5) that competes for BHRF-1 binding to
GPIIb/IIIa. Furthermore, only low background levels of either
protein bound when no GPIIb/IIIa was immobilized on the plate.
These data show that BHRF-1-FVIII can be targeted to GPIII/IIIIa
through the peptide targeting domain.
[0066] Because unconjugated FVIII was not removed from the
preparations of BHRF1-FVIII, experiments were performed to
determine the amount of unconjugated FVIII present. BHRF1-FVIII
activity was depleted using beads containing excess levels of
immobilized GPIIb/IIIa. Roughly 80% of the activity of BHFR1-FVIII
can be depleted, indicating about 20% of the FVIII activity in the
preparation came from unconjugated FVIII.
Example 4
[0067] In vitro Whole Blood Clotting Activity Assay with
BHRF-1-FVIII and FVIII
[0068] To assess the effect of platelet binding of BHRF-1-FVIII on
hemostatic activity, its activity was compared to that of
unconjugated FVIII using a Rotational Thromboelastometry
(ROTEM.RTM., Pentapharm GmbH) system as described in Landskroner,
et al., (Haemophilia 11:346-352, 2005). Unlike measures of clotting
activity such as the Coatest.RTM. chromogenic assay or the
activated partial thromboplastin time (aPTT) assay, the ROTEM.RTM.
assay depends on the function of the platelets and therefore, can
show effects of BHRF-1-FVIII binding to platelets. To perform the
assay, citrated hemophilia A mouse whole blood was mixed with an
equal dose of BHRF-1-FVIII (1 mIU) or unconjugated FVIII (based on
the Coatest.RTM. chromogenic assay) at room temperature. Samples
were recalcified by dispensing 300 .mu.L treated blood with an
automated pipette into ROTEM.RTM. cups with 20 .mu.L CaCl.sub.2
(200 mmol) without exogenous activator (NATEM). Measurement was
started immediately after the last pipetting and blood clot
formation was continuously monitored for 2 hours (7200 seconds) at
37.degree. C.
[0069] ROTEM.RTM. analysis parameters for hemostasis include
Clotting Time (CT), the time required to obtain clot firmness of 2
mm following the initiation of measurement, Clot Formation Time
(CFT), the time from clot firmness of 2 mm till clot strength of 20
mm, and .alpha.-angle, the velocity of clot formation.
[0070] As shown in FIG. 5, BHRF-1-FVIII required less time to form
a clot in the ROTEM.RTM. assay than an equal dose (based on a
chromogenic assay) of unconjugated FVIII, indicating a higher
efficiency of clotting. The difference in CT was about 400 seconds,
which corresponds to roughly 2-3 fold more FVIII activity, based on
FVIII standard curves.
[0071] Hemostatic activity and pharmacokinetic parameter of
targeted coagulation factors can be assessed in vivo using the
hemophilia A mouse model. Targeted coagulation factors can be
administered by tail vein intravenous injection. At multiple time
points after the treatment, blood will be collected in % sodium
citrate and hemostatic activity will be measured using ROTEM over
48 hours post infusion period which is equivalent to >6
half-life of FVIII (t.sub.1/2) in mice.
Example 5
[0072] In vitro Binding Assay to Human and Mouse Platelets
Binding of FVIII-BHRF-1 to human platelets
[0073] Human platelets were obtained from Allcells (Emeryville,
Calif.) at 5.times.10.sup.9 platelets/tube in 14 mL plasma. The
platelets and all washes, buffers, reagents, and centrifuges were
warmed to room temperature and maintained at room temperature
during the course of the experiment. The wash buffer (WB) for the
platelets is Tyrode's buffer supplemented with 20 mM HEPES, 0.5%
BSA, and 50 ng/mL PGE1 and 2.5 U/mL apyrase, pH 7.4.
[0074] The cells were centrifuged at 700.times.g for 15 minutes at
25.degree. C., and then the supernatant was carefully removed and
14 mL WB was added. The cells were gently re-suspended in the WB
and centrifuged as described.
[0075] Following the second centrifugation, the supernatant was
removed and the platelets were re-suspended in 15 mL WB. At this
point, the cells were split into three equal aliquots of 5 mL each.
The three aliquots were centrifuged as described earlier, and then
the three platelet pellets were re-suspended in either:
[0076] A. 5 mL binding buffer+5 mg/mL BSA (BBB, 50 mM Tris, 100 mM
NaCl, 1 mM each CaCl.sub.2, MgCl.sub.2, and MnCl.sub.2)
[0077] B. 5 mL HemA plasma which lacks FVIII, but vWF is
present
[0078] C. 5 mL immuno-depleted plasma lacking both FVIII and
vWF.
[0079] For buffer (A) or plasma (B or C), the following conditions
were used:
[0080] 1. buffer/plasma alone+2.5 nM BHRF-1-FVIII (containing about
20% uncongugated FVIII (see Example 3))
[0081] 2. buffer/plasma+platelet+2.5 nM BHRF-1-FVIII (containing
about 20% uncongugated FVIII)
[0082] 3. buffer/plasma alone+2.5 nM recombinant FVIII
[0083] 4. buffer/plasma+platelet+2.5 nM recombinant FVIII
[0084] For each condition 1-4, 100 .mu.L A, B, or C was pipetted
into a microfuge tube at room temperature, then the BHRF-1-FVIII or
unconjugated FVIII was added to the tube. The tubes were incubated
at 37.degree. C. for 1.5 hours (without shaking) Following the
incubation period, the tubes were centrifuged at maximum speed
(16,000 rpm) for 5 minutes to pellet the platelets. The supernatant
was collected to assay for FVIII activity. The amount of activity
in the supernatant reflects the amount of unbound FVIII or
BHRF-1-FVIII. The data demonstrate binding of the BHRF1-FVIII to
human platelets in all conditions (shown in FIG. 6). Since the
BHRF-1-FVIII contains roughly 20% unconjugated FVIII for conditions
A and C, the data indicate that a high percentage of conjugate was
bound. There was no binding of FVIII observed for conditions A and
B, while 35% of the FVIII activity was bound in condition C. The
figure also shows the level of FVIII activity remaining for
condition C corrected for the 35% non-specific binding of FVIII
were observed for this condition (i.e., the starting FVIII activity
was reduced by 35% to calculate the percentage bound).
Binding of FVIII-BHRF-1 to mouse platelets
[0085] BHRF-1-FVIII also bound to mouse platelets as shown in FIG.
7. A similar binding assay was performed as described for human
platelets except that citrated mouse blood was centrifuged
200.times.g for 15 minutes to harvest platelet rich plasma (PRP).
The PRP was diluted with citrate wash buffer (11 mM glucose, 128 mM
NaCl, 4.3 mM NaH.sub.2PO.sub.4, 7.5 mM Na.sub.2HPO.sub.4, 4.8 mM
Na-citrate, 2.4 mM citric acid, 0.35% BSA, pH 6.5)+50 ng/mL PGE1,
and washed twice in citrate wash buffer+50 ng/mL PGE1 (by
centrifuging at 1200.times.g for 10 minutes). The platelets were
finally re-suspended in binding buffer (50 mM Tris, 100 mM NaCl, 1
mM each CaCl.sub.2, MgCl.sub.2, and MnCl.sub.2)+5 mg/mL BSA.
Un-conjugated FVIII and BHRF-1-FVIII were added to the platelets
and after 2 hours at 37.degree. C., the platelets were removed by
centrifugation, and the unbound FVIII activity in the supernatant
determined.
[0086] As shown in the FIG. 7, 59% of the activity of unconjugated
FVIII bound to the platelets. To calculate the percentage of the
added BHRF-1-FVIII activity binding to platelets through the BHRF-1
peptide, the amount of starting FVIII activity was corrected by 59%
to reflect the level of non-specific binding of FVIII (not
occurring through the peptide). The corrected value for
BHRF-1-FVIII was 31% unbound (69% bound). When 100 uM integrilin
was added to complete for peptide binding, unbound activity rose to
82% unbound (18% bound) (also corrected for nonspecific FVIII
binding). These data demonstrate that BHRF-1-FVIII can bind to
mouse platelets through the BHRF-1 targeting domain.
Example 6
[0087] Pharmacokinetic Study
[0088] The level of FVIII in blood at various times after injection
into hemophilia A mice is determined using a whole blood
coagulation assay such as ROTEM.RTM. described above, which
reflects FVIII activity in both plasma and bound to cells (e.g.,
platelets).
Example 7
[0089] Chromogenic Assay for the Assessment of FVIII Activity
[0090] FVIII activity of purified proteins and conjugates was
assessed using the Coatest.RTM. SP assay kit (Chromogenix,
Lexington, Mass.). The assay was performed following the
manufacturer's instructions in a 96-well plate format. Briefly,
diluted samples containing FVIII or conjugate were combined in
order with a mixture of activated FIX/FX/phospholipid, followed by
25 mM CaCl.sub.2 and chromogenic substrate S-2765/I-2581. Between
each reagent addition, the samples were incubated at 37.degree. C.
for 5 minutes. After the final addition of chromogenic substrate,
the reaction was stopped after 5 minutes with 20% acetic acid and
the plate absorbances were read at 405 nm, normalized against a 490
nm background. Sample absorbances were calibrated against a
WHO/NIBSC plasma-derived FVIII standard curve with an operating
range of 0.3-40 mIU/mL.
Example 8
[0091] In vivo Efficacy Assay in Hemophilic Mice
[0092] To show the efficacy of targeted FVIII molecules in
promoting blood clotting and to assess the duration of these
effects, the tail clip injury or tail vein transection models which
use hemophilic (HemA) mice, can be used as described below.
Tail Clip Injury Model
[0093] Test samples are administrated to the mice via a tail vein
injection. Following administration, the mice are anesthetized
intraperitoneal (IP) with ketamine/xylazine (100 mg/kg, 10 mg/kg).
When the animals are fully anesthetized, the tails are placed
individually in 13 mL 37.degree. C. pre-warmed saline for
approximately 10 minutes. A tail cut is made with a sharp scalpel
and the tail is placed immediately in a new tube with 9 mL
37.degree. C. warm saline. Blood is collected continuously for 30
minutes. Blood loss volume is determined either by weight gain of
the blood collection tube or determined by the optical density of
the blood/saline mixture in the blood collection tube.
Tail Vein Transection
[0094] HemA male mice are randomized into different treatment
groups by their body weight. Mice are dosed by tail vein injection
24 hours prior to the tail vein transaction. Before the tail vein
transection, mice are anesthetized (IP) with a cocktail containing
50 .mu.g/kg of ketamine and 1 mg/kg of medetomidine. The tail is
marked at a diameter of 2.7 mm using a french catheter. The
anesthetic effect of medetomidine is reversed with 1 mg/kg of
atipamezole by IP injection. The tail vein is transected with a
scalpel blade. The tail is then submerged into 37.degree. C. saline
tube, and the tube is rotated to rinse away the blood from the cut.
When the saline becomes too opaque to visualize, it is replaced
with a new tube until the tail stops bleeding. The time it takes to
stop bleeding is recorded as the acute clotting time. The mouse is
then returned to its individual clean cage with white paper bedding
placed on top of a 4.times.8 inch heating pad. The time to re-bleed
and moribund is monitored hourly for the next 9-11 hours for
excessive blood loss.
Example 9
[0095] Recombinant Expression of Targeted FVIII
[0096] In one embodiment, HKB11 cells are grown in suspension
culture on an orbital shaker (100-125 rpm) in a 5% CO.sub.2
incubator at 37.degree. C. in a protein-free media and maintained
at a density between 0.25 and 1.5 .times.10.sup.6 cells/mL. HKB11
cells for transfection are collected by centrifugation then
resuspended in an expression medium such as FreeStyle.TM. 293
Expression Medium (Invitrogen, Carlsbad, Calif.) at
1.1.times.10.sup.6 cells/mL. The cells are seeded in 6-well plates
(4.6 mL/well) and incubated on an orbital rotator (125 rpm) in a
37.degree. C. CO.sub.2 incubator. For each well, 5 .mu.g plasmid
DNA is mixed with 0.2 mL Opti-MEM.RTM. I medium (Invitrogen,
Carlsbad, Calif.). For each well, 7 .mu.L 293fectin.TM. reagent
(Invitrogen, Carlsbad, Calif.) is mixed gently with 0.2 mL
Opti-MEM.RTM. I medium and incubated at room temperature for 5
minutes. The diluted 293fectin.TM. is added to the diluted DNA
solution, mixed gently, incubated at room temperature for 20-30
minutes, and then added to each well that has been seeded with
5.times.10.sup.6 (4.6 mL) HKB11 cells. The cells are then incubated
on an orbital rotator (125 rpm) in a CO.sub.2 incubator at
37.degree. C. for 3 days after which the cells are pelleted by
centrifugation at 1000 rpm for 5 minutes and the supernatant is
collected.
[0097] Stable transfection of HKB11 cells is obtained using the
following procedure. HKB11 cells are transfected with plasmid DNA
using 293fectin.TM. reagent as described in transient transfection.
The transfected cells are split into 100-mm culture dishes at
various dilutions (1:100, 1:1000, 1;10,000) and maintained in
DMEM-F12 medium supplemented with 5% FBS and 200 ug/mL hygromicin
(Invitrogen, Carlsbad, Calif.) for about 2 weeks. Individual single
colonies are picked and transferred into 6-well plates using
sterile cloning disks (Scienceware.RTM., Sigma-Aldrich, St. Louis,
Mo.). The clones are established and banked. These clones are
screened for high expression of the fusion protein by FVIII
activity assays (e.g., Coatest.RTM. and aPTT assays) as well as by
FVIII ELISA.
[0098] Factor VIII activity levels in culture supernatants and
purification fractions may be determined using a commercial
chromogenic assay kit (Coatest.RTM. SP4 FVIII, Chromogenix,
Lexington, Mass.) in a 96-well format as described above. Factor
VIII coagulation activity may also be determined using an aPTT
assay in FVIII-deficient human plasma by an Electra.RTM. 1800C
automatic coagulation analyzer (Beckman Coulter, Fullerton,
Calif.). Briefly, three dilutions of supernatant samples in
coagulation diluent are created by the instrument and 100 .mu.L is
then mixed with 100 .mu.L FVIII-deficient plasma and 100 .mu.L
automated aPTT reagent (rabbit brain phospho lipid and micronized
silica, Biomerieux, Durham, N.C.). After the addition of 100 .mu.L
25 mM CaCl.sub.2 solution, the time to clot formation is recorded.
A standard curve is generated for each run using serial dilutions
of the same purified FVIII used as the standard in the ELISA
assay.
[0099] While the present invention has been described with
reference to the specific embodiments and examples, it should be
understood that various modifications and changes may be made and
equivalents may be substituted without departing from the true
spirit and scope of the invention. The specification and examples
are, accordingly, to be regarded in an illustrative rather then a
restrictive sense. Furthermore, all articles, patent applications
and patents referred to herein are incorporated herein by reference
in their entireties.
* * * * *