Patent Application
20130280162
The invention relates to inhibitors of the urokinase-type plasminogen activator receptor (uPAR). The generated inhibitors are bivalent uPAR-ligands containing the receptor binding domains of the extracellular protease urokinase-type plasminogen activator (uPA) and of the extracellular matrix protein vitronectin (VN), in different configurations, linked by a scaffold. The binding of such inhibitors to uPAR results in a complex where the binding sites for both uPA and VN are occupied contemporarily, thus efficiently blocking both the proteolytic and signaling activities of the receptor.
The urokinase plasminogen activator receptor (uPAR, also named CD87) is a membrane glycoprotein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Extensive in vitro, in vivo and clinical evidence suggests that uPAR plays important functions in wide range of pathological processes including tumor growth, invasion and metastasis, inflammatory diseases and viral infections. Drugs interfering effectively with uPAR-function may therefore provide novel therapeutical regimens in a variety of pathological conditions.
The molecular interaction between uPAR and its two well-established ligands, the serine protease urokinase (uPA) and the extracellular matrix (ECM) protein vitronectin (VN), is required for the activity of uPAR in the regulation extracellular proteolysis, cell adhesion, migration, invasion and proliferation (Blasi & Carmeliet, 2002; Smith & Marshall, 2010). Binding of uPA to uPAR promotes extracellular proteolysis by accelerating cell-surface plasminogen activation while the binding of VN enforces cell adhesion to the ECM enhancing migratory and proliferative signaling through integrin-dependent activation of the p130Cas and ERK1/2 signaling pathways. The importance of the interaction between uPA and uPAR in vivo has been intensively studied, and usually confirmed, using a variety of specific antagonists including antibodies, recombinant proteins, synthetic peptides as well as low molecular weight compounds. Although the importance of the interaction with VN is well documented to be crucial for the signaling activity of uPAR (Madsen et al, 2007; Smith et al, 2008), the importance of this interaction in vivo has never been addressed. In a xenograft mouse model of tumor growth using human cancer cells over-expressing different uPAR-variants the author has recently demonstrated that the interaction with VN is indeed required for the activity of uPAR in accelerating tumor growth (manuscript in preparation) supporting the assumption that this interaction is indeed a relevant anti-cancer target.
Several international applications disclose peptides ligand of urokinase receptor, such as WO01/17544. In particular, WO97/35969 discloses peptides that are capable of binding to uPAR and to inhibit the binding of an integrin and vitronectin. The document does not refer to uPA binding.
In addition, WO2008/073312 relates to urokinase-type plasminogen activator receptor epitope and monoclonal antibodies derived therefrom. The document discloses antibodies, and antigen-binding fragments thereof, specific for urokinase-type plasminogen activator receptor (uPAR) and their use for the treatment or prevention of cancer. In particular, the disclosed antibodies are specific for a particular epitope on uPAR.
WO 2005116077 identifies antibodies or other ligands specific for the binary uPA-uPAR complexes, for ternary complexes comprising uPA-uPAR and for complexes of uPAR and proteins other than uPA such as integrins. The antibodies inhibit the interaction of uPA and uPAR with additional molecules with which the complexed interact. Such antibodies or other ligands are used in diagnostic and therapeutic methods, particularly against cancer.
Tressler R J et al., (APMIS. 1999 January; 107(1):168-73) disclose urokinase receptor antagonists based on the growth factor domains of both human and murine urokinase. Such antagonists show sub-nanomolar affinities for their homologous receptors. Further modification of these molecules by preparing fusions with the constant region of human IgG has led to molecules with high affinities and long in vivo half-lives. Smaller peptide inhibitors have been obtained by a combination of bacteriophage display and peptide analogue synthesis. All of these molecules inhibit the binding of the growth factor domain of uPA to the uPA receptor and enhance binding of the uPA receptor to vitronectin.
To address the in vivo importance of the uPAR: VN-interaction in disease state, and to transfer this knowledge to the clinic, potent and specific drugs are required. The present invention describes the engineering, expression, purification and characterization of an uPAR:VN-antagonist that is about one thousand times more potent than the currently available inhibitors.
The present invention concerns the conception, construction and validation of a novel type of inhibitor of the urokinase-type plasminogen activator receptor (uPAR). The generated inhibitor molecules (named uPAR-lock and uPAR-lockV2, also uPAR-lock molecules) are a bivalent uPAR-ligands containing the receptor binding domains of the extracellular protease urokinase-type plasminogen activator (uPA) and of the extracellular matrix protein vitronectin (VN) positioned in close proximity on a common scaffold. Binding of such inhibitors to uPAR results in a complex where the binding sites for both uPA and VN are occupied contemporarily and efficiently, thus blocking both the proteolytic and signaling activities of the receptor.
The inhibitor molecule of the present invention represents a potent antagonist of the physical and functional uPAR/VN-interactions and uPAR/uPA interactions.
It is very advantageous in that it targets the uPA-binding pocket in uPAR without stimulating VN-binding to receptor. Indeed, most, if not all, known drugs binding to the uPA-binding pocket of uPAR are agonists of the uPAR:VN-interaction and therefore inducing signaling. By contrast, the molecule of the instant invention uPAR-lock blocks the signaling.
In addition, there is no species-specificity barrier for the use of uPAR-lock molecules A main problem in the evaluation of drugs against the uPA:uPAR interaction is that they display profound species-specificity making it difficult to test the compound reliably in xenograft models. The specificity of uPAR-lock can easily be “set” by changing the species origin of the effector domain(s).
The present antagonist molecule displays excellent drug-properties such as long in vivo half-life due to the Fc-tag. In addition, it is constituted of human sequences, thus it is non-immunogenic. uPAR-lock molecules were designed to function as a blocking agent. Its high affinity and specificity for uPAR and the presence of the Fc-moiety make the molecule suitable to be used for uPAR-targeted diagnosis and/or therapy by for instance conjugation with appropriate effector molecules such as radionuclide, toxins etc.
Thus, the present antagonist molecule has versatile clinical applications.
It is therefore the object of the present invention a dimeric molecule comprising two polypeptides selected from the group of:
Preferably, in the above dimeric molecule:
The dimeric molecule of the invention may be obtained by any means known in the art.
A derivative may be a polypeptide with a longer or shorter sequence, i.e. modified to be resistant to enzymes, etc. . . . .
In the dimeric molecule of the invention the GFD domain preferably consists essentially of aa. 8 to aa. 48 of SEQ ID No. 1, more preferably it consists of aa. 1 to aa. 48 of SEQ ID No. 1.
In the dimeric molecule of the invention the SMB domain preferably consists of aa. 1 to 41 of SEQ ID No. 2.
In a preferred embodiment the first and the second polypeptide:
More preferably, the SMB domain and the GFD domain are linked by a first linker peptide.
Said first linker peptide preferably consists essentially of the sequence of SEQ ID No. 3.
In the dimeric molecule of the invention each of first and second polypeptide are preferably linked to the molecular scaffold by means of a second linker peptide.
Said linker peptide preferably consists essentially of the sequence of SEQ ID No. 4.
The molecular scaffold of the dimeric molecule of the invention is preferably an immunoglobulin constant region (Fc), a leucine zipper, a chemical or a peptide linker.
In an embodiment of the invention each of heavy chain constant regions of Fc has essentially the sequence
In a preferred embodiment the dimeric molecule of the invention consists essentially of a first monomer of sequence of SEQ ID No. 6, and of a second monomer of sequence of SEQ ID No. 7.
In another preferred embodiment, the first and the second monomer of the dimeric molecule of the invention have the sequence of SEQ ID No. 8.
Another object of the invention is the above dimeric molecule for medical use, preferably for use as treatment of cancer.
In a preferred embodiment the dimeric molecule of the invention is conjugated to a therapeutic agent, wherein the therapeutic agent is preferably a radionuclide or a toxin.
A further object of the invention is the above dimeric molecule for use in a diagnostic method, preferably for use in the diagnosis of a uPAR-mediated pathology or tumor.
Other objects of the invention are a method of treatment of cancer comprising the administration to a subject in need thereof of a therapeutically effective amount of the dimeric molecule of the invention, a pharmaceutical composition comprising the dimeric molecule of the invention and appropriated diluents or excipients. Said pharmaceutical composition can further comprise another therapeutic agent, preferably a radionuclide or a toxin.
Another object of the invention is a kit for the diagnosis of a uPAR-mediated pathology or tumor comprising the dimeric molecule of the invention.
The invention will be now described by non limiting examples referring to the following figures:
The function of uPAR in extracellular proteolysis mediated by uPA-binding can be competitively inhibited by the receptor binding of the growth factor-like domain of uPA (GFD). The function of uPAR in signal transduction mediated by VN-binding can be competitively inhibited by the somatomedin B domain of VN (SMB). In the following D1, D2 and D3 are the uPAR domains.
(A) The GFD and SMB Domains as Competitive uPAR Antagonists
Blocking of uPAR-function using mixtures of isolated GFD and SMB requires two consecutive first-order inter-molecular binding reactions (1 and 2) and may follow two routes (A and B) depending on whether it is GFD or SMB that binds the receptor first. The overall stability of the ternary uPAR:GFD:SMB complex, and therefore of receptor inhibition, is limited by the weakest of the second binding reactions, which in this case is SMB-binding (KD=360 nM). Experimentally determined equilibrium constants for the discrete binding reactions, when known, are from Gårdsvoll et al. (Gardsvoll & Ploug, 2007).
(B) Forced Proximity Engineering of a Molecular Scaffold Containing Both the GFD and SMB Domains (Named “uPAR-Lock”)
When GFD and SMB are forced in close proximity by attachment to a common scaffold (uPAR-lock) receptor inhibition follows the same sequence of binding reactions. Importantly, however, the second binding reactions (A2 and B2) are now zero-order (i.e. concentration independent) intra-molecular reorganizations. In this scenario, the efficiency of receptor inhibition is thus only limited by the strongest of the initial binding reactions, which in this case is the binding of the GFD-moiety (expected KD ˜0.29 nM). Consequently, it is predicted that an uPAR-lock, made with a suitable scaffold, will be a far stronger (>1000-fold) antagonist of uPAR-function than mixtures of GFD and SMB.
(C) Scaffolds
Several different types of scaffolds can be envisaged for making an uPAR-lock. GFD and SMB molecules may be attached to the constant regions of immunoglobulin heavy chain (Fc) that form covalent dimers. The GFD and SMB domains may also be tagged with leucine zipper sequences modified to form hetero-dimers (Moll et al, 2001). Also it is possible that the GFD and SMB domains may be engineered into a single polypeptide using appropriate linker regions.
(A) Crystal structure of the ternary complex between uPAR (in grey), the receptor binding domain of uPA (GFD, in black) and the receptor binding domain of VN (SMB, in white). N-terminal residues of GFD and SMB (Gln2 and Pro8) and C-terminal residues of GFD, SMB and uPAR (Lys48, Pro41, Asp274) are indicated. Note that the C-terminal residues of GFD (Lys48) and of SMB (Pro41) are distant only 18.9 Å and have the same polarity pointing away from the receptor and away from the presumed membrane anchorage location highlighted by the C-terminal residue of uPAR (Asp274) in the structure.
(B) Crystal structure of a dimeric human IgG heavy-chain constant region (Fc)2. N-terminal residue (bottom figure, Pro238) and C-terminal residue (top figure Ser444). Note that the N-terminal residues of the two polypeptides are spaced similarly to the C-terminal residues of GFD and SMB bound to uPAR. Two residues (Tyr407 and Thr366, in black) that may be manipulated to favor hetero-dimerization (Ridgway et al, 1996) are shown in the right and left side of the figure. The structures are from protein database (PDB) entries 3BT2 and 1H3X and were elaborated with the MacPyMOL software.
96-well plates coated with soluble uPAR (5 nM) were incubated with serial dilutions of the conditioned medium from Phoenix cells co-transfected with GFD/FcK and SMB/FcH (i.e. uPAR-lock). Bound uPAR was detected using secondary reagents detecting human Fc. Total binding to uPAR coated wells (squares), non-specific binding to uncoated wells (triangles) and specific binding (circles) are shown.
(B) Cartoon of the Predicted uPAR-Lock Structure and the Appearance of the Purified Protein by SDS-PAGE.
uPAR-lock is a covalent (disulfide bonded) hetero-dimer between GFD/FcK (FcK=Fc “Knob”=Fe carrying a Thr366->Tyr substitution) and SMB/FcH (FcH=Fc “Hole”=Fc carrying a Tyr407->Thr substitution). 3 μg of purified protein was separated by 12% SDS-PAGE under non-reducing (left) and reducing (right) conditions and the gel stained with colloidal Comassie stain.
(A) Fc-Tagged uPAR Bind with High Affinity to Immobilized uPA.
Immobilized uPA was incubated with increasing concentrations of uPAR tagged with a mouse Fc (uPAR/mFc). After washing bound uPAR/mFc was quantified using a biotinylated anti-mouse Fc antibody and Europium labeled streptavidin. Note that uPAR/mFc display specific high-affinity binding to immobilized uPA.
(B) uPAR-Lock Inhibit uPAR Binding to Immobilized uPA.
Immobilized uPA was incubated with uPAR/mFc (1 nM) in the presence of increasing concentrations of uPAR-lock (diamonds) or uPA (circles). The amount of uPAR/mFc bound to the immobilized uPA was quantified as above. Note that both uPAR-lock and uPA are competitive antagonists of uPAR/mFc binding to immobilized uPA.
(C) High-Affinity Binding of uPAR to Immobilized VN Requires uPA.
Immobilized VN was incubated with uPAR/mFc (5 nM) mixed with increasing concentrations of uPA. After washing, bound uPAR/mFc was quantified as above. Note that uPA is required for uPAR/Fc binding to immobilized VN.
(D) uPAR-Lock Inhibit uPA-Induced uPAR Binding to VN.
Immobilized VN was co-incubated with uPAR/mFc (1 nM), uPA (5 nM) and increasing concentrations of uPAR-lock (diamonds) or uPA (circles). After washing bound uPAR/mFc was quantified as above. Note that uPAR-lock, in contrast to uPA, is an antagonist of uPAR binding to immobilized VN.
Mock-transfected and 293/uPART54A cells in the presence or absence of 50 nM uPAR lock were seeded (20.000/well) in 96-well E-plates coated with VN. Cell adhesion was quantified by measuring the impedance (cell index) every two minutes for 3 hours in a real time cell analyzer (RTCA) instrument. When basal cell adhesion (mediated by the αvβ5 integrin, (Madsen et al, 2007)) reached plateau the wells were added uPA to a final concentration of 10 nM to induce uPART54A binding to VN and the recordings continued. Note that uPAR-lock completely inhibits the uPA-induced increase of cell adhesion of 293/uPART54A cells (compare the curves after uPA-addition). uPAR-lock does not affect integrin mediated adhesion (compare curves before uPA-addition) and uPA does not modulate the adhesion of cells transfected with empty pcDNAS/FRT-TO vector (293/mock).
As described above uPAR-lock is hetero-dimer between Fc-tagged GFD and SMB domains. The GFD/GFD and SMB/SMB hybrids are constructed in the same way as uPAR-lock but has either GFD or SMB domains on both polypeptides. For some experiments a mixture of GFD/GFD and SMB/SMB were used as this has the same domain composition as uPAR-lock but with the GFD and SMB domains located on separate scaffolds.
(B) Physical appearance of uPAR-lock and control proteins. Three micrograms of the purified proteins was separated by 10% SDS-PAGE under non-reducing (left) and reducing (right) conditions and the gel stained with colloidal Comassie stain.
(C) Increasing concentrations of uPAR-lock (circles), GFD/GFD (squares), SMB/SMB (triangles tip up) and GFD/GFD+SMB/SMB (triangles tip down) were allowed to bind to immobilized uPAR. Bound protein was detected using secondary reagents detecting human Fc. Note that the strongest binding is seen with uPAR-lock.
(D) Inhibition of uPAR/mFc binding to immobilized uPA by uPAR-lock and control proteins. Immobilized uPA was incubated with mixtures of uPAR/mFc (1 nM) and increasing concentrations of uPAR-lock and bound uPAR/mFc quantified using the assay described in
293/uPAR (A) and 293/uPART54A (B) cells (20.000/well) were seeded in a VN-coated 96-well E-plate in the absence (black) or presence of uPAR-lock (red), GFD/GFD (yellow), SMB/SMB or GFD/GFD+SMB/SMB (blue) and allowed to adhere. Two hours after seeding wells were added uPA to 10 nM and cell adhesion measurements continued for another two hours. Note that uPAR-lock inhibit uPA-independent uPAR-mediated cell adhesion to VN (compare red and black curves before uPA-addition in panel 7A) as well as uPA-induced adhesion (compare red and black curves after uPA-addition in panels 7A and 7B). In contrast both GFD/GFD and GFD/GFD+SMB/SMB are strong agonists of uPAR-mediated VN adhesion (compare yellow and green with black curves before uPA-addition in panel 7A and 7B). SMB/SMB is largely inactive.
293/uPAR cells were seeded in 12-well plates in complete serum containing medium and allowed to adhere over night. The next day the medium was replaced with complete medium without (left) or containing uPAR-lock (center) or GFD/GFD+SMB/SMB (both at 20 nM, right) and transferred to a time-lapse microscope. Random cell migration was recorded as previously described (Madsen et al, 2007) and quantified by manual cell tracking using the software ImageJ. Each dot represents a single cell. Mean migration speeds (+/−95% confidence intervals) are shown. The data were analyzed by non-parametrical analysis and corrected for multiple comparisons (*** P<0.001 **P<0.01 and ns P>0.05). Note that uPAR-lock dramatically inhibits the migration of 293/uPAR cells.
(A) Cartoon illustrating the structure of the homodimeric uPAR-lock variants containing GFD and SMB domains within a single polypeptide chain. As described above, uPAR-lock is a disulfide-linked heterodimer with the GFD and SMB located on two different polypeptides and tagged with human Fc constant regions containing Knob and hole mutations to favor heterodimerization. In contrast, GFD-SMB/mFc and SMB-GFD/mFc are homodimers containing both the SMB and GFD domains on the same polypeptide chain. (B) 293 cells expressing human uPAR were seeded in an 96-well E-plates coated with VN and transferred to a real time cell analyzer instrument (RTCA, xCELLigence, SP Roche Corp.). The electric impedance (termed cell index, CI), a measure of cell adhesion, was recorded at regular intervals. When the CI reached a plateau, indicative of complete cell adhesion, the wells were added a 3-fold dilution curve of SMB-GFD/mFc to the final concentrations indicated in the graph. The E-plate was returned to the instruments and the impedance measurements continued at regular intervals. The time point at which the inhibitor were added (T=0) and the time point (T=1 h) used for calculation of dose response curves shown in panel C are indicated by stippled vertical lines. The curves show the normalized cell index (NCI, Y-axis) as a function of time (X-axis). All cell indexes were normalized to the cell index measured immediately prior to inhibitor addition. (C) To determine IC50 values, the NCI measured one hour after reagent addition were calculated in % of the NCI for vehicle treated cells at the same time point (ΔNCI, Y-axis) and graphed in function of inhibitor concentration (X-axis). Sigmoidal dose response curves (variable slope) were fitted using the Prism 5 software suite and their derived IC50 values are indicated.
(A) Cartoon illustrating the structure of the heterodimeric uPAR-lock and homodimeric uPAR-lockV2, as well as variant of these carrying a single amino acid substitution (D22A) in the SMB domain that impairs the interaction of this domain with uPAR (Okumura Y, et al. J Biol Chem 2002). uPAR-lockV2 is identical to the SMB-GFD/mFc shown in
Effect of uPAR-lockV2 on prostate cancer growth in vivo. Male Balb C nu/nu mice were inoculated with (1×106) PC-3 cells through the s.c. route. Animals were treated with vehicle (PBS), 10.0 mg/kg of control mouse immunoglobulin or uPAR-lockV2 through the i.p. route. Tumors were measured twice weekly, and tumor volume was determined as described in Materials and Methods. Significant differences from control are represented by asterisks (*P<0.05, **P<0.01 and ***P<0.001).
Male athymic nu/nu mice were inoculated subcutaneously with PC-3 cells and treated by bi-weekly injections with PBS or 10.0 mg/kg of uPAR-lockV2 via intraperitoneal route. Eight weeks after xenografting, the tumors were harvested and subjected to immunohistochemical analysis (Panel A) as described in the materials and methods section. Ki-67 and Caspase-3 stainings are shown and nuclei are counterstained with Dapi. Quantification of the data is shown in Panel B.
Mice carrying PC-3 tumors were injected with Alexa488 labeled uPAR-lockV2 or Alexa488 labeled mouse IgG and 24 hours later tumors were excised and analyzed by fluorescent microscopy. Evident areas of fluorescence can be observed in tumors from animals injected with labeled uPAR-lockV2 (outlined areas in right panel) while similar areas are not observed in tumors from mice treated with labeled mouse IgG. Representative micrographs are shown.
Amino acid sequence of human uPA with the signal peptide (Met−20-Gly−1) in cursive, the mature protein (Ser1-Leu411) in bold and the uPAR-binding growth factor-like domain (GFD, Ser1-Lys48) in bold/underlined:
MRALLARLLLCVLVVSDSKG
SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHC
EIDKSK
TCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNY
CRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGKKPSSPPEELKFQCGQKTLRPRFKIIG
GEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSR
LNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYN
DPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQ
WKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKE
ENGLAL
Amino acid sequence human VN with the signal peptide (Met−19-Ala−1) in cursive, the mature protein (Asp1-Leu459) in bold and the uPAR-binding somatomedin-B domain (SMB, Asp1-Pro41) bold/underlined:
MAPLRPLLILALLAWVALA
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKP
QV
TRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPVLKPEEE
APAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFRGQYCYEL
DEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGVLDPDYPRNIS
DGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEECEGSSLSAVFEHF
AMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPS
LAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGANNYDDYRMD
WLVPATCEPIQSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL
Amino acid sequence of human uPAR with the signal peptide (Met−22-Gly−1) in cursive, a C-terminal peptide (Ala284-Thr313) removed during synthesis upon addition of the glycolipid membrane anchor attached to Gly283 in cursive and underlined, and the mature protein (Leu1-Gly283) in bold:
MGHPPLLPLLLLLHTCVPASWG
LRCMQCKTNGDCRVEECALGQDLCRTTIVRLWEEGEELEL
VEKSCTHSEKTNRTLSYRTGLKITSLTEVVCGLDLCNQGNSGRAVTYSRSRYLECISCGSSD
MSCERGRHQSLQCRSPEEQCLDVVTHWIQEGEEGRPKDDRHLRGCGYLPGCPGSNGFHNN
DTFHFLKCCNTTKCNEGPILELENLPQNGRQCYSCKGNSTHGCSSEETFLIDCRGPMNQCLV
ATGTHEPKNQSYMVRGCATASMCQHAHLGDAFSMNHIDVSCCTKSGCNHPDLDVQYRSG
AAPQPGPAHLSLTITLLMTARLWGGTLLWT
The expression vectors for Fc-tagged SMB and GFD are based on the pFRT/TO-Fc plasmid (Madsen et al, 2007) however a number of modifications were introduced to facilitate the shuffling of different coding regions as well as to improve protein yield and allow for the removal of the Fc-tag from the recombinant proteins by specific protease cleavage. Firstly, an XhoI restriction site located in the vector sequence downstream of the Fc coding region was destroyed by site-directed mutagenesis using oligos dXu/dXd. Secondly, a linker encoding a cleavage sequence for the PreScission protease made by annealing oligos PreF/PreR was inserted in the XhoI site located at the signal peptide/Fc junction. To remove the introns present in the Fc region of the construct, which was found to increase the yield of recombinant protein, the vector was transfected into CHO cells, RNA extracted, reverse transcribed, and the cDNA amplified with oligos hVNukpn/FcNr. The PCR product was digested Kpn1/NotI and used to replace the corresponding fragment of the parental vector generating pFRT/TO-Fc. For transient protein expression the Fc cassette was transferred KpnI/NotI to the pEGFP-N1 vector (Clontech Inc.) generating pN1-Fc. Knob and hole mutations (T366Y and Y407T, (Ridgway et al, 1996)) were introduced in the Fc regions by site-directed mutagenesis using oligo pairs FcKnobF/FcKnobR and FcHoleF/FcHoleR yielding vectors pN1-FcK and pN1-FcH, respectively. Sequences encoding the signal peptide (the negative amino acids refers to signal peptide sequence) and SMB domain of VN (Met−19 to Pro41, MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC KP (aa 1-60 of SEQ ID No. 10), signal peptide in cursive were generated by amplifying a VN cDNA with oligos hVNukpn/SMBRV2 and cloned KpnI/XhoI in pN1-FcK and pN1-FcH. A sequence encoding the signal peptide and GFD domain of uPA (amino acids Met−20 to Lys48, MRALLARLLLCVLVVSDSKGSNELHQVPSNCDCLNGGTCVSNKYF SNIHWCNCPKKFG GQHCEIDKSK (aa 1-68 of SEQ ID No. 9), signal peptide in cursive was generated by amplification of a human uPA cDNA using oligos ATFkpnF/GFDRV and cloned as described for the SMB. This cloning strategy generates mature fusion proteins composed of GFD (residues 1-48, (SEQ ID No. 1)) or SMB (residues 1-41, (SEQ ID No. 2)) connected to hinge and Fc regions of a human IgG1 by a GSGLELEVLFQGPIE (SEQ ID No. 4) linker. The expression vector for Fc-tagged uPAR was generated by amplification of a human uPAR cDNA with oligos URfSK/uPARXd and cloning the product KpnI/XhoI in pFRT/TO-Fc. The expression vector for uPAR tagged with a murine immunoglobulin heavy chain constant region (mFc) was generated by assembling (uPAR cDNA, amplified URfSK/UpreR2D, digested KpnI/XhoI) and an IgG1 cDNA (clone IRAVp968B035D, obtained from imaGenes GmbH, amplified mFcU/mFcD, digested XhoI/NotI) in pEGFP-N1 (digested KpnI/XhoI). The resulting mature chimeric protein (uPAR/mFc) is composed of human uPAR residues 1-277, a LEVLFQGPLEAGAG (SEQ ID No. 36) linker and amino acids 216-441 of the mouse immunoglobulin heavy chain (numbered according to (Adetugbo, 1978)). The expected sequence of all coding regions was confirmed by sequencing.
Composition of uPAR-Lock
The composition of uPAR-lock constructed as described above is a molecule, in particular a disulphide-linked heterodimer composed of the two polypeptides, GFD/FcK and SMB/FcH, with the following amino acid composition (N to C-terminal, IUPAC):
SNELHQVPSN
CDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHC
EIDKSK
GSGLELE
VLFQGPIE
In bold: Human uPA (amino acids 1-48)* (corresponding to SEQ ID No. 1),
in cursive: Artificial linker region ** and
in bold cursive: Human immunoglobulin (IgG) hinge and constant region (Fc) containing the “Knob” substitution (Y407T, underlined)***
*Shorter and longer pieces of uPA may also work. A sequence comprising amino acids 11-42 (underlined) may represent the minimal functional sequence.
**Different length and sequences of the linker regions may work equally well or better. The minimal length may be zero (i.e. no linker).
***The “Knob” substitution is utilized in this study to increase the quantity of heterodimers formed during expression but is not predicted to have any effect on the inhibitory quality of the heterodimer.
DQES
CKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAEC
KP
GSGLELEVLFQGPIE
.
In bold: Human VN (amino acids 141)* (corresponding to SEQ ID No. 2),
in cursive: Artificial linker region** and
in bold cursive Human immunoglobulin (IgG) hinge and constant region (Fc) containing the “Hole” substitution (T366Y, underlined)***
*Shorter and longer pieces of VN may also work. A sequence comprising amino acids 5-39 (underlined) may represent the minimal functional sequence.
**Different length and sequences of the linker regions may work equally well or better. The minimal length may be zero (i.e. no linker).
***The “Hole” substitution is utilized in this study to increase the quantity of heterodimers formed during expression but is not predicted to have any effect on the inhibitory quality of the heterodimer.
Phoenix cells (semi-confluent 10 cm plates) were washed once with pre-warmed serum-free DMEM and added 8 ml of OptiMEM (Invitrogen). For uPAR-lock (i.e. GFD/SMB-FcK/H) cultures were transfected with a mixture of pN1-GFD/FcK and pN1-SMB/FcH (3+3 μg/10 plate) using the Fugene transfection reagent (Roche) according to the manufactures instructions. The control proteins GFD/GFD and SMB/SMB were expressed by co-transfecting pN1-GFD/hFcK+pN1-GFD/hFcH and pN1-SMB/hFcK+pN1-SMB/hFcH, respectively. The transfected cultures were left for 6-8 days after which supernatants were collected and filtered (0.45 μm). Proteins were purified on Protein A Sepharose, eluted using 0.1 M Glycine pH 2.8, 0.5 M NaCl and dialyzed extensively against PBS.
96-well immuno plates (NUNC MaxiSorb, blackwell) were coated with pro-uPA or VN (100 μl, 10 nM) diluted in coating buffer (50 mM sodium carbonate, pH 9.6) at 4° C. ON. Plates were washed with wash buffer (phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T)) and non-specific binding sites blocked (0.15 ml/well) with blocking buffer (PBS containing 2% bovine serum albumin (BSA)) for 1-2 hour at RT. After washing with PBS-T, wells were incubated with uPAR/mFc (1 nM) in the presence or absence of the agonists and antagonists to be tested prepared in dilution buffer (PBS containing 1% BSA). The binding was allowed to occur for 1-2 hours at RT and the plates washed three times with wash buffer. Bound uPAR/mFc was detected by sequential incubations with a biotinylated goat anti-mouse Fc antibody (Sigma) and Eu3+ labeled streptavidin (Perkin Elmer). Bound Eu3+ was quantified by dissociation-enhanced time-resolved fluorescence measurement using an Envision Xcite plate reader (Perkin Elmer) using the DELFIA protocol.
The 293/uPAR, 293/uPART54A and 293/mock cell lines were generated by stable transfection of HEK293 Flp-In T-REx cells (Invitrogen Corp.) with the pcDNAS/FRT-TO expression vector containing a wild-type human uPAR cDNA (293/uPAR), an uPAR cDNA containing the Thr54Ala substitution (293/uPART54A) or the empty expression vector (293/mock) as described in detail previously (Madsen et al. 2007).
Cells were seeded in the wells of E-Plates (Roche) and cell adhesion was monitored at regular intervals using a real time cell analyzer (RTCA, xCELLigence SP, Roche). The RTCA system measures the electrical impedance across interdigitated microelectrodes integrated on the bottom of tissue culture 96-well E-Plates. The presence of the cells on top of the electrodes affect the local ionic environment at the electrode/solution interface, leading to an increase in the electrode impedance. The stronger the cell adhesion the larger the increase in electrode impedance.
Time-lapse live-cell imaging was performed at 37° C., 5% CO2 with an inverted Olympus IX80 microscope equipped with an incubation chamber (OKOlab) to control CO2 and temperature. Cells were plated in 12 well plates (Nunc) at the confluence of 1×105 cell/well. Time-lapse imaging was performed in serum-containing growth medium. Cells were viewed through 10× (uPlan FLN 10× Ph1, N.A. 0.30; Olympus) objective lenses and pictures were taken every 5 minutes for 5 h. The acquisition system includes a digital camera (Hamamatsu Orca-ER) and System Control Software Olympus ScanR. Adjustment of brightness/contrast, smoothening and sharpness of images was done using ImageJ 1.42q and always applied to the entire image. Cell migration speed was quantified with ImageJ 1.42q using the plug-in “manual tracking”. In the experiment randomly chosen cells were tracked and their average migration speed throughout the experiment calculated.
The expression vectors for recombinant proteins tagged with a mouse IgG constant region (pFRT/TO-mFc) was generated by replacing (Xho1/Not1) the human Fc region of pFRT/TO-Fc with the mouse Fc region taken from the uPAR/mFc expression vector. To generate the GFD-SMB chimera a human uPA cDNA was amplified with oligos ATFkpnF/GLINKR and a human VN cDNA with oligos SLINKF/SMBRV2. The two PCR products were purified and co-amplified with oligos ATFkpnF/SMBRV2. To generate the SMB-GFD chimera a human VN cDNA was amplified with oligos hVnUkpn/SLINKR and a human uPA cDNA with oligos GLINKF/GFDRV. The two PCR products were purified and co-amplified with oligos VnUkpn/GFDRV. The GFD-SMB and SMB-GFD chimeras were cloned Kpn1/Xho1 into pFRT/TO-mFc to generate expression vectors encoding GFD-SMB/mFc and SMB-GFD/mFc. The expression vector encoding the SMB-GFD chimera tagged with a human Fc (SMB-GFD/Fc, uPAR-lockV2), was generated by cloning the SMB-GFD chimera Kpn1/Xho1 into pFRT/TO-Fc.
The pFRT/TO-GFD-SMB/mFc, pFRT/TO-SMB-GFD/mFc and pFRT/TO-SMB-GFD/Fc expression vectors were transfected into CHO Flp-In cells (Invitrogen Corp.) and the recombinant proteins expressed under serum-free conditions as previously described (Madsen et al., JCB 2007). The recombinant chimeras were purified from the conditioned media by standard Protein A affinity chromatography and dialyzed extensively against PBS.
Six-week-old male Balb C nu/nu mice were obtained from Charles River. Before inoculation, PC-3 cells growing in serum-containing medium were washed with phosphate buffered saline (PBS), harvested by trypsinization, and pelleted at 1200 rpm for 7 minutes. Cell (1.0×106) were resuspended in 200 μl of PBS with 20% Matrigel. Animals were anesthetized by intraperitoneal (i.p) injection of Avertin and 1.0×106 cells were inoculated subcutaneously (s.c.) using a 26-gauge needle into the right flank of anesthetized mice. 5 days after xenografting the animals were randomized into 2 control groups, where animals were treated twice a week i.p. with vehicle (n=5, PBS) non-immune mouse IgG1 (n=5, 10 mg/kg), and an experimental group, where animals were treated with uPAR-lock V2 (n=5, 10 mg/kg). The animals were monitored twice a week for 7 weeks for tumor development and growth. Tumor volume was determined according to the formula: tumor volume=shorter diameter2×longer diameter/2. Two mice, that did not develop palpable tumors, (one from the IgG control group and one from uPAR-lock V2 group) were excluded from the analysis. There was no significant difference between tumor growth in PBS and IgG treated animals (data not shown) and the data from these mice were pooled (n=9) for the comparison with the experimental uPAR-lock V2 group (n=4). Results were analyzed as the mean±SE, and comparisons of the experimental data were analyzed by unpaired, two-tailed, equal variance, t-test.
Composition of GFD-SMB/mFc, SMB-GFD/mFc and SMB-GFD/hFc (uPAR-LockV2)
The composition of the above constructs is a molecule, in particular a disulphide-linked homodimer composed of the two polypeptides GFD-SMB/mFc or SMB-GFD/mFc or SMB-GFD/hFc, with the following amino acid composition (N to C-terminal, IUPAC):
GSGGGG
QESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKP
GSGLEAGA
G
PRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDV
EVHTAQTKPREEQFNSTERSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRP
KAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGS
YFVYSKLNYQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.
GFD-SMB/mFc is composed of residues 1-49 of human uPA (GFD, plain text), a GGGGSGGGGSGGGG (SEQ ID No. 3) linker (underlined), residues 2-41 of human VN (SMB, in bold), a GSGLEAGAG (aa 104-112 of SEQ ID No. 34) linker (underlined cursive) and the heavy chain constant region from a mouse immunoglobulin (mFc, cursive).
SNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSK
GSGLEAGAG
PRDCGC
KPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQT
KPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTI
PPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLN
VQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.
SMB-GFD/mFc is composed of residues 1-41 of human VN (SMB, plain text), a GGGGSGGGGSGGGG (SEQ ID No. 3) linker (underlined), residues 8-48 of human uPA (GFD, in bold), a GSGLEAGAG (aa 104-112 of SEQ ID No. 34) linker (underlined cursive) and the heavy chain constant region from a mouse immunoglobulin (mFc, cursive).
SMB-GFD/hFc (uPAR-LockV2)
KSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
GFD-SMB/hFc (uPAR-lockV2) is composed of residues 1-41 of human VN* (SMB, plain text) (corresponding to SEQ ID No. 2), a GGGGSGGGGSGGGG (SEQ ID No. 3) linker **(underlined), residues 8-48 of human uPA***(GFD, in bold) (corresponding to aa 8-48 of SEQ ID No. 1), a GSGLELEVLFQGPIE (SEQ ID No. 4) linker **(underlined cursive) and the heavy chain constant region from a human immunoglobulin (hFc, cursive).
*Shorter and longer pieces of VN may also work. The sequence consisting of amino acids 5-39 (grey shadow) may represent the minimal functional sequence.
**Different length and sequences of the linker regions may work equally well or better. The minimal length may be zero (i.e. no linker).
***Shorter and longer pieces of uPA may also work. A sequence comprising amino acids 11-42 (bold and underlined) may represent the minimal functional sequence.
For immunohistochemical analysis, primary tumors were excised, fixed in 4% paraformaldehyde (Formalin) and embedded in optimal cutting temperature (OCT) resin (Killik, BIO-OPTICA). Tissue blocks were sectioned at 8 μm and mounted onto positively charged glass slides for immuno-staining. For Ki-67 staining, sections were incubated with acetone at 4° C. for 1 minute. Slides were washed with PBS followed by blocking in pre-incubation buffer (PBS with 6% BSA and 10% FBS) for 1 h at RT. Slides were incubated with Ki-67 antibody (diluted 1:500) overnight at 4° C. followed by washing with PBS. For detection anti-rabbit Cy3 (1:200) and DAPI (1:2500) were used. Slides were mounted with Vectamount AQ. For detection of apoptotic cells, sections were incubated with 80% ethanol at room temperature for 1 minute. Slides were washed with PBS followed by blocking in pre-incubation buffer for 1 h at RT. Primary antibody (Cleaved caspase-3, 1:200) incubation was done overnight at 4° C. followed by washing with PBS. Detection was done as for Ki-67 stained slides. For quantification of cell proliferation and apoptosis, a total of 24 sections per animal were analyzed at 10× magnification, respectively. Data are shown as the average number of positive cells per field.
For tumor imaging experiments, uPAR-lockV2 and mouse IgG were labeled with Alexa488 dye according to the manufactures instructions. Mice carrying PC-3 tumors (8 weeks post xenografting) were injected with 50 μg labeled protein via intraperitoneal route and the tumors harvested 24 hours later. Tumor tissues were processed as described for immunohistochemical analysis.
Rationale of uPAR-Lock.
The growth factor-like domains of uPA (GFD) and the somatomedin B domain of VN (SMB) contains all the uPAR-binding determinants of the intact molecules, but lack their biological activity in extracellular proteolysis and cell signaling. Consequently, these domains are specific competitive antagonists of the uPAR:uPA and uPAR:VN interactions, respectively. Complete inhibition of both the uPA and VN mediated biological activities of uPAR requires the occupancy of both binding sites on the receptor and this may be achieved using the isolated GFD and SMB domains as illustrated in
To significantly improve the efficacy of using GFD and SMB to block uPAR-function, the author hypothesized that forcing proximity between the two domains by attachment to a common scaffold may result in a compound (named uPAR-lock) with vastly improved inhibitory properties as compare to the isolated domains (
The GFD/SMB-scaffold may be generated in different ways as shown in
Construction of an uPAR-Lock
Inspection of the crystal structure of the ternary complex between uPAR, the aminoterminal fragment of uPA (ATF) and the somatomedin B domain of VN (Huai et al, 2008) reveals that the peptide backbone of uPA and VN are closely located at some positions. In particular Lys48 in uPA and Pro41 in VN are only distant about 19 Å (
To join GFD and SMB onto a common scaffold the author choose the constant region (Fc) of human IgG as these form stable dimers with the two N-termini located in proximity (
To favor hetero-dimerization between the Fc-tagged GFD and SMB the author modified the Fc-regions to carry either “knob” (T366Y, FcK) or “hole” (Y407T, FcH) substitutions as described previously (Ridgway et al, 1996). Co-expression in mammalian cells of the chimeric proteins GFD/FcK and SMB/FcH results in the predominant (>90%) production of GFD/FcK-SMB/FcH hetero-dimers (i.e. uPAR-lock) as well as minor quantities (total <10%) of the (GFD/FcK)2 and (SMB/FcH)2 homo-dimers and GFD/FcK and SMB/FcH monomers.
uPAR-Lock Binds uPAR Specifically
To confirm the receptor binding activity of uPAR-lock, immobilized soluble uPAR were incubated with a dilution series of conditioned medium of Phoenix cells co-transfected with equal amounts of GFD/FcK and SMB/FcH expression vectors. After washing, bound uPAR-lock was detected using sequential incubations with a biotinylated anti-human Fc antibody and Eu-labeled streptavidin. As shown in
Purification of uPAR-Lock
To better characterize uPAR-lock the protein was purified from the conditioned medium of transfected cells by standard Protein A affinity chromatography. When analyzed by SDS-PAGE (
uPAR-Lock is a Competitive Antagonist of the uPA/uPAR-Interaction
To test the activity of uPAR-lock as an antagonist of the uPAR:uPA interaction the author utilized a binding assay in which purified uPAR tagged with a mouse Fc (uPAR/mFc) is allowed to bind to immobilized uPA (
uPAR-Lock is a Potent Antagonist of the uPAR/VN-Interaction
To test the activity of uPAR-lock as an antagonist of the uPAR:VN-interaction the author utilized a binding assay in which uPAR/mFc is allowed to bind immobilized VN in the presence of uPA (
uPAR-Lock is a Potent Functional Inhibitor of uPAR-Mediated Cell Adhesion to VN
To evaluate the activity of uPAR-lock in inhibiting uPAR-signaling in live cells the author quantified cell adhesion to VN coated wells by impedance measurements using a real-time cell analyzer (RTCA, xCELLigence SP, Roche) (
Forced Proximity Between GFD and SMB Contributes to the Activity of uPAR-Lock in Antagonizing the uPA: uPAR-Interaction
The rationale behind the construction of uPAR-lock predicts that the forced proximity generated between the GFD and SMB domains by attachment to a common scaffold is essential to its potent antagonistic activity. To address this prediction experimentally, the author constructed variants of uPAR-lock having identical scaffolds but carrying either two GFD domains (named GFD/GFD) or two SMB domains (named SMB/SMB) as shown in
Forced Proximity Between GFD and SMB is Essential for the Functional Antagonistic Activity of uPAR-Lock on uPAR-Mediated Cell Adhesion
To determine the importance of close proximity between GFD and SMB for the inhibitory activity of uPAR-lock on uPAR-mediated cell adhesion to VN, the author measured the changes in impedance in the process of 293/uPAR (
Forced Proximity Between GFD and SMB is Essential for the Functional Antagonistic Activity of uPAR-Lock on uPAR-Mediated Cell Migration
Downstream of cell adhesion, the expression of uPAR in 293 cells also stimulates random cell migration on serum-coated surfaces (Madsen et al, 2007). Consistently, the author found that uPAR-lock highly significantly inhibits basal migration of 293/uPAR cells (
uPAR-Lock V2
Variants of uPAR-lock containing the SMB and GFD domains in a single polypeptide may possibly be generated and are likely to have several advantages. Firstly, the manufacture of these is less complicated as only a single polypeptide has to be expressed. Secondly, the number of SMB and GFD domains is always equal when present in a single polypeptide chain preventing the formation of undesired, non-inhibitory and/or agonistic variants like the SMB/Fc and GFD/Fc which will be present in low levels in uPAR-lock preparations requiring heterodimerization.
With this goal in mind, we constructed chimeras in which the SMB and GFD domains are engineered into a single polypeptide chain and tagged with a C-terminal mouse Fc tag. Two variants were made. The first (SMB-GFD/mFc) is composed of an N-terminal SMB domain, a linker, the GFD domain and a C-terminal mouse immunoglobulin constant region (mFc). The second (GFD-SMB/mFc), has a similar organization but the relative positions of the SMB and GFD domains are inverted. A cartoon illustrating the structures of these molecules is shown in
As evident from the experiment shown in
Together these data show that uPAR-lockV2 is superior to uPAR-lock
Inhibition of uPAR-function by uPAR-lock and uPAR-lockV2 is predicted to require the contemporary binding of both the SMB and GFD domains to the receptor and this was largely documented for uPAR-lock in
uPAR-Lock V2 Inhibits Tumor Growth In Vivo
To determine the potential anti-tumor activity of uPAR-lock in vivo we conducted studies using a prostate cancer xenograft model. In this model, one million PC3 cells were inoculated in the right flank of male Balb C nu/nu mice through subcutaneous route. The xenografted animals were treated bi-weekly with uPAR-lockV2 by intraperitoneal injections and the volume of the tumors monitored by calibration. As shown in
Reduced Cell Proliferation and Enhanced Apoptosis in PC-3 Tumors of Animals Treated with uPAR-Lock
To investigate the biological reason for the reduced PC-3 tumor growth in animals treated with uPAR-lock we conducted immunohistochemistry analysis of sections of tumors taken from animals 8 weeks after xenografting (
Tumor Imaging and Drug-Delivery Using uPAR-Lock
In addition to its direct activity in reducing cancer growth, uPAR-lock may potentially also be used for the imaging tumors and/or as a drug delivery vehicle. To address this possibility directly, we xenografted mice with PC-3 cells and allowed the primary tumors to grow for 8 weeks without any pharmacological intervention. We then injected tumor-bearing animals with 50 micrograms of Alexa488-labeled uPAR-lockV2 or Alexa488-labeled mouse IgG via intraperitoneal route. Twenty-four hours after the injection of labeled proteins the tumors were excised, fixed, embedded, sectioned and inspected by microscopy for the presence of green fluorescence signal in the tumor tissue. As shown in
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2011/073577 | 12/21/2011 | WO | 00 | 7/3/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61426236 | Dec 2010 | US |
