Patent application title: Targeted Coagulation Factors and Method of Using the Same
Richard Feldman (El Cerrito, CA, US)
Ji-Yun Kim (Berkeley, CA, US)
Haiyan Jiang (San Francisco, CA, US)
Kirk Mclean (Orinda, CA, US)
Junliang Pan (Moraga, CA, US)
Glenn Pierce (Rancho Santa Fe, CA, US)
James Wu (El Cerrito, CA, US)
Xiao-Yan Zhao (Union City, CA, US)
BAYER HEALTHCARE LLC
IPC8 Class: AA61K3836FI
Publication date: 2011-03-31
Patent application number: 20110077202
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.
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.
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
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
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.
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 α1 (dsPA α1) 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 α1 was shown to bind to thrombin-activated human and dog platelets (Dong, et al., Thromb. Haemost. 92:956-965, 2004).
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.
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.
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.
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.
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.
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).
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.
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.
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
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
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.
FIG. 2: Structures of modified cyclic peptide integrilin, "BHRF-1" (A) and "BHRF-3" (B), for linking to FVIII through the B-domain cysteine.
FIG. 3: Binding affinity of BHRF-1 and BFRH-3 to immobilized GPIIa/IIIb.
FIG. 4: BHRF-1-FVIII binding assay to immobilized GPIIa/IIIb.
FIG. 5: In vitro clotting activity of BHRF-1-FVIII as compared with FVIII.
FIG. 6: In vitro binding of BHRF-1-FVIII to human platelets.
FIG. 7: In vitro binding of BHRF-1-FVIII to mouse platelets.
DESCRIPTION OF THE INVENTION
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.
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).
"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.
"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.
"FIX" means coagulation factor IX, which is also known as human clotting factor IX, or plasma thromboplastin component.
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.
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.
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.
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.
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.
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
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.
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 (Cmax 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.
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.
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.
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.
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).
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.
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).
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.
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.
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).
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.
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
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., 20th 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.
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.
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.
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.
As used herein, "patient" refers to human or animal individuals receiving medical care and/or treatment.
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.
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.
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.
Modified RGD Peptides with High Affinity for GPIIb/IIIa Binding
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.
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×2 μg/mL, diluted in Buffer A (20 mM Tris pH 7.5, 0.15 M NaCl, and 1 mM each of MgCl2, CaCl2, and MnCl2). After overnight incubation at 4° C., the plate was blocked for 1 hour at 30° C., with 3.5% BSA in Buffer B (50 mM Tris pH 7.5, 0.1 M NaCl, and 1 mM each of MgCl2, CaCl2, and MnCl2). 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° 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° 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 IC50 values determined using a 4-parameter logistic fit.
The modified integrilin peptide (BHRF1) is then coupled with FVIII via the cysteine (Cys) residue located in the B-domain of FVIII.
Coupling GPIIb/IIIa Binding Peptides to FVIII
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
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®-15 cartridges (Millipore, Billerica, Mass.), spun at 2000×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® 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 CaCl2 and 100 ppm Tween®-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° 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
To couple the modified integrilin peptide BHRF-1 to FVIII, 0.294 mg of the peptide (M. W. 1225) was added to 48 μL dry dimethyl sulfoxide (DMSO) to make a 5 mM stock solution. This stock solution (34.4 μL) 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.
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 (IC50=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
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.
BHRF-1-FVIII Binds to Immobilized GPIIb/IIIa
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 NaCl2, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2 and 1 mg/mL BSA). The unbound protein was removed by washing three times with binding buffer. Assay buffer (25 μL) was added to the plate, and FVIII activity was determined using a chromogenic assay kit (Coatest® 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.
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.
In vitro Whole Blood Clotting Activity Assay with BHRF-1-FVIII and FVIII
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®, Pentapharm GmbH) system as described in Landskroner, et al., (Haemophilia 11:346-352, 2005). Unlike measures of clotting activity such as the Coatest® chromogenic assay or the activated partial thromboplastin time (aPTT) assay, the ROTEM® 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® chromogenic assay) at room temperature. Samples were recalcified by dispensing 300 μL treated blood with an automated pipette into ROTEM® cups with 20 μL CaCl2 (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° C.
ROTEM® 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 α-angle, the velocity of clot formation.
As shown in FIG. 5, BHRF-1-FVIII required less time to form a clot in the ROTEM® 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.
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 (t1/2) in mice.
In vitro Binding Assay to Human and Mouse Platelets
Binding of FVIII-BHRF-1 to human platelets
Human platelets were obtained from Allcells (Emeryville, Calif.) at 5×109 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.
The cells were centrifuged at 700×g for 15 minutes at 25° 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.
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:
A. 5 mL binding buffer+5 mg/mL BSA (BBB, 50 mM Tris, 100 mM NaCl, 1 mM each CaCl2, MgCl2, and MnCl2)
B. 5 mL HemA plasma which lacks FVIII, but vWF is present
C. 5 mL immuno-depleted plasma lacking both FVIII and vWF.
For buffer (A) or plasma (B or C), the following conditions were used:
1. buffer/plasma alone+2.5 nM BHRF-1-FVIII (containing about 20% uncongugated FVIII (see Example 3))
2. buffer/plasma+platelet+2.5 nM BHRF-1-FVIII (containing about 20% uncongugated FVIII)
3. buffer/plasma alone+2.5 nM recombinant FVIII
4. buffer/plasma+platelet+2.5 nM recombinant FVIII
For each condition 1-4, 100 μL 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° 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
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×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 NaH2PO4, 7.5 mM Na2HPO4, 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×g for 10 minutes). The platelets were finally re-suspended in binding buffer (50 mM Tris, 100 mM NaCl, 1 mM each CaCl2, MgCl2, and MnCl2)+5 mg/mL BSA. Un-conjugated FVIII and BHRF-1-FVIII were added to the platelets and after 2 hours at 37° C., the platelets were removed by centrifugation, and the unbound FVIII activity in the supernatant determined.
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.
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® described above, which reflects FVIII activity in both plasma and bound to cells (e.g., platelets).
Chromogenic Assay for the Assessment of FVIII Activity
FVIII activity of purified proteins and conjugates was assessed using the Coatest® 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 CaCl2 and chromogenic substrate S-2765/I-2581. Between each reagent addition, the samples were incubated at 37° 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.
In vivo Efficacy Assay in Hemophilic Mice
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
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° 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° 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
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 μg/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° 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 4×8 inch heating pad. The time to re-bleed and moribund is monitored hourly for the next 9-11 hours for excessive blood loss.
Recombinant Expression of Targeted FVIII
In one embodiment, HKB11 cells are grown in suspension culture on an orbital shaker (100-125 rpm) in a 5% CO2 incubator at 37° C. in a protein-free media and maintained at a density between 0.25 and 1.5 ×106 cells/mL. HKB11 cells for transfection are collected by centrifugation then resuspended in an expression medium such as FreeStyle® 293 Expression Medium (Invitrogen, Carlsbad, Calif.) at 1.1×106 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° C. CO2 incubator. For each well, 5 μg plasmid DNA is mixed with 0.2 mL Opti-MEM® I medium (Invitrogen, Carlsbad, Calif.). For each well, 7 μL 293fectin® reagent (Invitrogen, Carlsbad, Calif.) is mixed gently with 0.2 mL Opti-MEM® I medium and incubated at room temperature for 5 minutes. The diluted 293fectin® 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 5×106 (4.6 mL) HKB11 cells. The cells are then incubated on an orbital rotator (125 rpm) in a CO2 incubator at 37° C. for 3 days after which the cells are pelleted by centrifugation at 1000 rpm for 5 minutes and the supernatant is collected.
Stable transfection of HKB11 cells is obtained using the following procedure. HKB11 cells are transfected with plasmid DNA using 293fectin® 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®, 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® and aPTT assays) as well as by FVIII ELISA.
Factor VIII activity levels in culture supernatants and purification fractions may be determined using a commercial chromogenic assay kit (Coatest® 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® 1800C automatic coagulation analyzer (Beckman Coulter, Fullerton, Calif.). Briefly, three dilutions of supernatant samples in coagulation diluent are created by the instrument and 100 μL is then mixed with 100 μL FVIII-deficient plasma and 100 μL automated aPTT reagent (rabbit brain phospho lipid and micronized silica, Biomerieux, Durham, N.C.). After the addition of 100 μL 25 mM CaCl2 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.
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.
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