Patent Application
20180251745
The present invention relates to the fields of medicine and hematology. More specifically, the invention provides novel coagulation Factor X/Xa agents and methods of using the same to modulate the coagulation cascade in patients in need thereof.
Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
The enzymes of coagulation are trypsin-like enzymes that belong to the S1 peptidase family of proteases that bear a chymotrypsin-like fold. The coagulation proteases contain catalytic domains that are highly homologous to each other and to the ancestral serine proteases of digestion. The structural homology/identity is so great (>70%) that residues in the catalytic domains of the coagulation enzymes are numbered according to the corresponding residues in chymotrypsinogen.
The coagulation enzymes circulate in blood as inactive precursors, zymogens, that require proteolytic cleavage for activation. The zymogens possess ˜10,000-fold or less proteolytic activity when compared to the serine proteases produced following activation. Initiation of coagulation at the site of vascular damage leads to a series of reactions in which a zymogen is converted to a protease through specific proteolytic cleavage and forms the enzyme for the successive reaction. This culminates in blood cell activation and the conversion of soluble fibrinogen to insoluble fibrin and hence the formation of the clot. Excess proteases are removed by reaction with circulating protease inhibitors that act as “suicide” substrates or those that recognize the active enzymes. Thus, proteolytic activation of the coagulation zymogens is a key regulatory feature of the coagulation cascade.
Although some of the coagulation zymogens are cleaved at two or more sites in their respective activation reactions, formation of the protease requires cleavage at a single site. Cleavage at this site and its structural consequences are considered in the most facile way using the homologous numbering system based on chymotrypsinogen and the extensive structural work done with trypsinogen and trypsin. The conversion of the zymogen to serine protease requires cleavage following Arg15 (typically the bond between Arg15 and Ile16) which typically removes an activation peptide and exposes a new N-terminus in the catalytic domain beginning with Ile16. One example is the conversion of factor X to factor Xa (see
The structural changes outlined above provide a molecular explanation for the conversion of a precursor zymogen to an active serine protease. However, unlike trypsin which is fully active following cleavage at Arg15, many of the coagulation enzymes act very poorly on their protein substrates. Even though they generally possess fully functional active sites and can cleave small peptidyl substrates, efficient cleavage of the biological substrate often requires a cofactor protein (
Depending on the state of the patient it may be desirable to develop altered coagulation cascade proteins which possess enhanced or reduced coagulation function. It is an object of the invention to provide such proteins for use as therapeutics.
In accordance with the present invention, compositions and methods are provided for influencing regulatory sites in the FX zymogen→protease transition pathway thereby driving production of a more “zymogen-like” FXa species. The compositions and methods of the invention are effective to modulate hemostasis in patients in need thereof.
In one embodiment, a variant Factor X/Factor Xa zymogen/protease which modulates hemostasis is provided. Preferably, the variant zymogen protease is encoded by SEQ ID NO: 2, wherein nucleotides 1684-1695 of SEQ ID NO: 2 can be any amino acid with the proviso that nucleotides 1684-1886 do not encode Val or Ala. More preferably, the variant zymogen/protease contains at least one modification in SEQ ID NO: 1 selected from the group consisting of a) Ile at position 16 is Leu, Phe, Asp or Gly; b) Val at position 17 is Leu, Ala, or Gly and c) Asp at position 194 is Asn or Glu. Nucleic acids encoding the variant zymogen/proteases of the invention are also disclosed as are methods of use thereof. Such nucleotides may optionally encode an intracellular PACE/furin cleavage site.
In yet another embodiment, a nucleic acid having the sequence of SEQ ID NO: 2, wherein the nucleotides at positions 1684-1695 encode the amino acids selected from the group consisting of Leu-Val-Gly, Gly-Val-Gly, Ile-Ala-Gly, Phe-Val-Gly and Ile-Gly-Gly, said nucleic acid optionally comprising nucleotides at position 2233-2235 which encode an amino acid selected from the group consisting of Asn or Glu.
A pharmaceutical composition comprising the Factor Xa variant of the invention in a biologically compatible carrier is also provided. Another preferred aspect of the invention includes methods for the treatment of a hemostasis related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of the variant Factor X/Xa zymogen/protease containing pharmaceutical compositions described herein. Such methods should have efficacy in the treatment of disorders where a pro-coagulant is needed and include, without limitation, hemophilia A and B, hemophilia A and B associated with inhibitory antibodies, coagulation factor deficiency, vitamin K epoxide reductase Cl deficiency, gamma-carboxylase deficiency, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation treatment disorders, Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency.
Certain zymogen/protease variants may be useful in the treatment of disorders where anti-coagulation is desired. Such disorders include, without limitation, thrombosis, thrombocytopenia, stroke, and coagulapathy.
Another aspect of the invention, includes host cells expressing the variant zymogen/proteases of the invention in order to produce large quantities thereof. Methods for isolating and purifying the zymogen protease variants are also disclosed.
Proteolysis is an essential aspect of blood coagulation and underlies many of the mechanisms regulating normal hemostasis. Procofactors and zymogens cannot participate to any significant degree in their respective macromolecular enzymatic complexes. This indicates that proteolytic activation must result in appropriate structural changes that lead to the expression of sites which impart enzyme, substrate and cofactor binding capabilities. While procofactor and zymogen activation has been intensively studied, the relationship between proteolysis and the expression of binding sites which impart function is incompletely understood. The present invention provides model compositions and systems which elucidate the molecular mechanisms underlying the expression of macromolecular binding interactions that accompany transitions from the zymogen state.
Factor X (FX)1 is a vitamin K-dependent two-chain glycoprotein which plays a central role in blood coagulation (
I. Definitions:
Various terms relating to the biological molecules of the present invention are used hereinabove and also throughout the specification and claims.
The phrase “variant zymogen/protease” refers to a modified FX zymogen or FXa protease which has been genetically altered such that its protease activity when converted to FXa is reduced or “zymogen-like” in the absence of specific cofactors (e.g., the binding affinity for the active site is lower than that observed in the wild type molecule. Notably, this affinity/activity is restored in the presence of the proper co-factors which include, without limitation factor Va. Preferred sites for amino acid alterations in the parent FX molecule include substitution of the isoleucine at position 16, substitution of the valine at position 17 and substitution of the aspartic acid at position 194, with the proviso that the amino acid at position 16 is not valine or alanine.
The phrase “hemostasis related disorder” refers to bleeding disorders such as hemophilia A and B, hemophilia A and B patients with inhibitory antibodies, deficiencies in coagulation Factors, VII, IX and X, XI, V, XII, II, von Willebrand factor, combined FV/FVIII deficiency, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e. FXa inhibitors); and platelet disorders such as, Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency. A hemostasis related disorder can also include bleeding related to thromboic disorders such as deep venous thrombosis, thrombosis associated with cardiovascular disease states or malignancies, thrombosis resulting from in-dwelling catheters or other invasive surgical procedures and thrombosis associated with autoimmune diseases such as lupus. The zymogen/protease variants could also provide necessary hemostasis for patients with disseminated intravascular coagulation or consumptive coagulopathies arising from a variety of disease states.
With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′ directions) in the naturally occurring genome of the organism from which it originates. For example, the “isolated nucleic acid” may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote.
With respect to RNA molecules of the invention, the term “isolated nucleic acid” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form (the term “substantially pure” is defined below).
With respect to protein, the term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.
The term “promoter region” refers to the transcriptional regulatory regions of a gene, which may be found at the 5′ or 3′ side of the coding region, or within the coding region, or within introns.
The term “vector” refers to a small carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell where it will be replicated. An “expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell.
The term “operably linked” means that the regulatory sequences necessary for expression of a coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.
The term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
The phrase “consisting essentially of” when referring to a particular nucleotide sequence or amino acid sequence means a sequence having the properties of a given SEQ ID NO:. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.
The term “oligonucleotide,” as used herein refers to primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application for which the oligonucleotide is used.
The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
The probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
The term “specifically hybridize” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
The term “primer” as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to act functionally as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3′ terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product.
The primer may vary in length depending on the particular conditions and requirements of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3′ hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
The term “percent identical” is used herein with reference to comparisons among nucleic acid or amino acid sequences. Nucleic acid and amino acid sequences are often compared using computer programs that align sequences of nucleic or amino acids thus defining the differences between the two. For purposes of this invention comparisons of nucleic acid sequences are performed using the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wisconsin. For convenience, the default parameters (gap creation penalty=12, gap extension penalty=4) specified by that program are intended for use herein to compare sequence identity. Alternately, the Blastn 2.0 program provided by the National Center for Biotechnology Information(found on the world wide web at ncbi.nlm.nih.gov/blast/; Altschul et al., 1990, J Mol Biol 215:403-410) using a gapped alignment with default parameters, may be used to determine the level of identity and similarity between nucleic acid sequences and amino acid sequences.
II. Preparation of Variant Zymogen-Protease Encoding Nucleic Acid Molecules and Polypeptides
A. Nucleic Acid Molecules
Nucleic acid molecules encoding the variant zymogen/proteases of the invention may be prepared by using recombinant DNA technology methods. The availability of nucleotide sequence information enables preparation of isolated nucleic acid molecules of the invention by a variety of means. For example, nucleic acid sequences encoding a zymogen/protease polypeptide may be isolated from appropriate biological sources using standard protocols well known in the art.
Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in a plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, Calif.), which is propagated in a suitable E. coli host cell. Alternatively, the nucleic acids may be maintained in vector suitable for expression in mammalian cells. In cases where post-translational modification affects zymogen/protease function (e.g., Factor Xa), it is preferable to express the molecule in mammalian cells.
In one embodiment, the nucleic acids encoding the factor X zymogen variants may be further modified via insertion of an intracellular proteolytic cleavage site. In order to express “activated” zymogen-like FXa variants in mammalian cells, an intracellular proteolytic cleavage site can be inserted between positions Arg15 and 16 in the variant FX zymogen. Such cleavage sites include: Arg-Lys-Arg or Arg-Lys-Arg-Arg-Lys-Arg (SEQ ID NO: 3). These cleavage sites are efficiently recognized by proteases (PACE/furin-like enzymes) within the cell and are removed. This results in a processed variant FX(a) in which the heavy chain on the molecule begins now begins at position 16. Introduction of this cleavage site at said position will allow for the intracellular conversion of FX to FXa.
In another embodiment, the entire 52 amino acid activation peptide can be removed and the intracellular protease cleavage site can be introduced in its place which will result in variant FXa.
Ultimately these types of modifications allow for secretion of the “active” processed form of variant FX from a cell that expresses the modified variant FX. Secretion of the cleaved factor obviates a need for proteolytic cleavage during blood clotting or following the isolation of the protein.
Variant zymogen/protease-encoding nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded. Thus, this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention. Such oligonucleotides are useful as probes for detecting zymogen/protease expression.
B. Proteins
A full-length or variant zymogen/protease polypeptide of the present invention may be prepared in a variety of ways, according to known methods. The protein may be purified from appropriate sources, e.g., transformed bacterial or animal cultured cells or tissues which express zymogen/protease, by immunoaffinity purification. However, this is not a preferred method due to the low amount of protein likely to be present in a given cell type at any time.
The availability of nucleic acid molecules encoding a variant zymogen/protease polypeptide enables production of zymogen/protease using in vitro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such as pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocyte lysates. In vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wis. or BRL, Rockville, Md.
Alternatively, according to a preferred embodiment, larger quantities of zymogen/protease may be produced by expression in a suitable prokaryotic or eukaryotic expression system. For example, part or all of a DNA molecule encoding variant Factor Xa for example, may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli or a mammalian cell such as CHO or Hela cells. Alternatively, in a preferred embodiment, tagged fusion proteins comprising zymogen/protease can be generated. Such zymogen/protease-tagged fusion proteins are encoded by part or all of a DNA molecule, ligated in the correct codon reading frame to a nucleotide sequence encoding a portion or all of a desired polypeptide tag which is inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli or a eukaryotic cell, such as, but not limited to, yeast and mammalian cells. Vectors such as those described above comprise the regulatory elements necessary for expression of the DNA in the host cell positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include, but are not limited to, promoter sequences, transcription initiation sequences, and enhancer sequences.
Variant zymogen/protease proteins, produced by gene expression in a recombinant prokaryotic or eukaryotic system may be purified according to methods known in the art. In a preferred embodiment, a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or nickel columns for isolation of recombinant proteins tagged with 6-8 histidine residues at their N-terminus or C-terminus. Alternative tags may comprise the FLAG epitope, GST or the hemagglutinin epitope. Such methods are commonly used by skilled practitioners.
Zymogen/protease proteins, prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such proteins may be subjected to amino acid sequence analysis, according to known methods.
As discussed above, a convenient way of producing a polypeptide according to the present invention is to express nucleic acid encoding it, by use of the nucleic acid in an expression system. A variety of expression systems of utility for the methods of the present invention are well known to those of skill in the art.
Accordingly, the present invention also encompasses a method of making a polypeptide (as disclosed), the method including expression from nucleic acid encoding the polypeptide (generally nucleic acid). This may conveniently be achieved by culturing a host cell, containing such a vector, under appropriate conditions which cause or allow production of the polypeptide. Polypeptides may also be produced in in vitro systems, such as in reticulocyte lysates.
III. Uses of Zymogen/Protease Proteins and Zymogen/Protease-Encoding Nucleic Acids
Variant zymogen/protease nucleic acids encoding polypeptides having altered protease activities may be used according to this invention, for example, as therapeutic and/or prophylactic agents (protein or nucleic acid) which modulate the blood coagulation cascade. The present inventors have discovered that factor X/Xa zymogen/protease molecules can increase coagulation and provide effective hemostasis.
A. Variant Zymogen/Protease Polypeptides
In a preferred embodiment of the present invention, variant zymogen/protease polypeptides may be administered to a patient via infusion in a biologically compatible carrier, preferably via intravenous injection. The variant zymogen/proteases of the invention may optionally be encapsulated into liposomes or mixed with other phospholipids or micelles to increase stability of the molecule. Zymogen/protease may be administered alone or in combination with other agents known to modulate hemostasis (e.g., Factor V, Factor Va or derivatives thereof). An appropriate composition in which to deliver zymogen/protease polypeptides may be determined by a medical practitioner upon consideration of a variety of physiological variables, including, but not limited to, the patient's condition and hemodynamic state. A variety of compositions well suited for different applications and routes of administration are well known in the art and are described hereinbelow.
The preparation containing the purified factor X/Xa analog contains a physiologically acceptable matrix and is preferably formulated as a pharmaceutical preparation. The preparation can be formulated using substantially known prior art methods, it can be mixed with a buffer containing salts, such as NaCl, CaCl2, and amino acids, such as glycine and/or lysine, and in a pH range from 6 to 8. Until needed, the purified preparation containing the factor X/Xa analog can be stored in the form of a finished solution or in lyophilized or deep-frozen form. Preferably the preparation is stored in lyophilized form and is dissolved into a visually clear solution using an appropriate reconstitution solution.
Alternatively, the preparation according to the present invention can also be made available as a liquid preparation or as a liquid that is deep-frozen.
The preparation according to the present invention is especially stable, i.e., it can be allowed to stand in dissolved form for a prolonged time prior to application.
The preparation according to the present invention which contains a factor X analog in combination with factor XIa or a derivative thereof which is able to activate the factor X analog into factor Xa or the factor Xa analog can be made available in the form of a combination preparation comprising a container that holds factor XIa which is immobilized on a matrix, potentially in the form of a miniature column or a syringe complemented with a protease, and a container containing the pharmaceutical preparation with the factor X analog. To activate the factor X analog, the factor X analog-containing solution, for example, can be pressed over the immobilized protease. During storage of the preparation, the factor X analog-containing solution is preferably spatially separated from the protease. The preparation according to the present invention can be stored in the same container as the protease, but the components are spatially separated by an impermeable partition which can be easily removed before administration of the preparation. The solutions can also be stored in separate containers and be brought into contact with each other only shortly prior to administration.
The factor X analog can be activated into factor Xa shortly before immediate use, i.e., prior to the administration to the patient. The activation can be carried out by bringing a factor X analog into contact with an immobilized protease or by mixing solutions containing a protease, on the one hand, and the factor X analog, on the other hand. Thus, it is possible to separately maintain the two components in solution and to mix them by means of a suitable infusion device in which the components come into contact with each other as they pass through the device and thereby to cause an activation into factor Xa or into the factor Xa analog. The patient thus receives a mixture of factor Xa and, in addition, a serine protease which is responsible for the activation. In this context, it is especially important to pay close attention to the dosage since the additional administration of a serine protease also activates endogenous factor X, which may shorten the coagulation time.
The preparation according to the present invention can be made available as a pharmaceutical preparation with factor Xa activity in the form of a one-component preparation or in combination with other factors in the form of a multi-component preparation.
Prior to processing the purified protein into a pharmaceutical preparation, the purified protein is subjected to the conventional quality controls and fashioned into a therapeutic form of presentation. In particular, during the recombinant manufacture, the purified preparation is tested for the absence of cellular nucleic acids as well as nucleic acids that are derived from the expression vector, preferably using a method, such as is described in EP 0 714 987.
Another feature of this invention relates to making available a preparation which contains a factor Xa analog with a high stability and structural integrity and which, in particular, is free from inactive factor X/Xa analog intermediates and autoproteolytic degradation products and which can be produced by activating a factor X analog of the type described above and by formulating it into an appropriate preparation.
The pharmaceutical preparation may contain dosages of between 10-1000 μg/kg, more preferably between about 10-250 μg/kg and most preferably between 10 and 75 μg/kg, with 40 μg/kg of the variant factor X polypeptide being particularly preferred. Patients may be treated immediately upon presentation at the clinic with a bleed. Alternatively, patients may receive a bolus infusion every one to three hours, or if sufficient improvement is observed, a once daily infusion of the variant factor Xa described herein.
B. Zymogen/protease-Encoding Nucleic Acids
Zymogen/protease-encoding nucleic acids may be used for a variety of purposes in accordance with the present invention. In a preferred embodiment of the invention, a nucleic acid delivery vehicle (i.e., an expression vector) for modulating blood coagulation is provided wherein the expression vector comprises a nucleic acid sequence coding for a variant zymogen/protease polypeptide, or a functional fragment thereof as described herein. Administration of zymogen/protease-encoding expression vectors to a patient results in the expression of zymogen/protease polypeptide which serves to alter the coagulation cascade. In accordance with the present invention, an zymogen/protease encoding nucleic acid sequence may encode an zymogen/protease polypeptide as described herein whose expression increases hemostasis. In a preferred embodiment, a zymogen/protease nucleic acid sequence encodes a human Factor Xa polypeptide variant.
Expression vectors comprising variant X/Xa zymogen/protease nucleic acid sequences may be administered alone, or in combination with other molecules useful for modulating hemostasis. According to the present invention, the expression vectors or combination of therapeutic agents may be administered to the patient alone or in a pharmaceutically acceptable or biologically compatible compositions.
In a preferred embodiment of the invention, the expression vector comprising nucleic acid sequences encoding the variant zymogen/protease variants is a viral vector. Viral vectors which may be used in the present invention include, but are not limited to, adenoviral vectors (with or without tissue specific promoters/enhancers), adeno-associated virus (AAV) vectors of multiple serotypes (e.g., AAV-2, AAV-5, AAV-7, and AAV-8) and hybrid AAV vectors, lentivirus vectors and pseudo-typed lentivirus vectors [e.g., Ebola virus, vesicular stomatitis virus (VSV), and feline immunodeficiency virus (FIV)], herpes simplex virus vectors, vaccinia virus vectors, and retroviral vectors.
In a preferred embodiment of the present invention, methods are provided for the administration of a viral vector comprising nucleic acid sequences encoding a variant zymogen/protease, or a functional fragment thereof. Adenoviral vectors of utility in the methods of the present invention preferably include at least the essential parts of adenoviral vector DNA. As described herein, expression of a variant zymogen/protease polypeptide following administration of such an adenoviral vector serves to modulate hemostasis. In the context of the variant Factor Xa described herein, such administration enhances the procoagulation activity of the protease.
Recombinant adenoviral vectors have found broad utility for a variety of gene therapy applications. Their utility for such applications is due largely to the high efficiency of in vivo gene transfer achieved in a variety of organ contexts.
Adenoviral particles may be used to advantage as vehicles for adequate gene delivery. Such virions possess a number of desirable features for such applications, including: structural features related to being a double stranded DNA nonenveloped virus and biological features such as a tropism for the human respiratory system and gastrointestinal tract. Moreover, adenoviruses are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis. Attesting to the overall safety of adenoviral vectors, infection with adenovirus leads to a minimal disease state in humans comprising mild flu-like symptoms.
Due to their large size (˜36 kilobases), adenoviral genomes are well suited for use as gene therapy vehicles because they can accommodate the insertion of foreign DNA following the removal of adenoviral genes essential for replication and nonessential regions. Such substitutions render the viral vector impaired with regard to replicative functions and infectivity. Of note, adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes.
For a more detailed discussion of the use of adenovirus vectors utilized for gene therapy, see Berkner, 1988, Biotechniques 6:616-629 and Trapnell, 1993, Advanced Drug Delivery Reviews 12:185-199.
It is desirable to introduce a vector that can provide, for example, multiple copies of a desired gene and hence greater amounts of the product of that gene. Improved adenoviral vectors and methods for producing these vectors have been described in detail in a number of references, patents, and patent applications, including: Mitani and Kubo (2002, Curr Gene Ther. 2(2):135-44); Olmsted-Davis et al. (2002, Hum Gene Ther. 13(11):1337-47); Reynolds et al. (2001, Nat Biotechnol. 19(9):838-42); U.S. Pat. No. 5,998,205 (wherein tumor-specific replicating vectors comprising multiple DNA copies are provided); U.S. Pat. No. 6,228,646 (wherein helper-free, totally defective adenovirus vectors are described); U.S. Pat. No. 6,093,699 (wherein vectors and methods for gene therapy are provided); U.S. Pat. No. 6,100,242 (wherein a transgene-inserted replication defective adenovirus vector was used effectively in in vivo gene therapy of peripheral vascular disease and heart disease); and International Patent Application Nos. WO 94/17810 and WO 94/23744.
For some applications, an expression construct may further comprise regulatory elements which serve to drive expression in a particular cell or tissue type. Such regulatory elements are known to those of skill in the art and discussed in depth in Sambrook et al. (1989) and Ausubel et al. (1992). The incorporation of tissue specific regulatory elements in the expression constructs of the present invention provides for at least partial tissue tropism for the expression of the variant zymogen/proteases or functional fragments thereof. For example, an E1 deleted type 5 adenoviral vector comprising nucleic acid sequences encoding variant zymogen/protease under the control of a cytomegalovirus (CMV) promoter may be used to advantage in the methods of the present invention.
Adenoviral vectors for recombinant gene expression have been produced in the human embryonic kidney cell line 293 (Graham et al., 1977, J. Gen. Virol. 36:59-72). This cell line is permissive for growth of adenovirus 2 (Ad2) and adenovirus 5 mutants defective in E1 functions because it comprises the left end of the adenovirus 5 genome and, therefore, expresses E1 proteins. E1 genes integrated into the cellular genome of 293 cells are expressed at levels which facilitate the use of these cells as an expression system in which to amplify viral vectors from which these genes have been deleted. 293 cells have been used extensively for the isolation and propagation of E1 mutants, for helper-independent cloning, and for expression of adenovirus vectors. Expression systems such as the 293 cell line, therefore, provide essential viral functions in trans and thereby enable propagation of viral vectors in which exogenous nucleic acid sequences have been substituted for E1 genes. See Young et al. in The Adenoviruses, Ginsberg, ed., Plenum Press, New York and London (1984), pp. 125-172.
Other expression systems well suited to the propagation of adenoviral vectors are known to those of skill in the art (e.g., HeLa cells) and have been reviewed elsewhere.
Also included in the present invention is a method for modulating hemostasis comprising providing cells of an individual with a nucleic acid delivery vehicle encoding a variant zymogen/protease polypeptide and allowing the cells to grow under conditions wherein the zymogen/protease polypeptide is expressed.
From the foregoing discussion, it can be seen that zymogen/protease polypeptides, and zymogen/protease polypeptide expressing nucleic acid vectors may be used in the treatment of disorders associated with aberrant blood coagulation.
C. Pharmaceutical Compositions
The expression vectors of the present invention may be incorporated into pharmaceutical compositions that may be delivered to a subject, so as to allow production of a biologically active protein (e.g., a variant zymogen/protease polypeptide or functional fragment or derivative thereof). In a particular embodiment of the present invention, pharmaceutical compositions comprising sufficient genetic material to enable a recipient to produce a therapeutically effective amount of a variant zymogen/protease polypeptide can influence hemostasis in the subject. Alternatively, as discussed above, an effective amount of the variant Factor X polypeptide may be directly infused into a patient in need thereof. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents (e.g., co-factors) which influence hemostasis.
In preferred embodiments, the pharmaceutical compositions also contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., 18th Edition, Easton, Pa. [1990]).
Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding, free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they may be placed in an appropriate container and labeled for treatment. For administration of zymogen/protease-containing vectors or polypeptides, such labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the capability of a skilled medical practitioner using the techniques and guidance provided in the present invention. Therapeutic doses will depend on, among other factors, the age and general condition of the subject, the severity of the aberrant blood coagulation phenotype, and the strength of the control sequences regulating the expression levels of the variant zymogen/protease polypeptide. Thus, a therapeutically effective amount in humans will fall in a relatively broad range that may be determined by a medical practitioner based on the response of an individual patient to vector-based zymogen/protease treatment.
D. Administration
The variant Factor X polypeptides, alone or in combination with other agents may be directly infused into a patient in an appropriate biological carrier as described hereinabove. Expression vectors of the present invention comprising nucleic acid sequences encoding variant zymogen/protease, or functional fragments thereof, may be administered to a patient by a variety of means (see below) to achieve and maintain a prophylactically and/or therapeutically effective level of the zymogen/protease polypeptide. One of skill in the art could readily determine specific protocols for using the zymogen/protease encoding expression vectors of the present invention for the therapeutic treatment of a particular patient. Protocols for the generation of adenoviral vectors and administration to patients have been described in U.S. Pat. Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242; and International Patent Application Nos. WO 94/17810 and WO 94/23744, which are incorporated herein by reference in their entirety.
Variant zymogen/protease encoding adenoviral vectors of the present invention may be administered to a patient by any means known. Direct delivery of the pharmaceutical compositions in vivo may generally be accomplished via injection using a conventional syringe, although other delivery methods such as convection-enhanced delivery are envisioned (See e.g., U.S. Pat. No. 5,720,720). In this regard, the compositions may be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intraarterially, orally, intrahepatically or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications. A clinician specializing in the treatment of patients with blood coagulation disorders may determine the optimal route for administration of the adenoviral vectors comprising zymogen/protease nucleic acid sequences based on a number of criteria, including, but not limited to: the condition of the patient and the purpose of the treatment (e.g., enhanced or reduced blood coagulation).
The present invention also encompasses AAV vectors comprising a nucleic acid sequence encoding a variant zymogen/protease polypeptide.
Also provided are lentivirus or pseudo-typed lentivirus vectors comprising a nucleic acid sequence encoding a variant zymogen/protease polypeptide
Also encompassed are naked plasmid or expression vectors comprising a nucleic acid sequence encoding a variant zymogen/protease polypeptide.
Proteolytic processing of precursor plasma proteins to affect activation is a hallmark of blood coagulation. The paradigm for this type of activation mechanism is the zymogen to protease transition in the chymotrypsin-like serine protease family. Bond cleavage at a highly conserved site (Arg15-Ile16; chymotrypsin numbering system) unmasks a new N-terminus which acts as an intramolecular ligand for Asp194 (
The basic principles of the zymogen to active enzyme transition for serine proteases at the structural level are well documented, particularly for chymotrypsin and trypsin and these examples serve as the paradigm for the serine protease family. General aspects can be summarized as follows (see
It is well known that the activation of FX results in major conformational changes in the serine protease domain which are accompanied by the ability of the protease to bind with much greater affinity to S1-directed probes and membrane-bound FVa (1-6). The overall molecular mechanism(s) which governs the transition of serine proteases is generally assumed to follow that of the trypsin system. However, this may not uniformly be the case. Single-chain tPA employs a different molecular strategy to maintain its zymogen-like state (8-11). Analysis of zymogen/protease pairs involved in blood coagulation, particularly FVII/FVIIa, indicates that several differences exist in this transition compared to the trypsin system (12). While several structural determinants on FXa are part of the so-called activation domain, it is currently unclear if formation of these sites is directly linked to the zymogen to protease transition. A recently described model of the zymogen FX suggests however that several of these elements may be disordered in the zymogen (13). Comparison of the zymogen model with the active enzyme reveals that residues making up the Ca2+ (Asp70-Glu80), Na+ (Ala183-Asp194; Gly219-Gly226) and autolysis loops (Thr144-Arg150) undergo major changes in their backbone positions upon the zymogen to protease transition. Since it is already well-documented, at least for trypsinogen/trypsin, that the S1 specificity site and formation of Ile16-Asp194 are allosterically linked, it is reasonable to hypothesize that other elements of the activation domain are also linked to the zymogen to protease transition. In the present example, we have designed experiments to test the hypothesis that destabilization of the Ile16-Asp194 internal salt bridge by making changes to position 16, 17, or 194 alters the active site cleft making the resulting variant “zymogen-like”. We also hypothesized that these changes would allosterically modulate FVa binding.
While there are several reports in the literature on the expression of rFX, most have relied on truncated versions or have not provided adequate characterization (15-20). Our initial attempts at expressing rFX in HEK 293 cells resulted in expression levels in the range of 1-2 mg rFX/L of conditioned media; however, only 10-40% of the material produced was found to be fully γ-carboxylated (21). The remaining material showed no γ-carboxylation. We took advantage of the different binding affinities of the vitamin K-dependent propeptides for the carboxylase and hypothesized that since the prothrombin propeptide exhibits the lowest affinity for the carboxylase, exchanging the propeptide of FX (highest affinity) with that of prothrombin may enhance γ-carboxylation by allowing for greater substrate turnover (22, 23). Using this new vector, stable transfectants of HEK 293 cells were selected, expanded, and rFX was purified by immunoaffinity chromatography. Phosphate elution from hydroxyapatite was used to separate carboxylated material away from uncarboxylated material. Our results, obtained now from over 30 stable cell lines indicate that on average ˜80-90% of the rFX is fully γ-carboxylated compared to 10-40% using the native FX propeptide. These results have recently been published and this strategy has subsequently been employed by at least one other laboratory (24,25). Thus, using this new expression system we are now producing milligram quantities (15-25 mg of fully γ-carboxylated rFX from ˜10 L of conditioned media) of protein for detailed structure/function studies.
Enzyme concentrations will be determined by active-site titration with ρ-nitrophenyl ρ-guanidinobenzoate (IIa) or fluorescein mono-ρ-guanidinobenzoate (FXa) (26, 27). FXa chromogenic substrate activity, in the presence or absence of various inhibitors, will be measured from initial rates of hydrolysis of Spectrozyme FXa, S-2222, or S-2765 as previously described (14). Kinetic parameters will be determined by least-squares fitting of the initial rate data to appropriate equations.
The FX mutants were generated using the Quick-change site-directed mutagenesis kit (Stratagene) and the entire FX cDNA was sequenced to verify the identity of the product. The various plasmids were transiently transfected into HEK 293 cells using Lipofectamine-2000. 48 hr post-transfection, media was collected and FX antigen levels were determine using a FX-specific ELISA and FXa activity was assessed by chromogenic assay prior to activation by RVV-X or by tissue factor-FVIIa.
Generation of FXa species distributed along the zymogen-protease transition pathway is outlined in Table 1. Transient transfection results indicate that we have generated a series of FXa variants with variable amounts of activity, ranging from ˜25% to <1%. We hypothesize that these differences in activity likely reflect FX variants which are shifted to varying degrees along the zymogen to protease transition. Stated differently, the Ile16-Asp194 internal salt bridge is stabilized to varying degrees depending upon the amino acid at positions 16, 17 or 194. We have chosen three of these variants (rFXaI16L, FXaI16G and FXaV17A) for further characterization.
a [FX] (nM)
b FXa Activity (nM)
c % wt
b FXa Activity (nM)
c % wt
a Antigen levels are calculated from a FX-specific ELISA and expressed as nM FX
b FXa activity levels are based on the rate of peptidyl substrate hydrolysis following activation by RVV-X or TF-FVIIa of a given FX variant and initial rates of hydrolysis are compared to FXa standard curve.
c % wt values are based upon comparison to wt-FXa activity levels. The values have been adjusted for antigen levels.
Stable cell lines in HEK293 cells were established and each of the zymogens were purified from 10 L of conditioned media (14, 24). The variants were activated with RVV-X and subsequently purified by gel filtration chromatography (14,24). SDS-PAGE of the variants prior to and following activation (reducing and non-reducing) is shown in
We first focused on assessing changes to the active site environment of each of the variants using specific probes that target this region of FXa. Kinetic studies using peptidyl substrates and active site directed probes revealed that FXaI16L and FXaV17A have an impaired ability to bind these probes (15 to 25-fold increase in the Km or Ki) while the rate of catalysis (kcat) was reduced by 3-fold compared to wild-type FXa (plasma-derived and recombinant) (Tables 2 and 3). Factor Xa I16G was not inhibited by any of the probes examined and its chromogenic activity was severely impaired (500 to 1000-fold) precluding calculation of kinetic parameters. These data are consistent with the idea that destabilization of internal salt-bridge formation (Ile16-Asp194), influences binding at the S1 specificity site. In contrast to these results, the assembly of FXaI16L and FXaV17A into prothrombinase almost completely restored the Km for peptidyl substrates while the kcat was still 3-fold reduced, indicating that FVa binding can rescue binding at the active site (Tables 2 and 3). Surprisingly even the Km value for I16G was almost completely restored (3-fold increased compared to wild-type FXa) when assembled in prothrombinase; however a 60-fold reduction in the kcat was found.
Consistent with these data, kinetic studies using prothrombin revealed that the Km values obtained for each of these variants assembled in prothrombinase were essentially equivalent to the wild-type enzyme, while the kcat values where reduced to a similar extent as for the chromogenic substrates (Table 4). Taken together, our results indicate that the zymogen to protease transition for FX not only influences the formation of the S1 site, but also contributes in a substantial way to the formation of a FVa binding site. Since direct binding of these FXa variants to FVa rescues binding at S1 site, allosteric linkage likely exists between these sites. Collectively these studies have illustrated a unique way to modify the zymogen to protease transition pathway and have revealed a possible way to develop zymogen-like forms of enzymes which are “activated” following strong ligand binding such as cofactor proteins.
The results obtained with the chromogenic substrate and the active site-directed inhibitors, indicate that the zymogen-like FXa variants bind to active site probes with reduced affinity. However, assembly of these variants into prothrombinase significantly improves the affinity for active site probes, suggesting that FVa binding can rescue binding at the active site. We next investigated whether the reverse is also true, that is occupation of the zymogen-like active site influences binding to FVa. In order to assess this hypothesis we measured the binding constants between FVa and FXaI16L and FXaV17A. To accomplish this we incubated FVa with a fluorescent derivative of inactive wtFXa in the presence of synthetic phospholipid vesicles and Ca2+ ions. The formation of the complex results in the increase of the fluorescent signal relative to fluorescent FXa alone. We then added increasing concentrations of a non-fluorescent FXa which, if it can bind FVa, will displace the fluorescent FXa, resulting in a decrease of the fluorescent signal. As a control we added S195A FXa as a competitor. This mutant is inactive because it is missing the Ser of the catalytic triad, but has a high affinity for FVa (Table 5). In contrast, when we added either FXaI16L or FXaV17A the affinity of these zymogen-like variants for FXa was significantly reduced compared to FXaS195A (Table 5). We next examined whether covalent occupation of the active site of FXaI16L could restore binding to FVa. To do this we modified the active site of wild-type FXa and FXaI16L with an irreversible inhibitor (EGR-chloromethyl ketone) and then repeated the experiment. The data show that that active site blocked FXaI16L bound FVa-membranes with the same affinity as wild-type active site blocked FXa. This indicates that occupation of the zymogen-like FXa active site has a direct influence on the binding to FVa.
Based on the observation that the zymogen-like FXa derivatives have poor reactivity with active-site directed probes and inhibitors in the absence of FVa, but apparently near normal activity when the variants are assembled in prothrombinase, we next evaluated the activity of FXaI16L in a plasma environment. Hemophilic A (data not shown) or B plasma was spiked with wild-type FXa to correct the clotting time (aPTT) of these plasmas; 0.1 nM wtFXa gave a clotting time of ˜32 sec. The addition of the same concentration of FXaI16L gave a clot time of ˜42 sec which is ˜50-70% of the activity relative to wtFXa, suggesting that this zymogen-like variant has almost normal clotting activity in plasma. Next we monitored the half-life of wild-type FXa and FXaI16L in hemophilia B plasma. The proteins were added to HB plasma and at different time points, an aliquot of the mixture was withdrawn and assayed in an aPTT-based assay. The results with HB plasma show that the relative residual activity of wild-type FXa was inhibited very rapidly (<2-min) (
We next evaluated the ability of zymogen-like FXaI16L to modulate hemostasis in a murine model of hemophilia (Schlachterman, et. al., 2005, J. Thromb. Haemost., 3, 2730-2737). The aPTT value of hemophilia B mice (C57BL/6) is approximately 50-55 sec. Factor XaI16L (200 μg/kg; n=7) or PBS (n=4) were injected via the tail vein of hemophilia B mice. At selected time points (5 and 30 min) blood was collected and an aPTT was performed on all samples. As shown in
To further test the effectiveness of FXaI16L in vivo, we examined whether this molecule could correct the bleeding time of hemophilia B mice following injury to the tail (Schlachterman, et. al., 2005, J. Thromb. Haemost., 3, 2730-2737). Blood loss was measured during a 10-min period after sectioning the distal part of the tail. In this type of assay, blood loss is minimal in normal wild-type Balb-c mice (n=7) and quite substantial in PBS injected (n=6) hemophilia B mice (Balb c) following the tail injury (
While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.
This application is a continuation application of U.S. patent application Ser. No. 15/229,922 filed Aug. 5, 2016, now U.S. Pat. No. 9,896,676 which is a divisional application of Ser. No. 13/726,187 filed Dec. 23, 2012, now U.S. Pat. No. 9,410,137 which is a divisional application of Ser. No. 12/093,783, filed Jul. 16, 2008 now U.S. Pat. No. 8,383,386, which is a National Phase application of PCT/US06/60927 filed Nov. 15, 2006, which in turn claims priority to U.S. Provisional Application 61/163,703 filed Mar. 26, 2005, the entire contents of each being incorporated herein by reference as though set forth in full.
This invention was made with government support under Grant No. PO1 HL-74124-01 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60736680 | Nov 2005 | US |
| Number | Date | Country | |
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| Parent | 13726187 | Dec 2012 | US |
| Child | 15229922 | US | |
| Parent | 12093783 | Jul 2008 | US |
| Child | 13726187 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15229922 | Aug 2016 | US |
| Child | 15899609 | US |
