Patent Application
20150098933
Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters or triglycerides that are internalized into the lysosome via low density lipoprotein (LDL) particles receptor-mediated endocytosis. Defects in LAL lead to a condition known as LAL deficiency characterized by the massive accumulation of cholesteryl esters (CE) and triglycerides (TG) in vital tissues of affected individuals. Wolman disease, a severe form of the LAL deficiency, typically leads mortality within one year of age due to complications associated with the massive accumulation of lipids in vital organs. Cholesteryl ester storage disease (CESD), a late onset LAL deficiency, can be identified in various stages of life with varying degrees of severity.
Human LAL (hLAL, EC 3.1.1.13) is encoded by the gene known as LIPA (NCBI Accession no. U08464.1) which contains nine coding exons localized in chromosome 10. The unprocessed full-length human LAL with its native signal peptide contains 399 amino acid residues. Allelic variations of human LIPA have been characterized and missense as well as nonsense and deletion mutations linked to WD and CESD have been identified (see, e.g., Lugowska et al., Lysosomal Acid Lipase Deficiency: Wolman Disease and Cholesteryl Ester Storage Disease, Lysosomal Storage Diseases (2012) Vol. 10:1-8).
Molecular and biochemical analyses of the LAL protein have suggested that two forms of human LAL may exist in human liver. A shorter form (41 kDa) of naturally occurring human LAL was detected along with a full-length hLAL (56 kDa) in human liver extracts (Ameis, D. et al., Eur. J. Biochem. 219:905-914, 1994). However, subsequent attempts to express and isolate the short form of LAL in vitro have consistently failed due to unknown biological mechanisms associated with post-translational modification of LAL (see, Zschenker et al. (2004)). Zschenker et al. (2004) concluded that the N-terminal region of LAL is essential for protein folding, stability, secretion and enzyme activity.
Enzyme replacement therapy for the treatment of LAL deficiency is currently being investigated as a viable treatment. Therefore, a need exists to provide various active forms of recombinant human LAL that could potentially minimize dosing amount and frequency and enhance the quality of life of patients with LAL deficiency.
The present invention is based on the discovery that while the N-terminal region of rhLAL is needed for proper expression and processing of the protein, it is not required for enzyme activity. A full-length rhLAL appears to undergo a post-translational modification (i.e., proteolytic cleavage) resulting in production of truncated hLAL protein missing the N-terminal portion. It is unexpected and surprising to discover that N-terminal truncated form of rhLAL (hereinafter, “TLAL”) having 323-371 amino acid residues of C-terminal portion exhibit equivalent or higher enzyme activity as compared to the full-length protein.
When recombinant human lysosomal acid lipase (rhLAL) is produced in mammalian cells, the cell culture medium contains not only LAL protein of the expected size (apparent molecular weight of 56 kDa by SDS polyacrylamide gel electrophoresis), but also a lower molecular weight (truncated) form (apparent molecular weight of 41 kDa on SDS-PAGE). Subsequent analyses revealed that this truncated rhLAL contains 324 amino acid residues of C-terminal portion of LAL and occurs by proteolysis of the 56 kDa LAL protein. TLALs have an N-terminal truncation up to 50, 60 or 70 amino acid residues.
Direct expression of a 41 kDA TLAL in mammalian cell line did not produce the protein above detectable ranges, suggesting the nascent LAL protein missing the N-terminal portion is rapidly degraded in the endoplasmic reticulum (ER), whereas inclusion of an N-terminal sequence enables the rhLAL to enter into the secretory pathway. However, production of the truncated form of rhLAL (TLAL) by proteolytic cleavage of the full-length rhLAL led to the discovery that the 41 kDa recombinant TLAL protein retained LAL activity, that is at least 50% or even higher than of that observed in the full-length 56 kDa rhLAL.
Accordingly, TLAL described herein is a recombinant protein that contains an N-terminal truncation from full-length LAL and retains enzymatic activity. In some aspects, TLAL exhibits enzyme activity at least 50%, 60%, 70%, 80%, 90% or 100% of the activity observed in a full-length rhLAL. In some aspects, TLAL has enzyme activity levels equivalent to or higher than that of a full-length LAL. The present invention provides a composition comprising an isolated TLAL, a mixture of the isolated rhLAL comprising a TLAL and at least one other form of rhLAL (e.g., full-length rhLAL or another TLAL). The present invention also provides a method of purifying TLAL from an isolated mixture comprising TLAL and at least one other form of rhLAL. The present invention also includes methods of purifying away a TLAL from an isolated mixture of rhLAL proteins. The present invention provides a pharmaceutical composition comprising TLAL and pharmaceutically acceptable carrier or excipient. Also included are methods of increasing LAL activity by N-terminal truncation of a full-length rhLAL and methods of producing a truncated rhLAL in mammalian cell culture.
In one aspect, the invention provides a composition comprising an isolated recombinant truncated lysosomal acid lipase (TLAL). In one embodiment, the TLAL has at least 90%, 95% or 99% identity to any one of the amino acid sequences set forth in SEQ ID NOs:10-58. In another embodiment, the TLAL is selected from the group consisting of SEQ ID NOs: 10-58. In one embodiment, the TLAL comprises the amino acid sequence set forth in SEQ ID NO:10. In another embodiment, the TLAL comprises the amino acid sequence set forth in SEQ ID NO:11. In one embodiment, the TLAL has an enzyme activity that is at least 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% of that of a full-length recombinant human LAL (rhLAL). In another embodiment, the TLAL has an enzyme activity that is at least equal to or higher than that of the full-length rhLAL.
In another aspect, the invention provides a composition comprising an isolated recombinant TLAL, wherein the TLAL comprises the amino acid sequence set forth in SEQ ID NO: 10. In one embodiment, the composition further comprises mammalian culture medium.
In another aspect, the invention provides a composition comprising an isolated recombinant TLAL, wherein the TLAL comprises the amino acid sequence set forth in SEQ ID NO: 11.
In another aspect, the invention provides a composition comprising an isolated mixture of rhLAL, the mixture comprising at least one form of recombinant TLAL. In one embodiment, the mixture further comprises a full-length rhLAL. In another embodiment, the recombinant TLAL comprises the amino acid sequence set forth in SEQ ID NO: 10. In yet another embodiment, the recombinant TLAL comprises the amino acid sequence set forth in SEQ ID NO: 11.
In another aspect, the invention provides a composition comprising an isolated recombinant human LAL, wherein the LAL is a fusion protein comprising a second moiety. In one embodiment, the second moiety is fused to the N-terminus of the LAL, the C-terminus of the LAL, or internally to any amino acid residue position between Pro31 and Gly77 of the LAL. In another embodiment, the second moiety is fused to the LAL recombinantly. In yet another embodiment, the second moiety is fused to rhLAL via a linker.
In another aspect, the invention provides a pharmaceutical composition comprising the composition of claim 1. In one embodiment, the pharmaceutical composition further comprises a buffer. In one embodiment, the pharmaceutical composition further comprises an excipient. In one embodiment, the pharmaceutical composition further comprises a salt.
In another aspect, the invention provides a method of purifying an rhLAL from an isolated mixture comprising a full-length rhLAL and a recombinant TLAL, the method comprising purifying away the recombinant full-length rhLAL or TLAL from the mixture. In one embodiment, the purification is performed using one or more methods selected from the group consisting of hydrophobic interaction chromatography, affinity chromatography, ion exchange chromatography, size-exclusion chromatography, selective precipitation, crystallization, and tangential flow filtration.
In another aspect, the invention provides a method for treating a LAL deficiency in a patient, the method comprising: administering to the patient a composition of the invention in an amount effective to treat a LAL deficiency. In one embodiment, the LAL deficiency is Wolman disease (WD). In another embodiment, the LAL deficiency is cholestryl ester storage disease (CESD). In another embodiment, the TLAL or rhLAL is administered intravenously. In another embodiment, the TLAL or rhLAL is administered via a pump. In another embodiment, the amount is in the range of 0.1 to 20 mg of TLAL or rhLAL per kg of body weight.
The present invention relates to a group of isolated truncated forms of recombinant human LAL (“TLAL”). Some TLAL proteins retain enzymatic activity and are capable of hydrolyzing cholesteryl esters (CE) and triglycerides (TG). The compositions described herein comprise one or more TLAL proteins or polypeptides. Proteins or polypeptides referred to herein as “isolated” are proteins or polypeptides that are purified to a state beyond that in which they exist in cells, and include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods, and also include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated. Further, proteins or polypeptides referred to herein as “recombinant” are produced by the expression of recombinant nucleic acids.
Positions of the amino acid residues of recombinant human LAL (rhLALs) are referred to as they are numbered from the N-terminal Met1 to the C-terminal Gln399 in unprocessed rhLAL. The corresponding amino acid residue positions from 1 through 399 are set forth in SEQ ID NO:1 (
All truncated forms of recombinant LAL (“TLALs”) described herein comprise a portion of full-length human LAL (“hLAL”).
In one embodiment, the TLAL described herein can be any one of TLAL having 315 to 371 amino acid residues long as set forth in SEQ ID NOs:2-58. In a preferred embodiment, the TLAL can be 323 to 371 amino acid residues long, having the amino acid sequence of the C-terminal region of hLAL (SEQ ID NOs:10-58).
In one embodiment, a TLAL comprises the C-terminal 323 amino acid residues of hLAL, and in another embodiment, consists of those 323 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Gly77 of hLAL. This truncated form is designated TLAL-G77 (SEQ ID NO:10).
In another embodiment, a TLAL comprises the C-terminal 324 amino acid residues of hLAL, and in another embodiment, consists of those 324 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Lys76 of hLAL. This truncated form is designated TLAL-K76 (SEQ ID NO:11).
Another recombinant TLAL described herein comprises the C-terminal 325 amino acid residues of hLAL, and in another embodiment, consists of those 325 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Asp75 of hLAL. This truncated form is designated TLAL-D75 (SEQ ID NO:12).
Another recombinant TLAL described herein comprises the C-terminal 315 amino acid residues of hLAL, and in another embodiment, consists of those 315 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Gln85 of hLAL. This truncated form is designated TLAL-Q85 (SEQ ID NO:2).
Another recombinant TLAL described herein comprises the C-terminal 316 amino acid residues of hLAL, and in another embodiment, consists of those 316 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Leu84 of hLAL. This truncated form is designated TLAL-L84 (SEQ ID NO:3)
Another recombinant TLAL comprises the C-terminal 317 amino acid residues of hLAL, and in another embodiment, consists of those 317 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Phe83 of hLAL. This truncated form is designated TLAL-F83 (SEQ ID NO:4).
Another recombinant TLAL comprises the C-terminal 318 amino acid residues of hLAL, and in another embodiment, consists of those 318 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Val82 of hLAL. This truncated form is designated TLAL-V82 (SEQ ID NO:5).
Another recombinant TLAL comprises the C-terminal 319 amino acid residues of rhLAL, and in another embodiment, consists of those 319 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Val81 of hLAL. This truncated form is designated TLAL-V81 (SEQ ID NO:6).
Another recombinant TLAL comprises the C-terminal 320 amino acid residues of hLAL, and in another embodiment, consists of those 320 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Pro80 of hLAL. This truncated form is designated TLAL-P80 (SEQ ID NO:7).
Another recombinant TLAL comprises the C-terminal 321 amino acid residues of hLAL, and in another embodiment, consists of those 321 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Lys79 of hLAL. This truncated form is designated TLAL-K79 (SEQ ID NO:8).
Another recombinant TLAL comprises the C-terminal 322 amino acid residues of hLAL, and in another embodiment, consists of those 322 amino acid residues. The N-terminus of this truncated form of rhLAL corresponds to Pro78 of hLAL. This truncated form is designated TLAL-P78 (SEQ ID NO:9).
The TLAL proteins described herein may contain a truncation at the C-terminus up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acid residues.
The present invention includes a composition of an isolated mixture of recombinant lysosomal acid lipase proteins comprising a TLAL and at least one more forms of recombinant human LAL (rhLAL). For example, the mixture of the LAL proteins can comprise at least one form of TLAL and at least one form of full-length LAL proteins. The mixture of the LAL proteins can be a mixture comprising two or more different forms of TLAL proteins described herein.
Without wishing to be tied with a theory, it is believed that some TLAL can be produced in mammalian cell culture that expresses a full-length rhLAL by a posttranslational modification (i.e., an intracellular protein maturation process). In one embodiment, the TLAL and the full-length hLAL are recombinant proteins produced from mammalian cell culture. The mixture comprising a full-length rhLAL and a TLAL can be obtained from mammalian cell culture expressing a full-length rhLAL which subsequently undergoes intracellular proteolytic cleavage. Accordingly, the present invention contemplates an isolated mixture comprising a full-length hLAL and a TLAL. The mixture can be obtained from conditioned medium of mammalian cell culture expressing the full-length LAL and be subjected to purification steps in which the various forms of LAL proteins can be further isolated and purified. In one particular embodiment, TLAL is purified away from another form of rhLAL protein contained in the mixture, e.g., the full-length rhLAL protein. In yet another embodiment, the full-length LAL protein is purified away from the TLAL in the mixture.
In one embodiment, an isolated mixture can comprise at least one form of TLAL selected from the group consisting of SEQ ID NOs: 2-58 and a full-length rhLAL. In one particular embodiment, the isolated mixture comprises TLAL-D75 (SEQ ID NO:12), TLAL-K76 (SEQ ID NO:13), or TLAL-G77 (SEQ ID NO:14) and any of the full-length rhLAL proteins described herein. Depending on the length of the signal peptide sequence cleaved off from the unprocessed rhLAL protein, the full-length human LAL present in the mixture can be 378 amino acid residues long (i.e., Ser22 through Gln399), 377 amino acid residues long (i.e., Gly23 through Gln399), 376 amino acid residues long (i.e., Gly24 through Gln399), 375 amino acid residues long (i.e., Lys25 through Gln399), 374 amino acid residues long (i.e., Leu26 through Gln399), 373 amino acid residues long (i.e., Thr27 through Gln399) or 372 amino acid residues long (i.e., Ala28 through Gln399).
In another embodiment, the isolated mixture comprising TLAL can contain at least two forms of TLAL. In one particular embodiment, the isolated mixture comprising TLAL can contain at least two forms of TLAL selected from the group consisting of TLAL-D75 (SEQ ID NO:12), TLAL-K76 (SEQ ID NO:13) and TLAL-G77 (SEQ ID NO:14).
The invention further includes fusion proteins, comprising an rhLAL (i.e., full-length LAL or TLAL) as a first moiety, linked to a second moiety that does not occur naturally in a human LAL. The second moiety can be an amino acid or polypeptide that are fused at the N-terminus of the rhLAL, the C-terminus of the rhLAL, or internally to any amino acid residue position from Pro31 to Ala90 of rhLAL (SEQ ID NO:1). As contemplated in the invention, the second moiety can be fused recombinantly or chemically (e.g., covalently) via a linker and/or spacer known in the art.
In one embodiment, the fusion protein comprises a TLAL or a full-length rhLAL and a second moiety comprising an affinity ligand, with or without a linker and/or spacer joining the first and the second moieties. The fusion protein comprising an affinity ligand can be produced in a host cell and can be purified from a cell lysate or from the culture medium, using a suitable affinity matrix that binds to the affinity ligand. A non-limiting example of such affinity ligand is the FLAG affinity sequence shown in
In one embodiment, the second moiety comprises a proteolytic cleavage sequence with or without a linker and/or spacer joining the first and the second moieties. The proteolytic cleavage sequence can be introduced at any desired position between Pro31 and Ala90 of rhLAL (SEQ ID NO:1), preferably between Pro31 and Gly77 of rhLAL. When a full-length LAL having an internal cleavage site is produced, a proteolytic reaction can be carried out in vitro to allow for cleavage of rhLAL at the desired site. In one specific embodiment, the cleavage sequence is an enterokinase (“ek”) linker (i.e., Asp-Asp-Asp-Asp-Lys: SEQ ID NO:64). Enterokinase is a protease which cleaves the protein after the lysine (K) residue at its cleavage site DDDDK (SEQ ID NO:64).
In some aspects, the fusion protein can be a TLAL protein or a full-length rhLAL containing one or more second moieties that function to target the protein to a specific cell, tissue or organ. In one embodiment, the fusion protein comprises a TLAL and a second moiety comprising a receptor ligand. The targeting moiety is capable of binding to a cell surface receptor present on a target cell, where it is internalized via endocytosis. Useful targeting moieties for directing the TLAL to the target cells include, but are not limited to, p97, insulin-like growth factor (IGF)-I, IGF-II, transferrin receptor ligand, RAP, ApoB, ApoE, aprotinin, lipoprotein lipase, low density lipoprotein receptor-related protein 1 (LRP-1), and variants, homologues or fragments thereof. These targeting moieties can increase rapid internalization, enhance pharmacokinetics, and direct proper targeting of the fusion protein to specific cells, tissues or organs.
In another embodiment, the fusion protein comprises a TLAL or a full-length LAL and a second moiety that functions to modulate serum half-life of the TLAL. For example, polyalkylene oxide conjugates of TLAL or full-length LAL protein are also a part of the invention. In one embodiment, polyethylene glycol (PEG) is conjugated to a TLAL at one or more sites, for example, through one or more epsilon amino groups of lysine residues, via a linker such as succinimidyl carbonate, thiazolidine thione, urethane or amide base linkers. Non-limiting examples of the second moiety that can be fused to the LAL protein by recombinant technology include a Fc region of human immunoglobulin, human serum albumin (HSA), transferrin, α2-macroglobulin, and variants, homologues or fragments thereof.
TLAL proteins with amino acid sequences corresponding to those allelic variants of hLAL are also part of the invention. TLAL proteins having an amino acid sequence not corresponding exactly to hLAL, but having one or more (for example, two, three, four or five) conservative amino acid substitutions are also included in the invention. Conservative amino acid substitutions are those substitutions that are made within these designated groups that allow for the retention of the acidic (Asp, Glu, Asn, Gln), basic (His, Lys, Arg), polar (Ser, Cys, Thr, Met), aromatic (Phe, Tyr, Trp) or hydrophobic (Gly, Ala, Val, Leu, Ile) properties of the amino acid side chain so long as such substitution allows for enzymatic activity of TLAL.
The present invention also contemplates modified rhLALs and modified TLALs that retain enzymatic activity. In one embodiment, a modified rhLAL comprises the amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:1. In another embodiment, a modified TLAL protein comprises the amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any of the amino acid sequences set forth in SEQ ID NOs:2-58.
In one embodiment of the invention, the isolated recombinant TLAL has an enzymatic activity of a lysosomal acid lipase (LAL). In another embodiment, the enzymatic activity of a LAL includes, but is not limited to, one or more enzymatic activities selected from the group consisting of hydrolysis of cholesteryl esters and hydrolysis of triglycerides. Assays for determining the enzymatic activity of a LAL are well known to one of ordinary skill in the art. For example, LAL enzymatic activity can be determined by using the fluorogenic substrate 4-methylumbelliferyl-oleate assay as described in Example 12 of WO/2011/133960 and Yan et al. (2006), American Journal of Pathology, 169(3):916-926, the entire contents of which are incorporated herein by reference. Cellular uptake of an rhLAL can be determined using macrophage and fibroblast cells according to the assay described in Example 13 of WO/2011/133960, the entire contents of which are incorporated herein by reference. Moreover, enzymatic activity of rhLAL can be assessed in vivo in LAL-deficient Yoshida Rats according to the assay described in Example 14 of WO/2011/133960 and Kuriyama et al. (1990), Journal of Lipid Research, vol. 31, p 1605-1611; Nakagawa et al. (1995), Journal of Lipid Research, vol. 36, p 2212-2218; and Yoshida and Kuriyama (1990), Laboratory Animal Science, vol. 40, p 486-489, the entire contents of each of which are incorporated herein by reference,
TLAL proteins of the present invention have an enzymatic level or activity that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or even 200% of the normal level of activity observed in a full-length rhLAL. The “normal level” as used herein refers to a level of LAL activity of the full-length hLAL in corresponding wild-type (i.e., LAL+/LAL+) cells or to a level of LAL activity of an unmodified full-length recombinant human LAL treated with or present in a similar condition.
In some embodiments, the TLAL of the present invention may achieve an enzymatic activity level that is increased by at least 1.25-fold, 1.5-fold, 1.75-fold, or even 2-fold, as compared to positive controls (e.g., a full-length rhLAL treated with or present in a similar condition). In some embodiments, TLAL of the present invention may achieve an enzymatic level or activity at least approximately 10 nmol/hr/mg, 20 nmol/hr/mg, 30 nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg, 80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200 nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400 nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg or 600 nmol/hr/mg in a target tissue.
TLAL can be produced from transgenic animals (e.g., cow, sheep, goat and birds), insect cells, plants, yeast or bacteria as known in the art using the methods described herein. Depending on a host cell and its biological mechanism, the TLAL can be produced directly from an unmodified coding sequence rhLAL or indirectly using proteolytic cleavage which results in deletion of a designed N-terminus region from a longer rhLAL produced.
In mammalian cell culture, recombinant human LAL (rhLAL) was observed as a 53 kDa band on SDS-PAGE (“SGP . . . ” in
Accordingly, the present invention includes recombinant methods to produce TLAL proteins, without requiring introduction of an artificial proteolytic cleavage site. TLAL can be produced from proteolysis of a full length form of hLAL that occurs in a mammalian cell culture (e.g., HEK293 cells). In particular embodiments, the TLAL proteins produced in cultured mammalian cells can be TLAL-K76 (SEQ ID NO:11) and/or TLAL-G77 (SEQ ID NO:10).
Not all TLAL proteins may be stable in all host cells. See, for example, Zschenker et al. (Zschenker, et al., J. Biochem. 136:65-72, 2004) describing the failure to achieve expression of a short form of LAL in Spodoptera frugiperda insect cells. Thus, where TLAL is not adequately expressed in certain host cells, a full-length or any rhLAL having an adequate length of the N-terminal region can be recombinantly modified to introduce an artificial proteolytic cleavage site within the N-terminal region to obtain a TLAL of a desired length.
In one embodiment, a full-length rhLAL comprising a proteolytic cleavage site can be expressed, isolated from host cell culture and subsequently subjected to proteolysis at the cleavage site to obtain the desired TLAL from the full-length rhLAL. A proteolytic cleavage site can be introduced at any desired position between Pro31 and Ala90 of rhLAL (SEQ ID NO:1). When a full-length LAL is produced with an internal cleavage site, a proteolytic reaction can be carried out in vitro to allow for cleavage of the full length proteins.
In one embodiment, the cleavage linker is an enterokinase (“ek”) recognition sequence (i.e., Asp-Asp-Asp-Asp-Lys; SEQ ID NO:64) which is also part of the FLAG affinity sequence. Enterokinase is a specific protease which cleaves the protein after the lysine (K) residue at its cleavage site DDDDK (SEQ ID NO:64).
Alternatively, the proteolytic linker can be the Factor Xa cleavage sequence (i.e., Ile-Glu/Asp-Gly-Arg; SEQ ID NO:65). Factor Xa cleaves a peptide containing the cleavage sequence of Ile-Glu/Asp-Gly-Arg (SEQ ID NO:65) after the arginine residue. A furin cleavage can be also used as a proteolytic linker. As the major processing enzyme of the secretory pathway, furin is a ubiquitous subtilisin-like proprotein convertase having the minimal cleavage site Arg-X-X-Arg′ (SEQ ID NO:66). Genenase I and its cleavage sequence can be also employed to produce TLAL by proteolysis. Genenase I cleaves a protein containing Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO:67) after the Tyr residue. Other proteases with specific cleavage sites known in the art can be employed without limitation to produce proteolytic cleavage of rhLAL to obtain a desired TLAL.
Suitable mammalian cells that can be used to obtain TLAL include primary cell cultures derived from a mammal at any stage of development or maturity. Mammalian cells also include cells of mammalian origin that have been transformed to divide for an unlimited number of generation, such as human embryonic kidney line (e.g., HEK293), human fibrosarcoma cell line (e.g., HT1080), human cervical carcinoma cells (HeLa), human lung cells (W138), human liver cells (Hep G2), human retinoblasts, BALB/c mouse myeloma line, COS-7, baby hamster kidney cells (e.g., BHK), Chinese hamster ovary cells (e.g., CHO+/−DHFR), mouse Sertoli cells (TM4), rat liver cells (BRL 3A), mouse mammary tumor (e.g., MMT-060562), TRI cells; MRC 5 cells, FS4 cells, monkey kidney cells (e.g., CV1, VERO-76), canine kidney cells (e.g., MDCK). Different host cells can be chosen to ensure its capacity to modify and process a TLAL protein.
The vector encoding the rhLAL protein and/or TLAL can be introduced into host cells for production of the TLAL protein. The host cells can be grown under conditions in which the coding sequence of the TLAL is expressed. The protein can be purified from the culture medium or from cell extracts, as appropriate. In one method, a coding region for a full-length hLAL (comprising cDNA or DNA synthetically produced) can be enzymatically introduced into a vector to operably link the coding region to a promoter and other control regions necessary for gene expression in a suitable protein expression system including, but not limited to, mammalian cells, plant cells, insect cells, yeast, or bacteria. For example, vectors available for use in mammalian host cells include any vector known in the art for expressing a protein in its corresponding expression system, e.g., pTT22 for HEK293 cells and SV40 for CV1 line. Methods for vector construction are generally known to those of ordinary skill in the art. (See, for example, Durocher et al., US Publication 2011/0039339A1).
A variety of expression vector/host systems can be utilized to express TLAL. The microorganisms include bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cells or plant cells infected with virus expression vectors (e.g., baculovirus). Furthermore, the present invention also contemplates TLAL proteins produced in any useful transgenic expression system including, without limitation, transgenic mammals, and in plant systems including tobacco plant and duck weed.
The control elements or regulatory sequences include those non-translated regions of the vector, enhancers, promoters, 5′ and 3′ untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including, tissue-specific, constitutive and inducible promoters, can be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferred. If a cell line that contains multiple copies of the sequence encoding TLAL is desired, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
The present invention also contemplates production of TLAL in a transgenic avian system. When an avian expression system is used, the vectors described in U.S. Pat. No. 6,730,822; U.S. Pat. No. 6,825,396; U.S. Pat. No. 6,875,588; U.S. Pat. No. 7,294,507; U.S. Pat. No. 7,521,591; U.S. Pat. No. 7,534,929; and U.S. Patent Publication No. 2006/0185024 are preferred. The present invention includes production of TLAL with or without a second moiety (e.g., targeting or proteolytic cleavage sequence). The avian expression vector can include one or more regulatory sequences such as oviduct-specific promoter, for example, and without limitation, ovomucoid promoters, ovalbumin promoters, lysozyme promoters, conalbumin promoters, ovomucin promoters, ovotransferrin promoters and functional portions of each of these promoters. Suitable non-specific promoters can include, for example and without limitation, cytomegalovirus (CMV) promoters, MDOT promoters and Rous-sarcoma virus (RSV) promoters, murine leukemia virus (MLV) promoters, mouse mammary tumor virus (MMTV) promoters and SV40 promoters and functional portions of each of these promoters. Non-limiting examples of other promoters which can be useful in the present invention include, without limitation, Pol III promoters (for example, type 1, type 2 and type 3 Pol III promoters) such as H1 promoters, U6 promoters, tRNA promoters, RNase MPR promoters and functional portions of each of these promoters. Typically, functional terminator sequences are selected for use in accordance with the promoter that is employed.
TLAL can be glycosylated on one, two, three, four, five or six Asn (N) sites for N-linked glycosylation depending on the length of the TLAL and the expression system in which it is produced. The full-length LAL has six potential glycosylation sites at Asn36, Asn72, Asn101, Asn161, Asn273 and Asn321. The invention includes all forms of glycosylated TLAL proteins, glycosylated at any or all of these sites. The present invention also contemplates an unglycosylated TLAL protein. In some embodiments, Asn72 may not be glycosylated (see for example, PCT/US2011/033699; WO/2011/133960). In one embodiment, the TLAL protein can comprise one or more glycan structures at the C-terminal Asn101, Asn161, Asn273 and Asn321 glycosylation sites. In another embodiment, the TLAL protein may have one or more glycan structures at the Asn36, Asn101, Asn161, Asn273 and Asn321 glycosylation sites. In yet another embodiment, the TLAL protein may contain one or more glycan structures at the Asn72, Asn101, Asn161, Asn273 and Asn321 glycosylation sites. In still another embodiment, the TLAL protein may contain one or more glycan structures at Asn36, Asn72, Asn101, Asn161, Asn273 and Asn321 glycosylation sites.
A TLAL can be purified to various grades of purity. The purity of TLAL in a composition is at least equal to or greater than 80%, 82%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% by total protein as determined by HPLC methods. The composition can be free of any detectable band of about 30 kDa to 37 kDa on SDS-PAGE. The composition can be free of any detectable band of about 50 kDa to 60 kDa on SDS-PAGE. A composition comprising TLAL can be purified to a grade that is essentially free of non-LAL protein. A composition comprising TLAL can also be purified to a state wherein the TLAL protein is essentially pure. A TLAL can be present in a solution or in a lyophilized preparation. The composition can be also present in varying proportions of TLAL to a full-length form (rhLAL), such as 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90% and 90% to 100%.
Pharmaceutical compositions comprising one or more forms of TLAL are contemplated in the invention. In some embodiments, the compositions comprising truncated LAL also comprise an aqueous or physiologically compatible fluid suspension or solution. Compositions of TLAL can comprise any nontoxic substance compatible with enzymatic activity and stability. Carrier substances can include sterile water, buffers such as sodium phosphate, potassium phosphate, and other physiologically acceptable buffers such as citrate, bicarbonate and acetate buffers, salts such as sodium chloride and potassium chloride, carbohydrates such as mannitol, maltose, sucrose, sorbitol, xylitol and glucose, alcohol, glycerol, amino acids, urea, EDTA, EGTA, polyvinylpyrollidone, polysaccharides, methylcellulose, sodium carboxymethyl cellulose, propylene glycol, polyethylene glycol, glycerol, ascorbic acid, lipids, phospholipids, buffering agents, dispersing agents, surfactants such as poloxamer 188, polysorbate 80, Cremophore-EL, Cremophore-R, labrofil, and the like. Compositions comprising TLAL can also comprise other proteins, such as full-length rhLAL, impurities not fully purified from the TLAL, or added proteins such as human serum albumin, collagen and gelatin. In some embodiments, the composition can contain benzyl alcohol, phenol or m-cresol. Compositions comprising any combination of the above are included in the invention.
In a specific example, recombinant human TLAL produced as disclosed herein, is employed in a pharmaceutical formulation wherein each 1 milliliter contains TLAL (e.g., 2 mg LAL), trisodium citrate dehydrate (e.g., 13.7 mg), citric acid monohydrate (e.g., 1.57 mg), and human serum albumin (e.g., 10 mg), and is formulated to an acidic pH such as 5.9±0.1.
Compositions comprising LAL can be lyophilized or stored as liquids. For preservation of activity in the lyophilized form, bulking agents such as glycine, mannitol, albumin and dextran can be useful. For cryoprotection, disaccharides, amino acids and polyethylene glycol can be useful. When sugars are included in the composition, the sugar can be included in the concentration of about 2% to about 40% weight to volume. In some embodiments, the composition comprising truncated LAL has a pH in the range of 4.0 to 6.0.
A TLAL or a composition comprising a TLAL can be used alone or in combination with other pharmaceutical agents or therapies in methods to treat a human with a deficiency of hLAL. Methods of administering a TLAL, dosing regimens, methods for evaluating the effectiveness of treatments, and pharmaceutical compositions and formulations for TLAL are as described for recombinant human LAL in U.S. patent application Ser. No. 13/229,558, filed Sep. 9, 2011, which is hereby incorporated by reference in its entirety.
Suitable formulations for administration may contain a pharmaceutical composition at various concentrations. In some embodiments, suitable formulations may contain a TLAL at a concentration up to about 500 mg/mL, e.g., up to about 400 mg/mL, up to about 300 mg/mL, about 250 mg/mL, up to about 200 mg/mL, up to about 150 mg/mL, up to about 100 mg/mL, up to about 90 mg/mL, up to about 80 mg/mL, up to about 70 mg/mL, up to about 60 mg/mL, up to about 50 mg/mL, up to about 40 mg/mL, up to about 30 mg/mL, up to about 25 mg/mL, up to about 20 mg/mL, up to about 10 mg/mL or up to about 5 mg/mL. In some embodiments, suitable formulations may contain a TLAL at a concentration ranging between about 1-500 mg/mL (e.g., about 1-250 mg/mL, about 1-200 mg/mL, about 1-150 mg/mL, about 1-100 mg/mL, about 10-100 mg/mL, about 1-80 mg/mL, about 1-70 mg/mL, about 1-60 mg/mL, about 1-50 mg/mL, about 1-40 mg/mL, about 1-30 mg/mL).
The present invention contemplates any route of administration which facilitates the uptake of TLAL into the lysosomes of pertinent cells, organs and tissues. In some embodiments, formulations suitable for intravenous administration can contain TLAL at a concentration of about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 50 mg/mL, about 75 mg/mL, about 100 mg/mL, about 150 mg/mL, about 200 mg/mL, about 250 mg/mL or 300 mg/ml.
The recombinant LAL proteins described herein that exhibit enzyme activity levels at least 50% of the full-length can be employed to treat LAL deficiency (e.g., Wolman disease and cholesteryl ester storage disease). The invention provides methods of treating human patients suffering from LAL deficiency comprising administering TLAL or LAL fusion protein to the patient, wherein the administration is sufficient to restore growth, to improve liver function, to reduce liver damage, to increase tissue levels of LAL, and/or increase LAL activity in the patient. For the treatment of LAL deficiency, generally, the amount of TLAL or LAL fusion protein administered can vary depending on known factors such as age, health, and weight of the recipient, type of concurrent treatment, frequency of treatment, and the like. Usually a dosage of active ingredient can be between about 0.01 and about 50 mg per kilogram of body weight. In one embodiment, dosage of LAL in accordance with the invention is between about 0.1 and 0.5 mg per kilogram of body weight. In one embodiment, the dose is between about 0.1 mg and about 5.0 mg per kilogram. In one embodiment, the dose is about 0.1 mg to about 5.0 mg per kilogram. In one embodiment, the dose is about 0.1 mg to about 10 mg per kilogram. In one embodiment, the dose is about 0.1 mg to about 20 mg per kilogram. In one embodiment the dose is about 0.1, about 0.2, about 0.25, about 0.30, about 0.35, about 0.40, about 0.45, about 0.50 mg per kilogram body weight. In one embodiment, the dose is between about 1 mg and about 5 mg per kilogram body weight. In one embodiment, the dose is about 1 mg per kilogram body weight. In one embodiment, the dose is about 3 mg per kilogram body weight. For example, 0.1 mg per kilogram of body weight, 0.2 mg per kilogram of body weight, 0.3 mg per kilogram of body weight, 0.4 mg per kilogram of body weight, 0.5 mg per kilogram of body weight, 1 mg per kilogram of body weight, 2 mg per kilogram of body weight, 3 mg per kilogram of body weight, 4 mg per kilogram of body weight, or 5 mg per kilogram of body weight can be administered.
In some aspects, the recombinant LAL proteins described herein can be administered to a human patient once in about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 30 days at a dose about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10 mg per kilogram of body weight. In one embodiment, the LAL protein is administered weekly, biweekly, or monthly.
In some aspects, the patient is subject to an escalating dosage regimen. For example, the patient can be administered initially with a dose, e.g., about 0.1 to about 0.5 mg per kilogram, for about two weeks or about one month and later with a high dose above 0.5 mg per kilogram of body weight. In some other aspects, the patient can be administered initially with a high dose and later with a dose lower than the initial dose during the treatment.
The therapeutic proteins can be injected by, for example, subcutaneous injections, intramuscular injections, and intravenous (IV) infusions or injections. In one embodiment, the TLAL or the LAL fusion protein is administered intravenously by IV infusion by any useful method. In one example, the TLAL or the LAL fusion protein can be administered by intravenous infusion through a peripheral line. In another example, the TLAL or the LAL fusion protein can be administered by intravenous infusion through a peripherally inserted central catheter. In another example, the TLAL or the LAL fusion protein can be administered by intravenous infusion facilitated by an ambulatory infusion machine attached to a venous vascular access port. In one embodiment, of intravenous infusion, the medication is administered over a period of 1 to 8 hours depending on the amount of medication to be infused and the patient's previous infusion-related reaction history, as determined by a physician skilled in the art. In another embodiment, the TLAL or the LAL fusion protein is administered intravenously by IV injection. In another embodiment, the TLAL or the LAL fusion protein can be administered via intraperitoneal injection. In still another embodiment, the TLAL is administered via a pharmaceutically acceptable capsule of the therapeutic protein. For example, the capsule can be an enteric-coated gelatin capsule.
In some embodiments, the therapeutic proteins are administered by infusion, and the infusion can occur over an extended time period, for example, 30 minutes to 10 hours. Thus, the infusion can occur, for example, over a period of about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours. The infusion can also occur at various rates. Thus, for example, the infusion rate can be between about 1 mL per hour and 20 mL per hour. In some embodiments, the infusion rate is between 5 mL and 10 mL per hour. In one embodiment, the infusion rate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mL per hour. In one embodiment, the infusion rate is between 0.1 and 5 mg/kg/hr. In one embodiment, the infusion rate is about 0.1, about 0.2, about 0.3, about 0.5, about 1.0, about 1.5, about 2.0, or about 3 mg/kg/hr.
Various methods of administering a TLAL protein, dosing regimens, methods for evaluating the effectiveness using a recombinant TLAL are as described for recombinant human LAL in U.S. patent application Ser. No. 13/229,558, filed Sep. 9, 2011, which is hereby incorporated by reference in its entirety.
HEK293E cells (human embryonic kidney cell line stably expressing EBNA-1 protein of Epstein-Barr virus) were transfected with pTT22-LAL (pTT22 with insertion of native human LAL coding region (SEQ ID NO:1); see Publication No. US 2011/0039339 for pTT22 vector) using polyethylenimine. The culture medium was supplemented with peptone TN1 24-48 hours post transfection. The culture remained at a cell density of about 2×106 cells/mL. Medium was collected 6 days after transfection. Protein was purified through a phenyl hydrophobic interaction (HIC) column as well as SP Sepharose™ column, and followed by purification on a S200 size exclusion chromatography (SEC) column.
Analysis of purified samples by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel stained with Coomassie blue showed the presence of two forms of rhLAL co-purified (
To determine whether the smaller protein was derived from rhLAL, proteins were analyzed on Western blot. The proteins were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane using a Pierce Fast Semi-Dry Blotter. The primary and secondary antibodies were rabbit α-LIPA pAb (Abnova catalog no. H00003988-R01) and goat α-rabbit-HRP IgG (H+L) pAb (Thermo Scientific Pierce catalog no. 31460), respectively. ECL Western Blotting Substrate (Thermo Scientific Pierce) was used on the membrane to allow for detection of chemiluminescence in an Alpha Innotech Gel Imaging Cabinet (Scion Corporation) (
Subsequently, the smaller LAL protein was purified from conditioned media containing both the long and the small form of rhLAL. N-terminal amino acid sequencing revealed that the larger rhLAL protein (53 kD) was indeed a full-length rhLAL having the N-terminal amino acid sequence of SGG and that the smaller form of rhLAL (41-43 kD) was a truncated LAL having the N-terminal amino acid sequence of KGP. The truncated rhLAL was named TLAL-K76, and confirmed to have the amino acid sequence of positions 76-399 of SEQ ID NO:1 (i.e., SEQ ID NO:11; see,
To determine whether the full-length rhLAL produced in HEK293 cells is capable of being internalized into target cells and enzymatically active, human macrophage (NR8383) cells were co-incubated with various concentration of the rhLAL-HEK (“SBC-102-HEK293”). rhLAL produced from transgenic chicken (“SBC-102-EW”) were used as positive control. Subcellular enzyme activity of LAL was measured (units/cell) as described in PCT/US2011/033699 (WO2011/133960). As shown in
The vector pTT22-EK-gp-LAL was constructed starting from pTT22-LAL. Forward and reverse primers (
HEK293 cells were transfected with the plasmid described above and grown in cell culture. Culture medium from ekLAL-HEK293 cells was adjusted to 20 mM Na3PO4, 137 mM NaCl, pH 6.0, final volume of 2 liters. The hydrophobic interaction column (HIC) (HiTrap Phenyl Sepharose 6 Fast Flow™, GE Healthcare™, Cat. No. 17-5193-01) base on a 90 μm matrix was loaded with samples and washed with 20 mM Na3PO4, 137 mM NaCl, pH 6.0, then washed with 5 mM Na3PO4 pH 6.0. Protein was eluted with 5 mM Tris, 50% propylene glycol, pH 7.2. Fractions containing protein with maximal enzymatic activity were used for further purification on an ion exchange (IEX) column (HiTrap SP Fast Flow™, GE Healthcare, Cat. No. 17-5157-01). SP Sepharose™ Fast Flow is a cation exchanger based on a 6% highly cross-linked beaded agarose matrix. The IEX column was loaded with the pooled fractions adjusted to 50 mM NaOAc, pH 5.0 and washed with buffer of the same salts. Proteins were eluted with 50 mM NaOAc, 35 mM CaCl2, pH 5.0. Pooled fractions from the IEX column were used for further purification on an α-FLAG affinity column (Anti-FLAG M2™ Affinity Gel, Sigma, Cat. No. A2220) equilibrated with TBS, pH 7.4, with elution using 1M Tris, pH 8.0. The affinity resin in the α-FLAG affinity column bound to the octapeptide DYKDDDDK (SEQ ID NO:60) of ekLAL. Fractions containing maximal activity of ekLAL were eluted, pooled and concentrated.
Enzymatic activity of ekLALs containing the octapeptide DYKDDDDK (SEQ ID NO:60) tag in the N-terminal region was measured as described the following section. Contrary to the findings by Zschenker et al. (2004) that the N-terminal region of LAL is essential for protein folding, stability, secretion and enzyme activity, ekLAL proteins containing the octapeptide in the N-terminal region (e.g., SEQ ID NOs:61 and 64) exhibited enzyme activity levels similar to that of the unmodified full-length wild-type rhLAL, suggesting that the mutations created by insertion of the octapeptide in the N-terminal region did not affect the enzyme activity of rhLAL.
Purified samples of ek tagged LAL (i.e., SEQ ID NO: 61 and 68) were added to rEK reaction buffer (5 μL of 10X EK buffer in 45 uL of dH2O). Enterokinase (1:20 units to protein ratio) was added to initiate digestion reactions, and the reaction pool was incubated at room temperature for 6 days. After digestion reaction, proteins were purified by the α-FLAG affinity column to separate the TLAL-HEK, from ekLAL-HEK (full-length). Various α-FLAG affinity column fractions were run on 12% SDS-PAGE (see, e.g.,
Enzyme activity of various forms of rhLAL including full-length rhLAL, rhLAL proteins containing the ek cleavage site (ekLALs), and TLAL was determined using the fluorogenic substrate 4-methylumbelliferyl-oleate assay essentially as described in Yan et al. (2006), American Journal of Pathology, Vol. 169, No. 3, p 916-926, the disclosure of which is incorporated herein by reference in its entirety.
A substrate stock solution of 2.5 mM 4-methylumbelliferyl oleate (4-MUO) in 4% Triton X-100 was prepared. The assay was performed in a microtiter plate, each well containing 62.5 μL of 0.2 M sodium citrate (pH 5.5) in 0.01% Tween80, 12.5 μL of LAL samples and 25 μL of the 2.5 mM 4-MUO. Change in fluorescence was monitored for 30 minutes at 37° C. using a Bio-Tek Synergy HT fluorometric microplate reader (excitation 360 nm and emission 460 nm). Prior to assay, samples containing rhLAL was diluted to a concentration that resulted in the reaction continuing linearly for at least 30 minutes. The reaction was stopped with 50 μL of 0.75 M Tris-HCl, pH 8.0 and the endpoint fluorescence signal was measured in the same plate reader used above (excitation 360 nm and emission 460 nm). Units of activity were determined using 4-methylumbelliferyl as a standard. One unit (U) is defined as the amount of enzyme which results in the formation of 1 μmole of 4-methylumbelliferyl/min under the assay conditions described above.
TLALs exhibited enzymatic activity levels that are similar to or higher than that of similarly treated ekLALs (see,
Each example in the above specification is provided by way of explanation of the invention, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications, combinations, additions, deletions and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment. It is intended that the present invention cover such modifications, combinations, additions, deletions, and variations.
All publications, patents, patent applications, internet sites, and accession numbers/database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.
This application claims the benefit of U.S. Provisional Application No. 61/605,850, filed Mar. 2, 2012. The entire teachings of the foregoing application are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/028688 | 3/1/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61605850 | Mar 2012 | US |
