WO2011075606A2 - Hyperglycosylated polypeptide variants and methods of use - Google Patents

Hyperglycosylated polypeptide variants and methods of use Download PDF

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WO2011075606A2
WO2011075606A2 PCT/US2010/060891 US2010060891W WO2011075606A2 WO 2011075606 A2 WO2011075606 A2 WO 2011075606A2 US 2010060891 W US2010060891 W US 2010060891W WO 2011075606 A2 WO2011075606 A2 WO 2011075606A2
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amino acid
polypeptide
hyperglycosylated
amino acids
expression cassette
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PCT/US2010/060891
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French (fr)
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WO2011075606A3 (en
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Jin Hong
Lawrence Blatt
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Alios Biopharma, Inc.
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Publication of WO2011075606A3 publication Critical patent/WO2011075606A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormones [GH] (Somatotropin)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/91—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation

Definitions

  • the present application relates to the fields of chemistry, biochemistry and medicine. More particularly, disclosed herein are hyperglycosylated polypeptide variants, pharmaceutical compositions that include one or more hyperglycosylated polypeptide variants, and methods of treating diseases and/or conditions with one or more hyperglycosylated polypeptide variants.
  • Polypeptides are used for a wide range of applications, including industrial uses, and human and veterinary therapy.
  • General recognized drawbacks to the use of many polypeptides include immunogenicity, susceptibility to proteolytic degradation by enzymes produced by the host, suboptimal pharmacokinetic properties such as stability and/or serum half-life, and the like.
  • hyperglycosylated polypeptide variant of a parent polypeptide wherein the hyperglycosylated polypeptide variant can be the parent polypeptide that has been modified to include a peptide extension inserted at a terminal region, where the peptide extension can be a peptide of 1-200 consecutive amino acids and can include at least two glycosylation sites, where the terminal region can be selected from the group consisting of an amino-terminal region that consists of the first 15 amino acids at the amino-terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide and a carboxy-terminal region that consists of the last 15 amino acids at the carboxy-terminus of the parent polypeptide.
  • the peptide extension can include at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites.
  • the parent polypeptide has been further modified to include at least one or more additional glycosylation sites through at least one amino acid substitution or at least one combination of amino acid substitutions in the amino acid sequence of the parent polypeptide.
  • compositions that can include one or more hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein, and a pharmaceutically acceptable carrier, excipient, or combination thereof.
  • Some embodiments disclosed herein relate to a method of ameliorating and/or treating a fibrotic disorder. Other embodiments disclosed herein relate to a method of ameliorating and/or treating cancer. Still other embodiments disclosed herein relate to a method of inhibiting the growth of a tumor. Yet other embodiments disclosed herein relate to a method of ameliorating and/or treating a viral infection.
  • Some embodiments disclosed herein relate to an expression cassette for expression a hyperglycosylated polypeptide, comprising a promoter operably linked to a nucleotide sequence encoding a signal peptide, a first extension sequence encoding a first peptide extension, and a gene encoding a biologically-active polypeptide, where the first peptide extension can be a peptide of 1-200 consecutive amino acids including at least two glycosylation sites.
  • the expression cassette can further include a second extension sequence encoding a second peptide extension.
  • the expression cassette can be provided without a gene encoding a biologically-active polypeptide, but instead having a restriction site allowing for insertion of any such desired gene.
  • the expression cassette can be in the form of a linear or circular DNA, for example, and may advantageously be in the form of a plasmid.
  • Some embodiments disclosed herein relate to an expression vector comprising one or more expression cassettes disclosed herein. Some embodiments disclosed herein relate to a recombinant host cell comprising one or more expression vectors and/or cassette disclosed herein.
  • Some embodiments disclosed herein relate to a method for producing a hyperglycosylated polypeptide variant of a parent polypeptide discosed herein.
  • hyperglycosylated polypeptide where the hyperglycosylated polypeptide comprises a biologically-active polypepide linked to a peptide extension, wherein the peptide extension is a peptide of 1 -200 consecutive amino acids and comprises at least two glycosylation sites, where the biologically-active polypeptide optionally comprises one or more glycosylation sites.
  • Figure 1 shows the amino acid sequence of human growth hormone 1 (hGH-1, SEQ ID NO: 1), wherein the signal peptide is shown in bold letters.
  • Figure 2 shows the amino acid sequence of human insulin-like growth factor 1A (hIGF- ⁇ , SEQ ID NO: 8), wherein the signal peptide is shown in bold letters.
  • Figure 3 shows the amino acid sequence of human granulocyte colony- stimulating factor (hG-CSF, SEQ ID NO: 15), wherein the signal peptide is shown in bold letters.
  • Figure 4 shows the amino acid sequence of human Erythropoietin (hEPO, SEQ ID NO: 22), wherein the signal peptide is shown in bold letters.
  • Figure 5 shows the amino acid sequence of human insulin (SEQ ID NO: 29), wherein the signal peptide is shown in bold letters.
  • Figure 6 are schematic diagrams showing some embodiments of the expression cassette for expressing a hyperglycosylated polypeptide.
  • Figures 7A-I show the structure and sequence of the expression cassette in expression vectors pC6-l-hGHl, pN6-l-hGHl, pN6-2-hGHl, pNlO-l-hGHl, pN10-C4-l- hGHl, pN10-C6-l-hGHl, P N14-l-hGHl, P N14(2)-l-hGHl, and P N14(3)-l-hGHl, respectively.
  • the main cassette cloning sites are the upstream Not I site and downstream Pme I or EcoR I site.
  • Figure 8 shows the structure and sequence of the expression cassette in expression vector pNlO-1-hIGFlA.
  • the main cassette cloning sites are the upstream Not I site and downstream EcoR I site.
  • Figure 9 shows the structure and sequence of the expression cassette in expression vector pNlO-l-hG-CSF.
  • the main cassette cloning sites are the upstream Not I site and downstream EcoR I site.
  • Figure 10 shows the structure and sequence of the expression cassette in expression vector pNlO-l-hEPO.
  • the main cassette cloning sites are the upstream Not I site and downstream EcoR I site.
  • Figure 11 shows the structure and sequence of the expression cassette in expression vector pN 10-1 -Insulin.
  • the main cassette cloning sites are the upstream Not I site and downstream EcoR I site.
  • Figure 12 A-E shows the western blot of various hyperlgyocylated variants of the parent interferon alfacon-1 (CIFN).
  • the interferons shown in Figure 12A are CIFN-31-102-108, CIFN-Nl-31-102-108, CIFN-N2-31-102- 108, CIFN-N3-31-102-108, and CIFN-N4-31-102- 108.
  • the interferons shown in Figure 12B are CIFN-102-138 and CIFN- 102-138-C2.
  • the interferons shown in Figure 12C are CIFN-N 14(3)- 102, CIFN-N 14(3)- 102- 138, CIFN-N14(3)-108-138, and CIFN-N 14(3)- 108.
  • the interferons shown in Figure 12D are CIFN-Nl l-31-102-138, CIFN-N8-31-102- 138, CIFN-N6-31-102- 138, and CIFN-N4-31- 102-138.
  • CIFN-N11-31-102-138, CIFN-N11-8-102-138 and CIFN-N6-31- 102-138 were digested with (+) or without (-) glycosidase PNGase F.
  • Figure 13 A-D shows the HCV replicon activities and interferon specific activities of CIFN and variouis hyperglycosylated variants of the parent CIFN.
  • Figure 13 A shows the HCV replicon activities of CIFN and CIFN-N14(3)-102.
  • Figure 13B shows the HCV replicon activities of CIFN-N14(3)-108 and CIFN-N14(3)-102-138.
  • Figure 13C shows the HCV replicon activity of CIFN-N14(3)-108-138.
  • Figure 13D shows the interferon specific activities of CIFN, CIFN-N14(3)-102, CIFN-N14(3)-102-138, CIFN-N14(3)-108, and CIFN-N14(3)-108-138.
  • Figure 14 shows the western blot of various hyperglycosylated variants of the parent human interferon beta (IFNB) having an N-terminal peptide extension with 10 glycosylation sites.
  • Variant EPO-N10 has the signal peptide from EPO (SEQ ID NO: 165)
  • variant CD33-N10 has the signal peptide from CD33 (SEQ ID NO: 164)
  • variant IFNB-N10 has the signal peptide from IFNB (SEQ ID NO: 168)
  • variant DS-N10 has an artificial signal peptide (SEQ ID NO: 163).
  • Figure 15 shows the western blot of IFNB and various hyperglycosylated variants of the parent IFNB.
  • the interferons shown in Figure 15A are IFNB-N10, IFNB-N8, IFNB-N6, and IFNB-N4.
  • IFNB-N10, IFNB-N8 and IFNB-N6 were digested with (+) or without (-) glycosidase PNGase F.
  • Figure 16 shows the western blot of hyperglycosylated variants of the parent human interferon alpha 1 (IFN al), the parent human interferon gamma (IFNG) and the parent human interferons lambdal (IFN ⁇ ), lambda2 (IFN ⁇ 2) and lambda3 (IFN ⁇ 3).
  • the interferon shown in Figure 16A is IFN Ocl-N10.
  • the interferons shown in Figure 16B are IFNG-N5 and IFNG-N10.
  • the interferons shown in Figure 16C are IFN ⁇ 1- ⁇ 16, IFN ⁇ 2- N16 and IFN ⁇ 3- ⁇ 16.
  • Figure 17 shows the western blot of hGHl-N6, a hyperglycosylated variant of the parent human growth horman 1 (hGHl).
  • Figure 18A shows a graph illustrating the IFN activity of mN14-CIFN- 108 in mouse plasma over time after a single subcutaneous injection in mice.
  • Figure 18B shows graphs illustrating the beta2-microglobulin protein level, and the induction of OAS1 mRNA in liver in mice over time after a single subcutaneous injection.
  • the term "hyperglycosylated polypeptide variant of a parent polypeptide” refers to a polypeptide variant that includes one or more additional glycosylation sites that are not present in a parent polypeptide.
  • the parent polypeptide has been modified to include a peptide extension inserted at a terminal region, where the peptide extension includes one or more glycosylation sites.
  • the parent polypeptide has been modified to include (1) one or more additional glycosylation sites in the amino acid sequence of the parent polypeptide, wherein the additional glycosylation site(s) are introduced by an amino acid substitution and/or a combination of amino acid substitutions, and (2) a peptide extension inserted at a terminal region, where the peptide extension includes one or more glycosylation sites.
  • the parent polypeptide has been modified only by inclusion of the one or more additional glycosylation sites and the peptide extension on either the carboxy or amino terminus of the parent polypeptide.
  • Each added glycosylation site may be an N-linked glycosylation site or an O-linked glycosylation site.
  • native glycosylation site refers to a glycosylation site that exists in a first naturally-occurring polypeptide and is introduced into a second naturally-occurring or a non-naturally occurring polypeptide at a homologous amino acid position.
  • a native glycosylation site can exist in one or more naturally-occurring polypeptides, and the native glycosylation site can be glycosylated or non-glycosylated in the first naturally-occurring polypeptide.
  • the glycosylation site N-L-S at the 25th, 26th, 27th amino acids of the sequence of interferon ocl4 can be introduced at amino acid positions 25, 26 and 27 of the sequence of interferon alfacon-1 as a native glycosylation site.
  • non-native glycosylation site refers to a glycosylation site that does not exist in any naturally-occurring polypeptide, as well as a glycosylation site that exists in a first naturally-occurring polypeptide and is introduced into a second naturally-occurring or a non-naturally occurring polypeptide at a non-homologous amino acid position.
  • the glycosylation site (N-L-S) at amino acid positions 25, 26 and 27 of the sequence of interferon ocl4 can be introduced at amino acid positions 101, 102 and 103 of the sequence of interferon alfacon-1 (D-E-S) as a non-native glycosylation site.
  • the glycosylation site (N-S-S) at amino acid positions 95, 96 and 97 of the sequence of interferon ocl4 can be introduced to a peptide extension that is inserted at the N-terminal region of human insulin as a non-native glycosylation site.
  • non-native and native glycosylation sites include N-linked glycosylation sites and O-linked glycosylation sites.
  • N-linked glycosylation sites include, for example, Asn-X-Ser/Thr (N-X-S/T), where the Asparagine (Asn) residue provides a site for N-linked glycosylation and X is any amino acid residue.
  • O-linked glycosylation sites include at least one serine or threonine residue.
  • a number of O-linked glycosylation sites are known in the art and have been described in, for example, Ten Hagen et al., J. Biol.
  • Standard techniques can be used to determine whether a polypeptide comprises N-linked and/or O-linked glycosylation. See, for example, R. Townsend and A. Hotchkiss, eds., Techniques in Glycobiology, Marcel Dekker Inc., 1997; and E. Hounsell, ed. Gly co analy is Protocols (Methods in Molecular Biology, Vol. 76), Humana Press, 1998. For example, the change in electrophoretic mobility of a protein before and after treatment with chemical or enzymatic deglycosylation can be used to determine the glycosylation status of a protein.
  • Enzymatic deglycosylation can be carried out using any of a variety of enzymes, including, but not limited to, peptide-N4-(N-acetyl- -D-glucosaminyl) asparagine amidase (PNGase F), endoglycosidase Fl, endoglycosidase F2, endoglycosidase F3, a(2 ⁇ 3,6,8,9) neuraminidase, and the like.
  • PNGase F peptide-N4-(N-acetyl- -D-glucosaminyl) asparagine amidase
  • F endoglycosidase Fl
  • endoglycosidase F2 endoglycosidase F3
  • SDS-PAGE sodium docecyl sulfate-polyacrylamide gel electrophoresis
  • a marked decrease in band width and change in migration position after treatment with PNGaseF is considered diagnostic of N-linked glycosylation.
  • the carbohydrate content of a glycosylated protein can also be detected using lectin analysis of protein blots (for example, proteins separated by SDS-PAGE and transferred to a support, such as a nylon membrane).
  • Lectins, carbohydrate-binding proteins from various plant tissues have both high affinity and narrow specificity for a wide range of defined sugar epitopes found on glycoprotein glycans. See Cummings, Methods in Enzymol, 1994, 230:66-86.
  • Lectins can be detectably labeled (either directly or indirectly), allowing detection of binding of lectins to carbohydrates on glycosylated proteins.
  • a lectin bound to a glycosylated protein can be easily identified on membrane blots through a reaction utilizing avidin or anti-digoxigenin antibodies conjugated with an enzyme such as alkaline phosphatase, ⁇ -galactosidase, luciferase, or horse radish peroxidase, to yield a detectable product. Screening with a panel of lectins with well-defined specificity can provide considerable information about a glycoprotein's carbohydrate complement.
  • the phrase "increased glycosylation" is used herein to indicate increased levels of attached carbohydrate molecules, normally obtained as a result of increased number or better utilization of glycosylation site(s).
  • the increased glycosylation may be determined by any suitable method known in the art for analyzing attached carbohydrate structures. For example, Western blot can be used to determine the amount of attached carbohydrates in a glycosylated polypeptide.
  • An amino acid residue "located close to" a glycosylation site is usually located in position -4, -3, -2, -1, +1, +2, +3, or +4 relative to the amino acid residue of the glycosylation site to which the carbohydrate moiety is attached, in particular in position -2, - 1, +1, +2, such as position -1 or +1, in particular position -1.
  • These positions may be modified to increase glycosylation at the glycosylation site.
  • the modification can be a substitution.
  • polypeptide refers to a polymer of amino acids.
  • a polypeptide can be of various lengths. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.
  • a polypeptide can be with or without N- terminal methionine residues.
  • a polypeptide may include post-translational modifications, for example, glycosylations, acetylations, phosphorylations and the like.
  • polypeptide examples include, but are not limited to, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, non-coded amino acids, etc.), polypeptides with substituted linkages, fusion proteins, as well as polypeptides with other modifications known in the art, both naturally occurring and non-naturally occurring.
  • polynucleotide and “nucleic acid molecule” are used interchangeably herein to refer to polymeric forms of nucleotides of any length. Thus, oligonucleotides are included within the definition of polynucleotide.
  • the polynucleotides may contain deoxyribonucleotides, ribonucleotides, and their analogs. Nucleotides may have any three-dimensional structure, and may perform various functions.
  • polynucleotide includes single-, double-stranded and triple helical molecules.
  • a polynucleotide can be a double-stranded DNA found, inter alia, in linear DNA molecules (for example, restriction fragments), viruses, plasmids, and chromosomes.
  • Oligonucleotide generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as “oligomers” or “oligos,” and can be isolated from genes, or chemically synthesized by methods known in the art.
  • Non-limiting embodiments of polynucleotides include: genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide can also include modified nucleic acid molecules, such as methylated nucleic acid molecules, nucleic acid molecules with modified purine or pyrimidine bases and nucleic acid molecule analogs.
  • Nucleic acids may be naturally occurring DNA or RNA, or may be synthetic analogs of DNA or RNA, such as those known in the art. Synthetic analogs include native structures that have been modified to include alterations in the backbone, sugars and/or heterocyclic bases.
  • modified backbone include phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters; boranophosphates; achiral phosphate derivatives, such as 3'-0'-5'-S- phosphorothioate, 3'-S-5'-0- phosphorothioate, 3 '-CH 2 -5' -O-phosphonate and 3'-NH-5'-0 phosphoroamidate; and peptide nucleic acids in which the entire ribose phosphodiester backbone is replaced with a peptide linkage.
  • modifications of the backbone, sugars and/or heterocyclic bases include
  • Percent (%) sequence identity with respect to polynucleotide or polypeptide sequences is defined as the percentage of bases or amino acid residues in a candidate sequence that are identical with the bases or amino acid residues in another sequence, after aligning the two sequences. Gaps can be introduced into the sequence alignment, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not considered as part of the sequence identity. Alignment for purposes of determining percent (%) sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer methods and programs such as BLAST, BLAST-2, ALIGN, FASTA (available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA), or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • GCG Genetics Computing Group
  • percent (%) amino acid sequence identity values may be obtained by using the WU-BLAST-2 computer program described in Altschul et al., Methods in Enzymology, 1996, 266:460-480.
  • Many search parameters in the WU-BLAST-2 computer program can be adjusted by those skilled in the art.
  • a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the amino acid sequence of a first protein of interest and the amino acid sequence of a second protein of interest as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the first protein of interest.
  • Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 described in Altschul et al., Nucleic Acids Res., 1997, 25:3389-3402.
  • NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, MD.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • Some embodiments disclosed herein relate to a hyperglycosylated polypeptide variant of a parent polypeptide that is the parent polypeptide that has been modified to include a peptide extension inserted at a terminal region, where the peptide extension includes one or more glycosylation sites.
  • peptide extension refers to any polymer of consecutive amino acid residues.
  • a peptide extension can be of various lengths, for example, ranging from a single amino acid residue to a peptide of 500 amino acid residues.
  • the peptide extension can be at least about 4 amino acids in length, alternatively at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 145 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 155 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 165 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 180 amino acids in length
  • the peptide extension can include any number of glycosylation sites. In some embodiments, the peptide extension can include one glycosylation site. In other embodiments, the peptide extension can include two, three or four glycosylation sites. In still other embodiments, the peptide extension can include five, six, seven, eight, nine, or ten glycosylation sites. In yet other embodiments, the peptide extension can include eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty glycosylation sites.
  • the peptide extension can include twenty- one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty- eight, twenty-nine, thirty glycosylation sites. In yet still other embodiments, the peptide extension can include thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, or more glycosylation sites. Each glycosylation site that is included in the peptide extension may be an N-linked glycosylation site or an O- linked glycosylation site.
  • the peptide extension can include at least one glycosylation site. In other embodiments, the peptide extension can include at least two, at least three or at least four glycosylation sites. In still other embodiments, the peptide extension can include at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites. In yet other embodiments, the peptide extension can include at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty glycosylation sites.
  • the peptide extension can include at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, at least twenty-six, at least twenty-seven, at least twenty-eight, at least twenty-nine, at least thirty glycosylation sites. In yet still other embodiments, the peptide extension can include at least thirty-one, at least thirty-two, at least thirty-three, at least thirty-four, at least thirty-five, at least thirty-six, at least thirty-seven, at least thirty-eight, at least thirty-nine, at least forty, or more glycosylation sites.
  • a glycosylation site that is included in the peptide extension may be a non- native glycosylation site that is present in the parent polypeptide or a non-native glycosylation site that is not present in the parent polypeptide.
  • the parent polypeptide is interferon ocl4
  • the glycosylation site N-L-S at amino acid positions 25, 26 and 27 of the sequence of interferon ocl4 can be introduced to the peptide extension inserted in the parent interferon ocl4 as a non-native glycosylation site.
  • the glycosylation site N-L-S at amino acid positions 25, 26 and 27 of the sequence of interferon ocl4 can be introduced to the peptide extension inserted in the parent hGH-1 as a non-native glycosylation site.
  • the peptide extension can be inserted at an amino-terminal and/or a carboxy-terminal region of the parent polypeptide. In some embodiments, the peptide can be inserted at an amino-terminal region of the parent polypeptide. In other embodiments, the peptide extension can be inserted at a carboxy-terminal region of the parent polypeptide. In still other embodiments, the peptide extension can be inserted at an amino-terminal region and a carboxy-terminal region of the parent polypeptide.
  • the terminal region of the parent polypeptide at which the peptide extension is inserted can be of various lengths.
  • the terminal region where the peptide extension is inserted at can be an amino-terminal region that consists of the first 15 amino acids at the amino terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide.
  • the terminal region where the peptide extension is inserted at can be an amino terminal region that consists of the first 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) at the amino terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide.
  • the peptide extension can be inserted before the first amino acid at the amino terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide. In still other embodiments, the peptide extension is inserted after the fifteenth amino acid at the amino terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide. In yet other embodiments, the peptide extension is not inserted before the first amino acid at the amino-terminus of the parent polypeptide. In yet still other embodiments, the peptide extension includes at least two glycosylation sites and is not inserted before the first amino acid at the amino-terminus of the parent polypeptide. In some embodiments, the peptide extension includes at least four glycosylation sites and is not inserted before the first amino acid at the amino-terminus of the parent polypeptide.
  • the peptide extension when the parent polypeptide is human growth hormone 1 (hGH-1), the peptide extension can be inserted between the 26th and the 27th amino acids of the parent hGH-1. In other embodiments, the peptide extension can be inserted in a position selected from between the 27th and the 28th amino acids, between the 28th and the 29th amino acids, between the 29th and the 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, between the 39th and the 40th amino acids, between the 40th and the 41st amino acids, between the 41st and the 42nd amino acids, between the 42nd and the 43rd amino acids,
  • the peptide extension when the parent polypeptide is human insulin-like growth factor 1A (hIGF- ⁇ ), the peptide extension can be located between the 21st and the 22nd amino acids of the parent hIGF- ⁇ . In other embodiments, the peptide extension can be inserted in a position selected from between the 22nd and the 23rd amino acids, between the 23rd and the 24th amino acids, the 24th and the 25th amino acids, between the 25th and the 26th amino acids, between the 26th and the 27th amino acids, between the 27th and the 28th amino acids, between the 28th and 29th amino acids, between the 29th and the 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, and between the 37th and
  • the peptide extension when the parent polypeptide is human G-CSF (hG- CSF), the peptide extension can be located between the 29th and the 30th amino acids of the parent hG-CSF. In other embodiments, the peptide extension can be inserted in a position selected from between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, between the 39th and the 40th amino acids, between the 40th and the 41st amino acids, between the 41st and the 42nd amino acids, between the 42nd and the 43rd amino acids, between the 43rd and the 44th amino acids, between the 44th and the 45th amino acids, and between the
  • the peptide extension when the parent polypeptide is human erythropoietin (hEPO), the peptide extension can be located between the 27th and the 28th amino acids of the parent hEPO. In other embodimetns, the peptide extension can be inserted in a position selected from between the 28th and the 29th amino acids, between the 29th and the 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, between the 39th and the 40th amino acids, between the 40th and the 41st amino acids, between the 41st and the 42nd amino acids, between the 42nd and the 43rd amino acids, between the 43
  • the peptide extension when the parent polypeptide is human insulin, can be located between the 24th and 25th amino acids of the parent human insulin. In other embodiments, the peptide extension can be inserted in a position selected from between the 25th and the 26th amino acids, between the 26th and the 27th amino acids, between the 27th and the 28th amino acids, between the 28th and 29th amino acids, between the 29th and 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, and betweent he 39th and the 40th amino acids of the parent human insulin.
  • the terminal region where the peptide extension is inserted at can be a carboxy-terminal region that consists of the last 15 amino acids at the carboxy- terminus of the parent polypeptide. In other embodiments, the terminal region where the peptide extension is inserted at can be a carboxy-terminal region that consists of the last 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) at the carboxy-terminus of the parent polypeptide. In some embodiments, the peptide extension can be inserted after the last amino acid at the carboxy-terminus of the parent polypeptide. In other embodiments, the peptide extension is not inserted after the last amino acid at the carboxy-terminus of the parent polypeptide.
  • the peptide extension includes at least two glycosylation sites and is not inserted after the last amino acid at the carboxy-terminus of the parent polypeptide. In still other embodiments, the peptide extension includes at least four glycosylation sites and is not inserted after the last amino acid at the carboxy-terminus of the parent polypeptide.
  • a "parent polypeptide” can be any polypeptide, including naturally-occurring and non-naturally occurring polypeptides.
  • a "naturally occurring polypeptide” refers to a polypeptide that can be found in nature as distinct from being artificially produced by man.
  • a polypeptide that is produced by an organism in nature, where the organism has not been intentionally modified by man is naturally occurring.
  • organisms that can produce naturally-occurring polypeptides include, but are not limited to, bacteria, such as gram-positive bacteria and gram-negative bacteria; archaea, such as euryachaeota and cre- archaeota; fungus, such as yeasts, molds, mushrooms; plants, such as seed plants, bryophytes, ferns, fern allies, and algae; and animals, including non-mammalian vertebrates such as birds (for example, turkey, chicken, and duck) and fish (for example, zebrafish), and mammalian vertebrates such as human, pig, mouse, dog, cat, horse, rat, cattle, and sheep.
  • bacteria such as gram-positive bacteria and gram-negative bacteria
  • archaea such as euryachaeota and cre- archaeota
  • fungus such as yeasts, molds, mushrooms
  • plants such as seed plants, bryophytes, ferns, fern allies, and algae
  • a naturally-occurring polypeptide can be at least about 50 amino acids in length, at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 180 amino acids in length, alternatively at least about 190 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 210 amino acids in length, alternatively at least about 220 amino acids in length, alternatively at least about 230 amino acids in length, alternatively at least about 240 amino acids in length, alternatively at least about 250 amino acids in length, alternatively at least about 260 amino acids in length, alternatively at least about 270 amino acids in length, alternatively at least about 280 amino acids in length, alternatively at least about 290 amino acids in length, alternatively at least about
  • non-naturally occurring polypeptide is used interchangeably with the term “synthetic polypeptide,” and refers to a polypeptide that cannot be found in nature and is artificially produced by man.
  • Non-naturally occurring polypeptides include, but are not limited to, hybrid polypeptides, consensus polypeptides, fusion polypeptides, recombinant polypeptides, and other non-naturally occurring variants of a naturally-occurring or a non-naturally occurring polypeptide.
  • the parent polypeptide can be a non-naturally occurring polypeptide.
  • the parent polypeptide can be a hybrid polypeptide.
  • the parent polypeptide can be a consensus polypeptide.
  • the parent polypeptide can be a fusion polypeptide.
  • the parent polypeptide can be a recombinant polypeptide.
  • a non-naturally occurring polypeptide can be of various lengths.
  • a non-naturally occurring polypeptide is at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 145 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 155 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 165 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 180 amino acids in length, alternatively at least about 190 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 210
  • hybrid polypeptide is a hybrid polypeptide having an amino acid sequence comprising discrete sub-sequences corresponding in amino acid identity and number to sub-sequences of more than one naturally-occurring and/or non-naturally occurring polypeptides.
  • Each of the naturally-occurring and/or non-naturally occurring polypeptides from which the discrete sub-sequences are derived from may be the same or different from each other.
  • a hybrid polypeptide can include discrete subsequences from two naturally-occurring and/or non-naturally polypeptides, where the two naturally-occurring and/or non-naturally polypeptides are different from each other.
  • a hybrid polypeptide can include discrete sub-sequences from three, four, five, six, seven, eight, nine, or ten naturally-occurring and/or non-naturally occurring polypeptides.
  • the discrete sub-sequences can be selected from naturally- occurring human growth hormone 1 (hGH-1, SEQ ID NO: 1), naturally-occurring human insulin-like growth factor 1A (hIGF- ⁇ , SEQ ID NO: 8), naturally-occurring human granulocyte colony-stimulating factor (hG-CSF, SEQ ID NO: 15), naturally-occurring human Erythropoietin (hEPO, SEQ ID NO:22), naturally-occurring human insulin (SEQ ID NO: 29), and the amino acid sequence of the hybrid polypeptide differs from each of the amino acid sequences of naturally-occurring hGH-1, naturally-occurring hIGF- ⁇ , naturally-occurring hG-CSF, naturally-occurring hEPO, and naturally-occurring human insulin, respectively.
  • hGH-1 naturally-occurring human insulin-like growth factor 1A
  • hG-CSF naturally-occurring human granulocyte colony-stimulating factor
  • the discrete sub-sequences can be selected from naturally-occurring IFN- a2b, naturally-occurring IFN-al4, naturally-occurring IFN- ⁇ , and naturally-occurring IFN- co, and the amino acid sequence of the hybrid interferon differs from each of the amino acid sequences of naturally occurring IFN-a2b, naturally-occurring IFN-al4, naturally-occurring IFN- ⁇ , and naturally-occurring IFN-co, respectively.
  • the discrete sub-sequences can be selected from naturally-occurring IFN-a2b, naturally-occurring IFN- al4, naturally-occurring IFN- ⁇ , Infergen® consensus IFN-a, and naturally-occurring IFN-co, and the amino acid sequence of the hybrid interferon differs from each of the amino acid sequences of naturally-occurring IFN-a2b, naturally-occurring IFN-a 14, naturally-occurring IFN- ⁇ , Infergen® consensus IFN-a, and naturally-occurring IFN-co, respectively.
  • a hybrid polypeptide can include, in the order from N-terminus to C-terminus, from about 2 to about 90, for example, from about 2 to about 5, from about 5 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from about 85 to about 90 contiguous amino acids of two, three, four, or five polypeptides selected from hGH-1 (SEQ ID NO: 1), hIGF- ⁇ (SEQ ID NO: 8), hG-CSF (SEQ ID NO: 15), hEPO (SEQ ID NO: 22), and human insulin (SEQ ID NO: 29).
  • hGH-1 SEQ ID NO: 1
  • a "consensus polypeptide” is a polypeptide having a consensus amino acid sequence that is derived by aligning the amino acid sequences of three or more naturally occurring and/or non-naturally occurring polypeptides, and identifying the amino acids that are shared by at least two of the naturally occurring and/or non-naturally occurring polypeptide sequences.
  • a consensus polypeptide can include a consensus amino acid sequence that is derived by aligning the amino acid sequences of three, four, five, six, seven, eight, nine, or ten naturally occurring and/or non-naturally occurring polypeptides, and identifying amino acids that are shared by at least two, three, five, six, seven, eight, nine, or ten of the naturally occurring and/or non-naturally occurring polypeptide sequences.
  • a consensus polypeptide can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-al4, and naturally-occurring human IFN- ⁇ .
  • a consensus polypeptide can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-al4, and naturally-occurring human IFN- col.
  • a consensus polypeptide can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN- ⁇ , and naturally-occurring human IFN-col.
  • a consensus polypeptide can be a sequence derived from aligning the sequences of naturally occurring human IFN-al4, naturally-occurring human IFN- ⁇ , and naturally-occurring human IFN-col.
  • a consensus polypeptide can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-al4, naturally-occurring human IFN- ⁇ , and naturally-occurring human IFN-col.
  • a consensus polypeptide can be a sequence derived from aligning the sequences of naturally-occurring human IFN- ⁇ , naturally-occurring human ⁇ - ⁇ 2, and naturally-occurring human ⁇ - ⁇ 3.
  • consensus polypeptide is Infergen® consensus IFN-a (Three Rivers Pharmaceuticals, Warrendale, PA).
  • a consensus sequence can be derived by including one or more consensus polypeptides, such as Infergen® consensus IFN-a, in the amino acid sequence alignment.
  • a "fusion polypeptide” is a polypeptide comprising a fusion partner, such as one or more heterologous peptides or polypeptides.
  • Suitable fusion partners include, but are not limited to, peptides and polypeptides that confer enhanced stability in vivo (for example, enhanced serum half-life); peptides and polypeptides that can provide ease of purification (for example, (His) n ) and the like; peptides and polypeptides that can provide for secretion of the fusion protein from a cell, and the like; peptides and polypeptides that can provide an epitope tag, such as GST, hemagglutinin (for example, CYPYDVPDYA), FLAG (for example, DYKDDDDK), c-myc (for example, CEQKLISEEDL), and the like; peptides and polypeptides that can provide a detectable signal, such as an enzyme that generates a detectable product
  • Various signal peptide sequences can be incorporated into a polypeptide as a fusion partner to provide for secretion of the fusion protein from a cell.
  • the fusion partner can be a signal peptide that provides for secretion from a mammalian cell.
  • signal peptides include, but are not limited to, a signal peptide from human insulin-like growth factor II (hIGF-II).
  • hIGF-II human insulin-like growth factor II
  • Methods of producing a fusion polypeptide comprising a hIGF-II signal peptide are described in U.S. Patent No. 7,396,811, the contents of which are hereby incorporated by reference in its entirety.
  • the fusion partner can be a bacterial secretion signal peptide.
  • Such signal peptides include, but are not limited to, the secretion signal of Braun's lipoprotein of E. coli, S. marcescens, E. amylosora, M. morganii, and P. mirabilis; the TraT protein of E. coli and Salmonella; the penicillinase (PenP) protein of B. licheniformis, B. cereus and S. aureus; pullulanase proteins of Klebsiella pneumoniae and Klebsiella aerogenese; E. coli lipoproteins lpp-28, Pal, RplA, RplB, OsmB, NIpB, and Orll7; chitobiase protein of V.
  • the fusion partner can be a yeast secretion signal peptide.
  • Secretion signal peptides that can be used in yeast are known in the art. See, for example, U.S. Patent No. 5,712,113, the contents of which are hereby incorporated by reference in its entirety.
  • a fusion polypeptide can further include a protease cleavage site that is positioned between the fusion partner and the remainder of the fusion polypeptide.
  • proteolytic cleavage sites that can be included in a fusion polypeptide disclosed herein include, but are no limited to, an enterokinase cleavage site: (Asp) 4 Lys; a factor Xa cleavage site: Ile-Glu-Gly-Arg; a thrombin cleavage site, such as Leu- Val-Pro-Arg-Gly-Ser; a renin cleavage site, such as His-Pro-Phe-His-Leu-Val-Ile-His; a collagenase cleavage site, such as X-Gly-Pro (where X is any amino acid); a trypsin cleavage site, such as Arg-Lys; a viral protease cleavage site, such
  • a "recombinant polypeptide” is a non-naturally occurring polypeptide that is produced utilizing recombinant techniques.
  • a recombinant polypeptide can be produced by genetic engineering techniques known in the art, such as recursive sequence recombination of nucleic acid segments, diversity generation methods (such as shuffling) of nucleotides, or manipulation of isolated segments of polynucleotides or polypeptides.
  • recombinant polypeptides are known in the art, including, but not limited to, recombinant human growth hormone (hGH) such as Genotropin available from Pflizer, New York, NY; recombinant human insulin-like growth factor 1 such as Increlex available from Tercica, Brisbane, CA; recombinant human G-CSF such as Neupogen® available from Amgen Inc., Thousand Oaks, CA; recombinant erythropoietin (EPO) such as Epogen ® available from Amgen Inc., Thousand Oaks, CA; and recombinant insulin such as NovoLog ® available from Novonordisk Inc., Princeton NJ.
  • hGH human growth hormone
  • hGH human growth hormone
  • hGH human growth hormone
  • hGH human growth hormone
  • human insulin-like growth factor 1 such as Increlex available from Tercica, Brisbane, CA
  • recombinant human G-CSF such as Neupogen® available from Amgen Inc., Thousand
  • the parent polypeptide can include one or more modified amino acids.
  • modified amino acid include, but are not limited to, a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, a carboxylated amino acid, a phosphorylated amino acid (for example, phosphotyrosine, phosphoserine, and phosphothreonine), an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, an amino acid conjugated to an organic derivatizing agent, and the like.
  • the parent polypeptide can be a naturally occurring or non-naturally occurring polypeptide that includes one or more polyethylene glycol (PEG) moieties.
  • PEG polyethylene glycol
  • the PEG molecule(s) can be conjugated to one or more amino acid side chains of the PEGlyated polypeptide.
  • the PEG can be coupled directly to a polypeptide without a linking group, or through an amino group, a sulfhydryl group, a hydroxyl group, or a carboxyl group.
  • Methods for attaching a PEG to a polypeptide are known in the art, and any known method can be used. See, for example, Park et al, Anticancer Res., 1981, 1:373-376; and U.S. Patent No.
  • the PEGylated polypeptide can contain a PEG moiety on only one amino acid. In other embodiments, a PEGylated polypeptide contains two, three, four, five, six, seven, eight, nine or ten PEG moieties on two, three, four, five, six, seven, eight, nine or ten different amino acid residues.
  • the parent polypeptide can be a PEGylated polypeptide that is PEGylated at or near the amino- terminus, where a peptide extension can be inserted at the carboxy-terminal region of the parent polypeptide.
  • the PEG moiety can be conjugated to the polypeptide at one or more amino acid residues from amino acid 1 through amino acid 4, or from amino acid 5 through amino acid 10.
  • the parent polypeptide can be a PEGylated polypeptide that is PEGylated at or near the carboxy-terminus, where a peptide extension can be inserted at the amino-terminal region of the parent polypeptide.
  • the PEGylated polypeptide is PEGylated at one or more amino acid residues at one or more residues from amino acid 100 through amino acid 114.
  • the addition of one or more pegylation sites can provide PEG-derivatized polypeptide with reduced serum clearance.
  • the PEG can be a monomethoxyPEG molecule (mPEG) that reacts with a primary amine group on the subject polypeptide.
  • mPEG monomethoxyPEG molecule
  • polypeptides can be a parent polypeptide.
  • these include a large number of biologically-active polypeptides, and preferably include those that are biologically active in humans and those that are biologically active in veterinary applications, including in mammals, birds, reptiles, amphibians, and fish, and those active in vertebrates in general.
  • a non-limiting list of examples of biologically-active polypeptides that are active in humans and other mammals includes, but is not limited to, naturally-occurring and non-naturally occurring growth hormones (GHs); naturally-occurring and non-naturally occurring insulin-like growth factors (IGFs); naturally-occurring and non-naturally occurring granulocyte colony-stimulating factors (G-CSFs); naturally-occurring and non-naturally occurring erythropoietin (EPO); naturally-occurring and non-naturally occurring insulin; naturally-occuring and non-naturally ouccuring antibodies, such as hybrid antibodies, chimeric antibodies, humanized antibodies, monoclonal antibodies; naturally-occurring and non-naturally occurring antigen binding fragments of an antibody (Fab fragments); naturally-occurring and non-naturally occurring single-chain variable fragments of an antibody (scFV fragments); naturally-occurring and non-naturally occurring blood factors such as ⁇ -globin, hemoglobin, tissue plasminogen
  • the parent polypeptide can be an insulin. In some embodiments, the parent polypeptide can be human insulin.
  • insulins include, but are not limited to, proinsulin, preproinsuhn, insulin, insulin analogs, and the like. Some examples of insulins are disclosed in U.S. Patent Nos. 5,474,978, 5,514,646, 5,504,188, 5,547,929, 5,650,486, 5,693,609, 5,700,662, 5,747,642, 5,922,675, 5,952,297, 6,034,054, and 6,211,144; and International Patent Publications Nos. WO 00/121197, WO 09/010645 and WO 90/12814, which hereby are incorporated by reference for the limited purpose of their disclosure of insulin.
  • non-naturally occurring insulins include synthetic insulins available under the tradenames of NovoLog ® , Novolin ® , Humulin ® , Humalog ® , Lantus ® , Lente ® , and Ultralente ® .
  • Insulin analogs include, but are not limited to, superactive insulin analogs, monomeric insulins, and hepatospecific insulin analogs.
  • Various examples of insulin analogs include, but are not limited to, insulin analogs available under the tradenames of Humalog ® ; Humalog ® Mix 50/50TM; Humalog ® Mix 75/25TM; Humulin ® 50/50; Humulin ® 70/30; Humulin ® L; Humulin ® N; Humulin ® R; Humulin ® Ultralente; Lantus ® ; Lente® Iletin ® II; Lente ® Insulin; Lente ® L; Novolin ® 70/30; Novolin ® L; Novolin ® N; Novolin ® R; NovoLogTM; NPH Iletin ® I; NPH-N; Pork NPH Iletin ® II; Pork Regular Iletin ® II; Regular
  • insulin analogs include, but are not limited to, acylated insulin, glycosylated insulin, and the like.
  • acylated insulin include those disclosed in U.S. Patent No. 5,922,675, for example, insulin derivatized with a C 6 -C2i fatty acid (such as myristic, pentadecylic, palmitic, heptadecylic, or stearic acid) at an a- or ⁇ - amino acid of glycine, phenylalanine or lysine.
  • human insulin can comprise any one of the amino acid sequences as set forth in GenBank under the following accession numbers: P01308, CAA00714; CAA00713; CAA00712; CAA01254; IHISA and IHISB; 1 HIQA and 1 HIQB; IHITA and 1HITB; 1 HLSA and 1HLSB; 1VKTA and 1VKTB; which hereby are incorporated by reference for the limited purpose of their disclosure of amino acid sequences of insulin.
  • the parent polypeptide can be an antibody.
  • antibodies include, but are not limited to, antibodies of various isotypes (for example, IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM); monoclonal antibodies produced by any means known to those skilled in the art, including an antigen-binding fragment of a monoclonal antibody; humanized antibodies; chimeric antibodies; single-chain antibodies; antibody fragments such as Fv, F(ab')2, Fab', Fab, Facb, scFv and the like; provided that the antibody is capable of binding to antigen.
  • antibodies that are specific for a cell surface receptor and that function as antagonists to the receptor, including, but not limited to, antibodies to TGF- ⁇ receptor; antibodies to TNF-a receptor; antibodies to VEGF receptor (see for example, U.S. Patent Nos.
  • antibodies to epidermal growth factor receptor antibodies specific for receptor ligands, including, but not limited to, antibodies to TGF- ⁇ , antibodies to TNF-a, antibodies to VEGF, and the like; antibodies specific for a tumor-associated antigen; antibodies specific for CD20; antibodies specific for epidermal growth factor receptor-2 (HER-2); antibodies specific for the receptor binding domain of IgE; antibodies specific for adhesion molecules, such as antibodies specific for a subunit (CD 11 a) of LFA-1; antibodies specific for ⁇ 4 ⁇ 7; and the like.
  • antibodies specific for receptor ligands including, but not limited to, antibodies to TGF- ⁇ , antibodies to TNF-a, antibodies to VEGF, and the like
  • antibodies specific for a tumor-associated antigen antibodies specific for CD20; antibodies specific for epidermal growth factor receptor-2 (HER-2); antibodies specific for the receptor binding domain of IgE; antibodies specific for adhesion molecules, such as antibodies specific for a subunit (CD 11 a) of LFA-1; antibodies specific for ⁇ 4 ⁇ 7; and
  • recombinant antibodies include, but are not limited to rituximab (a chimeric monoclonal antibody specific for CD20) available under the tradename of Rituxan , infliximab (a monoclonal antibody specific for TNF-oc) available under the tradename of Remicade , trastuzumab (a monoclonal antibody specific for HER- 2/neu receptor) avaible under the tradename of Herceptin ® , adalimumab (a human monoclonal antibody specific for TNF-oc) available under the tradename of HumiraTM, omalizumab (a humanized antibody specific for human immunoglobulin E (IgE)) available under the tradename of Xolair ® , tositumomab (a monoclonal antibody specific for CD20) available under the tradename of Bexxar ® , efalizumab (a humanized monoclonal antibody specific for CD 11 a) available under the tradename of
  • the parent polypeptide can be an antibody Fab fragment. In an embodiment, the parent polypeptide can be a Fab fragment of an antibody specific for TNF-a. In another embodiment, the parent polypeptide can be a Fab fragment of an antibody specific for HER-2 receptor. In still another embodiment, the parent polypeptide can be a Fab fragment of an antibody specific for VEGF. In other embodiments, the parent polypeptide can be an antibody scFV fragment. In an embodiment, the parent polypeptide can be an scFV fragment of an antibody specific for TNF-a. In another embodiment, the parent polypeptide can be an scFV fragment of an antibody specific for HER-2 receptor. In still another embodiment, the parent polypeptide can be an scFV fragment of an antibody specific for VEGF.
  • the parent polypeptide can be a blood factor.
  • blood factor include, but are not limited to, tissue plasminogen activators (TPA); ⁇ -globin; hemoglobin; coagulation factors such as Factor Vila, Factor VIII, and Factor IX; and the like.
  • TPA tissue plasminogen activators
  • ⁇ -globin ⁇ -globin
  • hemoglobin coagulation factors
  • coagulation factors such as Factor Vila, Factor VIII, and Factor IX
  • the amino acid sequences of various blood factors are publicly available.
  • human TPA can comprise any one of the amino acid sequences as set forth in GenBank under the following accession numbers: P0070, NP_127509, and NP- 000921
  • human Factor Vila can comprise the amino acid sequence set forth in GenBank Accession No.
  • human Factor IX can comprise any one of the amino acid sequences as set forth in GenBank under the following accession numbers P00740 and NP_000124; and human Factor VIII can comprise any one of the amino acid sequences as set forth in GenBank under the following accession numbers: AAH64380, AAH22513, and P00451 ; which hereby are incorporated by reference for the limited purpose of their disclosure of amino acid sequences of blood factors.
  • non-naturaly occurring blood factors include recombinant human factor Vila available under the tradename of NovoSeven ® , recombinant factor VIII available under the tradename of Kogenate , and recombinant human factor IX available under the tradename of BeneFIX .
  • CSFs Colony stimulating factors
  • the parent polypeptide can be a colony stimulating factor.
  • the parent polypeptide can be human granulocyte colony stimulating factor (hG-CSF).
  • the parent polypeptide can be human granulocyte-monocyte colony stimulating factor (hGM-CSF).
  • colony stimulating factors include, but are not limited to, granulocyte colony stimulating factor (G-CSF), such as NEUPOGEN filgrastim and NEULASTATM pegfilgrastim; granulocyte-monocyte colony stimulating factor (GM-CSF), such as LEUKINE ® sargramostim; macrophage colony stimulating factor (M-CSF); megakaryocyte colony stimulating factor (Meg-CSF); IL-3; and the like.
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte-monocyte colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • Meg-CSF megakaryocyte colony stimulating factor
  • IL-3 and the like.
  • the amino acid sequences of various blood factors are publicly available. For example, amino acid sequences of IL-3 are disclosed in U.S. Pat. Nos. 4,877,729 and 4,959,455, and International Patent Publication No.
  • amino acid sequences of human G-CSF are disclosed in U.S. Pat. No. 4,810,643; amino acid sequences of fusion proteins comprising IL-3 are disclosed in International Patent Publication Nos. WO 91/02754 and WO 92/04455; amino acid sequences of human G-CSF can be found under GenBank Accession Nos. P09919, NP_757374, P010219, and NP_000750; amino acid sequences of human GM-CSF can be found under GenBank Accession Nos. NP_000749 and P04141 ; amino acid sequences of IL- 3 can be found under GenBank Accession Nos.
  • AAH66272, AAH66273, and AAH66276; and amino acid sequences of M-CSF can be found under GenBank Accession Nos. AAA59572.1, AAB59527.1, AAA59573.1, AAB29303.1, 1HMCB, 1HCMB, and AAA64849.1. All patent references and GenBank records disclosed in this paragraph are hereby incorporated by reference for the limited purpose of their disclosure of amino acid sequences of clony stimulating factors.
  • the parent polypeptide can be a growth hormone. In some embodiments, the parent polypeptide can be human growth hormone 1 (hGH-1).
  • growth hormones include, but are not limited to, somatotropin; human growth hormones; growth hormone variants disclosed in U.S. Patent Nos. 6,143,523, 6,136,563, 6,022,711, and 5,688,666; fusion proteins comprising a growth hormone, such as those disclosed in U.S. Patent No. 5,889,144; growth hormone fragments that retain growth hormone activity; a growth hormone disclosed in U.S. Patent No. 6,387,879; and the like.
  • Non-limiting examples of growth hormones also include alternative forms of known growth hormones, including naturally-occurring derivatives, variants and metabolic products (for example, degradation products primarily of biosynthetic hGH and engineered variants of hGH produced by recombinant methods (see for example, U.S. Patent No. 6,348,444)).
  • Non-limiting examples of non-naturally occurring human growth hormone include synthetic human growth hormone available under the tradenames of Genotropin , Nutropin ® , Norditropin ® , Saizen ® , Serostim ® , and Humatrope ® .
  • hGH-1 can comprise an amino acid sequence as set forth in GenBnk Accession No. P01241. Growth factors
  • the parent polypeptide can be a growth factor.
  • the parent polypeptide can be a human insulin-like growth factor (hIGF).
  • the parent polypeptide can be human insulin-like growth factor 1A (hIGF- ⁇ ).
  • the parent polypeptide can be an erythropoietin (EPO).
  • the parent polypeptide can be a human erythropoietin (hEPO).
  • growth factors include, but are not limited to, keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), active fragments of a growth factor, fusion proteins comprising a growth factor, bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor- 9 (GDF-9), hepatoma derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF- ⁇ ), and the like.
  • KGF keratinocyte growth factor
  • SCF stem cell factor
  • FGF fibroblast growth factor
  • FGF such as basic FGF and acidic FGF
  • HGF hepatocyte growth factor
  • amino acid sequences of various growth factors are publicly available. For example, amino acid sequences of basic FGFs can be found under GenBank Accession Nos. AAB20640, AAA57275, A43498, and AAB20639; amino acid sequences of acidic FGFs can be found under GenBank Accession Nos. AAB29059, CAA46661, and 1605206A; amino acid sequences of human stem cell factor can be found under GenBank Accession Nos.
  • EPO erythropoietin
  • Eprex ® Epogen ® (epoetin-a), Aranesp ® (darbepoetin-a), NeoRecormon ® , and Epogin ® (epoetin- ⁇ ).
  • Amino acid sequence of human erythropoietin (hEPO) can be found under GenBank Accession Nos. P01588, CAA26095.1, AAA52400.1, and AAD 13964.1.
  • the parent polypeptide can be a soluble receptor.
  • soluble receptor polypeptides include, but are not limited to, soluble TNF-a receptors; soluble VEGF receptors; soluble interleukin receptors, such as soluble IL-1 receptors and soluble type II IL-1 receptors; soluble ⁇ / ⁇ T cell receptors; ligand-binding fragments of a soluble receptor, and the like.
  • Suitable soluble receptors bind a ligand that, under normal physiological conditions, binds to and activates the corresponding membrane- bound or cell surface receptor.
  • a suitable soluble receptor can function as a receptor antagonist, by binding the ligand that would ordinarily bind the receptor in its native (for example, membrane-bound) form.
  • amino acid sequences of various soluble receptors are publicly available.
  • amino acid sequences of soluble VEGF receptors can be found under GenBank Accession Nos. AAC50060, NP_002010, P17948, and P35968;
  • soluble VEGF receptors can be found described in U.S. Patent Nos. 6,383,486, 6,375,929, and 6,100,071 ;
  • soluble IL-4 receptors are described in U.S. Pat. No. 5,5102,905; and soluble IL-1 receptors are described in U.S. Patent Publication No. 20040023869; amino acid sequences of soluble TNF-a receptors can be found under GenBank Accession Nos. P19438 and P20333.
  • the parent polypeptide can be a chemokine.
  • chemokines include, but are not limited to, IP- 10, monokine induced by interferon-gamma (Mig), Groa/IL-8, RANTES, MIP-la, ⁇ - ⁇ , MCP-1, PF-4, and the like.
  • the amino acid sequences of various chemokines are publicly available. For example, amino acid sequences of IP- 10 are disclosed in U.S. Patent Nos. 6,491,906, 5,935,567, 6,153,600, 5,728,377, and 5,1024,292; amino acid sequences of Mig are disclosed in U.S. Patent No.
  • the parent polypeptide can be an angiogenic agent.
  • angiogenic polypeptides include, but are not limited to, vascular endothelial growth factors (VEGFs), including VEGF121, VEGF165, VEGF-C, VEGF-2, etc.; transforming growth factor-beta; basic fibroblast growth factor; glioma-derived growth factor; angiogenin; angiogenin-2; and the like.
  • VEGFs vascular endothelial growth factors
  • the amino acid sequences of various angiogenic agents are publicly available. For example, amino acid sequences of VEGF polypeptides are disclosed in U.S. Patent Nos.
  • amino acid sequences of VEGF-2 polypeptides are disclosed in U.S. Patent Nos. 5,726,152 and 6,608,182; amino acid sequences of glioma-derived growth factors having angiogenic activity are disclosed in U.S. Patent Nos. 5,338,840 and 5,532,343; amino acid sequences of angiogenin can be found under GenBank Accession Nos. AAA72611, AAA51678, AAA02369, AAL67710, AAL67711, AAL67712, AAL67713, and AAL67714.
  • the parent polypeptide can be a neuroactive peptide.
  • neuroactive polypeptides include, but are not limited to, nerve growth factor (NGF), bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagons, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, and the like.
  • NGF nerve growth factor
  • bradykinin bradykinin
  • cholecystokinin gastin
  • secretin secretin
  • polypeptides can be used as a parent polypeptide in the methods and/or compositions disclosed herein. Additional polypeptides include, but are not limited to, thrombolytic agents, atrial natriuretic peptides, glial fibrillary acidic protein, follicle stimulating hormone (FSH), human alpha- 1 antitrypsin, leukemia inhibitory factor (LIF), transforming growth factor (TGF), a luteinizing hormone, a macrophage activating factor, tumor necrosis factor (TNF), neutrophil chemotactic factor (NCF), nerve growth factor (NGF), tissue inhibitor of metalloproteinases; vasoactive intestinal peptide, angiotropin, fibrin, hirudin, relaxin, and the like.
  • FSH follicle stimulating hormone
  • LIF leukemia inhibitory factor
  • TGF transforming growth factor
  • NGF nerve growth factor
  • tissue inhibitor of metalloproteinases tissue inhibitor of metalloproteinases
  • the parent peptide is not an interferon. In some embodiments, the parent peptide is not a Type I interferon. In other embodiments, the parent peptide is not a Type II interferon. In still other embodiments, the parent peptide is not a Type III interferon. In yet other embodiments, the parent peptide is not a follicle-stimulating hormone (FSH), for example human FSH (hFSH). In yet still other embodiments, the parent peptide is not a cytokine. In yet still other embodiments, the parent peptide is not a growth hormone, for example, human growth hormone. Conversely, in other embodiments, the parent peptide or polypeptide is one of those mentioned in this paragraph.
  • FSH follicle-stimulating hormone
  • hFSH human FSH
  • the parent peptide is not a cytokine.
  • the parent peptide is not a growth hormone, for example, human growth hormone.
  • the parent peptide or polypeptide
  • a hyperglycosylated polypeptide variant of a parent polypeptide can be the parent polypeptide that has been modified to include a peptide extension inserted at a terminal region, where the peptide extension can include at least one glycosylation site.
  • the peptide extension can include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites.
  • the only modifications to the parent polypeptide are addition of the peptide extension to the amino or carboxy terminus and, optionally, addition of one or more glycosylation sites in the parent polypeptide.
  • the hyperglycosylated polypeptide can be of various lengths.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can be at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 145 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 155 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 165 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 175 amino acids in length, alternatively at least about 180 amino acids in length, alternatively at least about 185
  • the parent polypeptide can be a growth hormone (GH) and the peptide extension can be inserted between the 26th and the 27th amino acids of the parent growth hormone.
  • the parent polypeptide can be human growth hormone 1 (hGH-1) and the peptide extension can be inserted between the 26th and the 27th amino acids of the parent hGH-1.
  • the parent polypeptide can be an insulin-like growth factor.
  • the parent polypeptide can be human insulin-like growth factor 1A (hIGF- ⁇ ) and the peptide extension can be located between the 21st and the 22nd amino acids of the parent hIGF-lA.
  • the parent polypeptide can be a granulocyte colony-stimulating factor (G-CSF).
  • G-CSF granulocyte colony-stimulating factor
  • the parent polypeptide can be human G-CSF and the peptide extension can be located between the 29th and the 30th amino acids of the parent human G-CSF.
  • the parent polypeptide can be an erythropoietin.
  • the parent polypeptide can be human erythropoietin (EPO) and the peptide extension can be located between the 27th and the 28th amino acids of the parent human EPO.
  • EPO erythropoietin
  • the parent polypeptide can be an insulin.
  • the parent polypeptide can be human insulin and the peptide extension can be located between the 24th and the 25th amino acids of the parent human insulin.
  • the peptide extension can include an amino acid motif, where the amino acid motif can include one or more glycosylation sites.
  • the amino acid motif can be where B 1 is an amino acid residue; B 2 is a Serine (S) or a Threonine (T); and B 3 is a sequence of Zl amino acids, where Zl is an integer from 1 to 8 and each amino acid in the sequence B is independently an amino acid residue.
  • B 1 can be any amino acid. In other embodiments, B 1 can be any amino acid except for Proline (P). In an embodiment, B 1 can be a Valine (V). In another embodiment, B 1 can be an Isoleucine (I). In still another embodiment, B 1 can be a Glycine (G). In yet another embodiment, B 1 can be an Alanine (A).
  • B 2 can be a Serine (S). In other embodiments, B 2 can be a Threonine (T). [0113] In some embodiments, B 3 can be any amino acid. In an embodiment, B 3 can be a Valine (V). In another embodiment, B can be an Isoleucine (I). In still another embodiment, B 3 can be a Glycine (G). In yet another embodiment, B 3 can be an Alanine (A).
  • Non-limiting examples of the amino acid motif ⁇ 1 ⁇ 2 ⁇ 33 ⁇ 4 ⁇ include NI[T/S]V, NI[T/S]I, NI[T/S]A, NI[T/S]G, NA[T/S]V, NA[T/S]I, NA[T/S]A, NA[T/S]G, NV[T/S]V, NV[T/S]I, NV[T/S]A, NV[T/S]G, NG[T/S]V, NG[T/S]I, NG[T/S]A, NG[T/S]G, NI[T/S]VNI[T/S]V, NI[T/S]VNA[T/S]G, NI[T/S]VNV[T/S]V, NI[T/S]ANI[T/S]A, NI[T/S]ANI[T/S]G, and NV[T/S]ANG[T/T/S
  • NB B [B ]zi can be NITV. Sequences of some non-limiting examples of the amino acid motif NB 1 B 2 [B 3 ]zi are given in SEQ ID NOs: 183-238.
  • the amino acid motif included in the peptide extension can be [B 4 ]z 2 NB 5 B 6 [B 7 ]z3, where B 4 is a sequence of Z2 amino acids, Z2 is an integer from 1 to 8, and each amino acid in the sequence B 4 is independently an amino acid residue; B 5 is an amino acid residue; B 6 is a Serine (S) or a Threonine (T); B 7 is a sequence of Z3 amino acids, wherein Z3 is an integer from 1 to 8, wherein each amino acid in the ⁇
  • sequence B is independently an amino acid residue.
  • B 4 can be any amino acid.
  • B 4 can be a Valine (V).
  • B 4 can be an Isoleucine (I).
  • B 4 can be a Glycine (G).
  • B 4 can be an Alanine (A).
  • B 5 can be any amino acid. In an embodiment, B 5 can be any amino acid except for Proline (P). In an embodiment, B 5 can be a Valine (V). In another embodiment, B 5 can be an Isoleucine (I). In still another embodiment, B 5 can be a Glycine (G). In yet another embodiment, B 5 can be an Alanine (A).
  • B 6 can be a Serine (S). In other embodiments, B 6 can be a Threonine (T).
  • B 7 can be any amino acid.
  • B 7 can be a Valine (V).
  • B can be an Isoleucine (I).
  • B 7 can be a Glycine (G).
  • B 7 can be an Alanine (A).
  • B 7 can be GG.
  • B 7 can be ⁇
  • B can be GR.
  • Examples of the amino acid motif [B 4 ]z 2 NB 5 B 6 [B 7 ]z3 include, but are not limited to, INI[T/S]V, INI[T/S]I, VNI[T/S]A, VNI[T/S]G, VNI[T/S]GR, VNI[T/S]GG, VNI[T/S]GGG, INA[T/S]V, INA[T/S]G, GNA[T/S]I, INA[T/S]A, GNI[T/S]G, GNA[T/S]G, INA[T/S]G, ANA[T/S]G, ANV[T/S]V, VNV[T/S]I, VNV[T/S]A, ANI[T/S]V, VNV[T/S]G, ING[T/S]V, ANG[T/S]I, VNG[T/S]A, VNG[T/S]G, S
  • the amino acid motif [B 4 ] Z2 NB 5 B 6 [B 7 ]z3 can be VNITG. In other embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNITGG. In still other embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNITGGG. In yet other embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNISGR. In yet still other embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNITGVNISGR.
  • the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNITGG VNIS GR. In other embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNITGGGVNISGR.
  • These various motifs can be repeated in the peptide extension, either by repeating the same glycosylation motive, or by mixing various motifs in the peptide extension. In addition, the motifs can be directly linked to each other, or can be separated by additional amino acids acting as spacers between the motifs.
  • amino acids used as the spacers can be charged or uncharged, hydrophobic or hydrophilic or neither, and each spacer can be of any length desired, from zero to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more amino acids in length, including any intermediate values not specifically mentioned here.
  • Sequences of some non-limiting examples of the amino acid motif [B 4 ] Z2 NB 5 B 6 [B 7 ]z3 are given in SEQ ID NOs: 239-328.
  • the amino acid motif (or mixture of motifs), such as NB 1 B 2 [B 3 ]zi and [B 4 ]z 2 NB 5 B 6 [B 7 ]z3, can be present one or more times in the peptide extension.
  • the amino acid motif can be present once, twice, three times, or four times in the peptide extension.
  • the amino acid motif can be present five times, six times, seven times, eight times, nine times, or ten times in the peptide extension.
  • the amino acid motif can be present eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-one times, twenty-two times, twenty-three times, twenty-four times, twenty-five times, twenty-six times, twenty-seven times, twenty-eight times, twenty-nine times, thirty times, or more in the peptide extension.
  • the amino acid motif can be present at least once, at least twice, at least three times, or at least four times in the peptide extension. In still other embodiments, the amino acid motif can be present at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times in the peptide extension.
  • the amino acid motif can be present at least eleven times, at least twelve times, at least thirteen times, at least fourteen times, at least fifteen times, at least sixteen times, at least seventeen times, at least eighteen times, at least nineteen times, at least twenty times, at least twenty-one times, at least twenty- two times, at least twenty-three times, at least twenty-four times, at least twenty-five times, at least twenty-six times, at least twenty-seven times, at least twenty-eight times, at least twenty-nine times, at least thirty times, or more in the peptide extension.
  • the amino acid motif can include one N-linked glycosylation site. In other embodiments, the amino acid motif can include two, three, four, five, or six N-linked glycosylation sites. In some embodiments, the amino acid motif can include one O-linked glycosylation sites. In other embodiments, the amino acid motif can include two, three, four, five, or six O-linked glycosylation sites. In still other embodiments, the amino acid motif can include one, two, three, four, five, or six N-linked gltycosylation sites and/or one, two, three, four, five, or six O-linked glycosylation sites.
  • the amino acid motif can include at least one N- linked glycosylation site. In other embodiments, the amino acid motif can include at least two, at least three, at least four, at least five, or at least six N-linked glycosylation sites. In some embodiments, the amino acid motif can include at least one O-linked glycosylation sites. In other embodiments, the amino acid motif can include at least two, at least three, at least four, at least five, or at least six O-linked glycosylation sites.
  • the amino acid motif can include at least one, at least two, at least three, at least four, at least five, or at least six N-linked gltycosylation sites and/or at least one, at least two, at least three, at least four, at least five, or at least six O-linked glycosylation sites.
  • Non-limiting examples of peptide extensions include:
  • VNITG SEQ ID NO: l
  • VNITGVNITG SEQ ID NO: 2
  • VNITGVNITGVNITG SEQ ID NO:3
  • VNITGVNITGVNITGVNITG SEQ ID NO: 4
  • VNITGVNITGVNITGVNITGVNITG SEQ ID NO:5
  • VNITGVNITGVNITGVNITGVNITGVNITGVNITG SEQ ID NO:6
  • VNITGVNITGVNITG VNITG VNITG VNITGVNITG (SEQ ID NO: 7)
  • VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 8)
  • VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 9)
  • VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 10)
  • VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITG VNISGR SEQ ID NO: 17
  • VNITGGG (SEQ ID NO: 18)
  • VNITGGGVNITGGG (SEQ ID NO: 19)
  • VNITGGGVNITGGGVNITGGG SEQ ID NO: 20
  • VNITGGGVNITGGGVNITGGGVNITGGG SEQ ID NO: 21
  • VNITGGGVNITGGGVNITGGGVNITGGGVNITGGG SEQ ID NO: 22
  • VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGG SEQ ID NO: 23
  • VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGG SEQ ID NO: 24
  • VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITGVNISGR SEQ ID NO: 29
  • VNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITG G VNITGG VNITGG VNIS GR (SEQ ID NO: 30)
  • the parent polypeptide in addition to the peptide extension that is inserted at a terminal region, can be further modified to include at least one additional glycosylation site in the amino acid sequence of the parent polypeptide.
  • Each additional glycosylation site that is introduced in the amino acid sequence of the parent polypeptide can be by at least one amino acid substitution or at least one combination of amino acid substitutions.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can be a parent polypeptide that has been modified to include a peptide extension inserted at a terminal region and at least one additional glycoyslation site introduced to the amino acid sequence of the parent polypeptide.
  • the additional glycosylation sites can be introduced to the parent polypeptide through amino acid substitution(s) located in a region that consists of the amino acid residues after the first 15 amino acids at the amino-terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide and before the last 15 amino acids at the carboxy-terminus of the parent polypeptide.
  • the additional glycosylation sites can be introduced to the parent polypeptide through amino acid substitution(s) located in a region consisting of the first 15 amino acid residues at the amino- terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide.
  • the additional glycosylation sites can be introduced to the parent polypeptide through amino acid substitution(s) located in a region consisting of the last 15 amino acid residues at the carboxy-terminus of the parent polypeptide.
  • the parent polypeptide can be a growth hormone (GH), where the additional glycosylation sites can be introduced to the parent GH through amino acid substitution(s) located at a region consisting of the 15th to 180th amino acids, the 20th to 160th amino acids, the 30th to 140th amino acids, the 40th to 120th amino acids, or the 50th to 110th amino acids of the parent GH, where any signal peptide in the parent GH is excluded.
  • GH growth hormone
  • the parent polypeptide can be an insulin-like growth factor (IGF), where the additional glycosylation site(s) can be introduced to the parent IGF through amino acid substitution(s) located at a region consisting of the 16th to 130th amino acids, the 40th to 110th amino acids, the 50th to 100th amino acids, or the 60th to 80th amino acids of the parent IGF, where any signal peptide in the parent IGF is excluded.
  • IGF insulin-like growth factor
  • the parent polypeptide can be a G-CSF, where the additional glycosylation site(s) can be introduced to the parent G-CSF through amino acid substitution(s) located at a region consisting of the 16th to 170th amino acids, the 20th to 150th amino acids, the 25th to 130th amino acids, or the 30th to 110th amino acids in the parent G-CSF, where any signal peptide in the parent G-CSF is excluded.
  • the parent polypeptide can be an erythropoietin (EPO), where the additional glycosylation site(s) can be introduced to the parent EPO through amino acid substitution(s) located at a region consisting of the 16th to 160th amino acids, the 20th to 140th amino acids, the 25th to 120th amino acids, or the 30th to 100th amino acids in the parent EPO, where any signal peptide in the parent EPO is excluded.
  • EPO erythropoietin
  • the parent polypeptide can be an insulin, where the additional glycosylation site(s) can be introduced to the parent insulin through amino acid substitution(s) located at a region consisting of the 16th to 85th amino acids, the 20th to 148th amino acids, the 25th to 75th amino acids, or the 30th to 65th amino acids in the parent insulin, where any signal peptide in the parent insulin is excluded.
  • the parent polypeptide in addition to the peptide extension that is inserted at a terminal region, has been further modified to include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten additional glycosylation sites in the amino acid sequence of the parent polypeptide.
  • Each additional glycosylation site that is introduced to the parent polypeptide may be a native glycosylation site that is not present in the parent polypeptide, or a non- native glycosylation site.
  • the amino acid sequence of the parent polypeptide can be modified to include a native glycosylation site and a non-native glycosylation site In some embodiments, the amino acid sequence of the parent polypeptide can be modified to include at least two, three, four, five, six, seven, eight, nine, or ten native glycosylation sites from the parent polypeptide, and at least two, three, four, five, six, seven, eight, nine, or ten non-native glycosylation sites.
  • the amino acid sequence of the parent polypeptide can be modified to include an O-linked glycosylation site. In other embodiments, the amino acid sequence of the parent polypeptide can be modified to include an N-linked glycosylation. In other embodiments, the amino acid sequence of the parent polypeptide can be modified to include both O-linked and N-linked glycosylation.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can further include at least one native glycosylation site from the parent polypeptide that is glycosylated in the hyperglycosylated polypeptide variant, but is not glycosylated in the parent polypeptide.
  • a hyperglycosylated polypeptide variant of a parent polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-7, 9- 14, 16-21, 23-28, and 30-35.
  • a hyperglycosylated polypeptide variant of a parent polypeptide is a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 2-7, 9-14, 16-21, 23-28, and 30-35.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can have at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and
  • the glycosylation of hyperglycosylated polypeptide variants of a parent polypeptide is altered compared to that of the parent polypeptide. In some embodiments, the glycosylation pattern of hyperglycosylated polypeptide variants of a parent polypeptide is altered compared to that of the parent polypeptide. In some embodiments, the hyperglycosylated polypeptide variants of a parent polypeptide exhibit increased amount of glycosylation compared to the parent polypeptide.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can exhibit one or more of the following properties: increased serum half-life; reduced immunogenicity in vivo; increased functional in vivo half-life; increased stability; reduced degradation by gastrointestinal tract conditions; and improved water solubility.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can have an increased serum half-life compared to a naturally occurring polypeptide or compared to the parent polypeptide under substantial similar or the same conditions.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can have an increased AUC compared to a naturally occurring polypeptide or compared to the parent polypeptide under substantial similar or the same conditions.
  • serum half-life is used interchangeably herein with the terms “plasma half-life,” and “circulating half-life.”
  • the hyperglycosylated polypeptide variants of a parent polypeptide can have a serum half-life that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9- fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40- fold, at least about 50-fold, at least about 60-fold, at least about 70
  • the extent of the increase in serum half-life of a hyperglycosylated polypeptide variant of a parent polypeptide is determined by comparing the serum half-life of the hyperglycosylated polypeptide variant of the parent polypeptide to the serum half-life of the parent polypeptide in human blood or human serum in vivo.
  • the hyperglycosylated polypeptide variants of a parent polypeptide can have an AUC that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5- fold, at least 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least 9.5-fold, at least 10-fold, or more, greater than
  • a hyperglycosylated polypeptide variant of a parent polypeptide can be detectably labeled, and be administered to a subject (for example, an experimental non-human animal, or a human subject), and, at various time points following administration of the hyperglycosylated polypeptide variant, a blood sample is drawn and the amount of detectably labeled hyperglycosylated polypeptide variant in the blood sample can be determined.
  • the hyperglycosylated polypeptide variants of a parent polypeptide can be detected in the serum of the subject after at least about 3 days, at least about 5 days, at least about 7 days, at least about 9 days, at least about 11 days, at least about 13 days, at least about 15 days, at least about 17 days, at least about 19 days, at least about 21 days, at least about 23 days, at least about 25 days, at least about 27 days, at least about 29 days, at least about 31 days, at least about 33 days, at least about 35 days, at least about 37 days, at least about 39 days, at least about 41 days, at least about 43 days, at least about 45 days, at least about 47 days, at least about 49 days, at least about 51 days, or longer after administration.
  • the hyperglycosylated polypeptide variants disclosed herein can have various molecular weight.
  • the molecular weight of the hyperglycosylated polypeptide variants is at least 70 kD, at least 75 kD, at least 80 kD, at least 85 kD, at least 90 kD, at least 95 kD, at least 100 kD, at least 105 kD, at least 110 kD, at least 115 kD, at least 120 kD, at least 125 kD, or at least 130 kD.
  • the molecular weight of the hyperglycosylated polypeptide variants is in the range of about 70 kD to about 200 kD.
  • the molecular weight of the hyperglycosylated polypeptide variants is in the range of about 70 kD to about 150 kD. In still other embodiments, the molecular weight of the hyperglycosylated polypeptide variants is in the range of about 70 kD to about 100 kD. In yet still other embodiments, the molecular weight of the hyperglycosylated polypeptide variants is in the range of about 80 kD to about 100 kD.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can be prepared using conventional techniques, including chemical synthesis methods, production by standard recombinant techniques, and combinations thereof.
  • the hyperglycosylated polypeptide variant can be synthesized using an automated solid-phase tert-butyloxycarbonyl and benzyl protection strategy.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can also be synthesized by native chemical ligation using standard methods of chemical synthesis. The purity of synthesized polypeptides may be assessed by reverse-phase high performance liquid chromatography (HPLC) and isoelectric focusing. The primary structures of the ligands may be verified by Edman sequencing methods.
  • the term "host cell” includes an individual cell or cell culture, which can be or has been a recipient of any recombinant vector(s), or synthetic or exogenous polynucleotide.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a synthetic or exogenous polynucleotide.
  • the term "recombinant host cell” refers to a host cell that includes one or more recombinant vectors.
  • a host cell can be a prokaryotic cell.
  • a host cell can be a eukaryotic cell.
  • Non-limiting examples of recombinant vectors include propagation vectors and expression vectors.
  • construct refers to a recombinant nucleic acid that has been generated for the purpose of the expression of a specific nucleotide sequence(s), or is to be used in the construction of other recombinant nucleotide sequences.
  • An example of a construct is a recombinant DNA.
  • a vector refers to a polynucleotide construct, typically a plasmid or a virus, used to transmit genetic material to a host cell.
  • a vector can be an agent such as a plasmid, for example, a circular plasmid.
  • a vector as used herein can be composed of either DNA or RNA.
  • a vector is composed of DNA.
  • an expression vector refers to a polynucleotide construct that can express a gene in a host cell.
  • an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and a gene is said to be “operably linked to” the promoter.
  • DNA regulatory sequences and “regulatory elements,” used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a coding sequence and/or production of an encoded polypeptide in a host cell.
  • promoter is a nucleotide sequence that directs the transcription of a gene. Typically, a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of the gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals (including humans). A promoter can be inducible, repressible, and/or constitutive.
  • the term “enhancer” refers to a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
  • transformation refers to a permanent or transient genetic change induced in a cell following introduction of exogenous nucleic acid to the cell. Genetic modification can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.
  • operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter effects transcription or expression of the coding sequence.
  • hyperglycosylated polypeptide variants of a parent polypeptide described herein can be prepared by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide variant.
  • the codons can be selected such that they are favored in the host cell in which the recombinant polypeptide will be produced.
  • oligonucleotides coding for portions of the hyperglycosylated polypeptide variant may be synthesized and assembled by PCR techniques, ligation, ligation chain reaction (LCR), or any other method or procedure known to one skilled in the art.
  • the individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • the nucleotide sequence encoding the hyperglycosylated polypeptide variant can be inserted into a recombinant propagation vector to produce a sufficient amount of the polynucleotide encoding the amino acid sequence of a hyperglycosylated polypeptide variant.
  • the nucleotide sequence encoding the hyperglycosylated polypeptide variant can be inserted into a recombinant expression vector for production of the hyperglycosylated polypeptide variant in a host cell.
  • the polynucleotide encoding the amino acid sequence of a hyperglycosylated polypeptide variant of a parent polypeptide can be generated such that at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or more, of the codons are codons that are preferred in human sequences.
  • Table 1 Codon Usage in Human.
  • Threonine 5.68 ACU (22.4) ACC (40.5)
  • the polypeptide-encoding nucleic acid molecules can be propagated by placing a nucleotide sequence that encodes a hyperglycosylated polypeptide variant of a parent polypeptide in a recombinant propagation vector.
  • Various viral and non-viral vectors including plasmids, bacteriophages (for example, lambda, PI, Ml 3, etc.), cosmids, fosmids, PI -derived artificial chromosomes (PAC), bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), animal viruses, plant virus, or Human Artificial Chromosomes (HAC) may be used as propagation vectors.
  • the choice of vectors will depend on the type of cell in which propagation is desired and the purpose of propagation, and is within the knowledge of one skilled in the art. Propagation vectors that are useful for amplifying and making large amounts of the desired DNA sequence can be used.
  • an expression cassette for expressing a hyperglycosylated polypeptide variant can include a promoter operably linked to a nucleotide sequence encoding a signal peptide, a first extension sequence under the control of the promoter encoding a first peptide extension, and a restriction site allowing for insertion of a gene encoding a biologically-active polypeptide, where the first peptide extension can be a peptide of 1-200 consecutive amino acids and include at least two glycosylation sites.
  • the expression cassette upon insertion of a gene encoding a biologically-active polypeptide at the restriction site, directs expression of a fusion protein comprosiing the biologically-active polypeptide linked to the first peptide extension.
  • the restriction site can be either immediately upstream or downstream of the first extension sequence encoding the first peptide extension.
  • the first peptide extension can be a peptide of 1-180, 1-150, 10-140, 20-130, 30-120, 40-110, 50-100, 60-90, 70-80 consecutive amino acids.
  • the first peptide extension can include at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty glycosylation sites.
  • the expression cassette can further include a gene encoding a biologically-active polypeptide inserted at the restriction site.
  • the first extension sequence is located between the gene encoding the biologically-active polypeptide and the nucleotide sequence encoding the signal peptide.
  • the gene encoding the biologically-active polypeptide is located between the nucleotide sequence encoding the signal peptide and the first extension sequence.
  • the expression cassette can further include a second extension sequence encoding a second peptide extension.
  • the gene encoding the biologically-active polypeptide is located between the first extension sequence and the second extension sequence.
  • the second peptide extension can include at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty glycosylation sites.
  • the expression cassette can be in the form of a linear or circular DNA, for example, and may be in the form of a plasmid.
  • the expression cassette can further include a second extension sequence encoding a second peptide extension.
  • the gene encoding the parent polypeptide is located between the first extension sequence and the second extension sequence.
  • the second peptide extension can include at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites.
  • Figure 6 schematic diagrams of some embodiments of the expression cassette for expressing a hyperglycosylated polypeptide are shown.
  • Figure 6A shows an expression cassette comprising a promoter operably linked with a nucleotide encoding a signal peptide, a first extension sequence under control of the promoter encoding a first peptide extension, and a gene encoding a biologically-active polypeptide inserted at the restriction site that is downstream of the first extension sequence.
  • Figure 6B shows an expression cassette comprising a promoter operably linked with a nucleotide encoding a signal peptide, a first extension sequence under control of the promoter encoding a first peptide extension, a second extension sequence encoding a second peptide extension, and a gene encoding a biologically-active polypeptide inserted at the restriction site that is downstream of the first extension sequence and upstream of the second extension sequence.
  • Figure 6C shows an expression cassette comprising a promoter operably linked with a nucleotide encoding a signal peptide, a first extension sequence under control of the promoter encoding a first peptide extension, and a gene encoding a biologically-active polypeptide inserted at the restriction site that is upstream of the first extension sequence.
  • promoters can be used to drive expression of the hyperglycosylated variants of a parent polypeptide in the expression cassettes disclosed herein.
  • promoters include, but are not limited to, viral promoters, plant promoters and mammalian promoters.
  • viral promoters include, but are not limited to Cytomegalovirus (CMV) promoter, Simian virus 40 (SV40) promoter, the 35S RNA and 19S RNA promoters of cauliflower mosaic virs (CaMV) described in Brisson et al., Nature 1984, 310:511-514, and the coat protein promoter to tobacco mosaic virus (TMV) described in Takamatsu et al., EMBO J. 1987, 3:111.
  • CMV Cytomegalovirus
  • SV40 Simian virus 40
  • CaMV cauliflower mosaic virs
  • TMV tobacco mosaic virus
  • plant promoters include, but are not limited to, heat shock promoters, such as soybean hspl7.5-E or hspl7.3- B described in Gurley et al., Mol. Cell. Biol. 1986, 6:559-565.
  • mammalian promoters include, but are not limited to, human elongation factor loc-subunit (EFl-l oc) promoter, human ubiquitin C (Ubc) promoter, and murine phosphoglycerate kinase- 1 (PGK) promoter.
  • EFl-l oc human elongation factor loc-subunit
  • Ubc human ubiquitin C
  • PGK murine phosphoglycerate kinase- 1
  • signal peptides can be used in the expression cassettes and/or expression vectors disclosed herein.
  • signal peptides include, but are not limited to, the endogenous signal peptide for interferons, including the signal peptide of type I, II and III interferons; and the endogenous signal pepide for any known cytokine, such as the signal peptide of erythropoietin (EPO), TGF- ⁇ , TNF, ILl-oc, and ILl- ⁇ .
  • EPO erythropoietin
  • TGF- ⁇ erythropoietin
  • TNF ILl-oc
  • ILl- ⁇ ILl- ⁇
  • Nucleotide sequences of the non-limiting examples of signal peptides are given in SEQ ID NOs: 162-169.
  • SEQ ID NO: 162 shows the sequence of signal peptide of human insulin
  • SEQ ID NO: 163 shows the sequence of an artificial signal peptide
  • SEQ ID NO: 164 shows the sequence of signal peptide of human CD33
  • SEQ ID NO: 165 shows the sequence of signal peptide of human EPO
  • SEQ ID NO: 166 shows the sequence of signal peptide of human G- CSF
  • SEQ ID NO: 167 shows the sequence of signal peptide of human growth hormone 1
  • SEQ ID NO: 168 shows the sequence of signal peptide of human interferon beta
  • SEQ ID NO: 169 shows the sequence of signal peptide of human insulin like growth factor 1A.
  • an artificial signal peptide can be used in an expression cassette and/or expression vector to facilitate the secretion of the biologically active polypeptide expressed from the expression cassette and/or expression vector.
  • the signal polypeptide for a protein that is different from the biologically active polypeptide can be used in an expression cassette and/or expression vector to facilitate the secretion of the biologically active polypeptide expressed from the expression cassette and/or expression vector.
  • the native signal polypeptide for a biologically active polypeptide can be used in an expression cassette and/or expression vector to facilitate the secretion of the biologically active polypeptide.
  • a biologically active polypeptide can utilize an artificial signal peptide or a signal peptide from another molecule and get effectively secreted out of the host cells. In some instances, a biologically active polypeptide prefers its own native signal peptide for efficient secretion out of the host cells. In some embodiments, the biologically active polypeptide can be human growth hormone, interferon lambda 1, interferon lambda 2, or interferon lambda 3.
  • Expression cassettes disclosed herein can include a transcription initiation region and/or a transcriptional termination region.
  • transcription termination region include, but are not limited to, the Bovine growth hormone (BGH) polyA, SV40 polyA, and thymidine kinase (TK) polyA sites.
  • BGH Bovine growth hormone
  • SV40 polyA SV40 polyA
  • TK thymidine kinase
  • the cells containing the expression cassette and/or vector may be selected by means of a selectable marker.
  • selectable markers include, but are not limited to, antibiotic selection markers such as Neomycine resistance gene, kanamycin resistance gene, gentamycin resistance gene, and Zeocin resistance gene.
  • Expression cassettes may be introduced into a variety of vectors suitable for eukaryotic host cell expression, such as plasmid; HAC; YAC; vectors derived from animal viruses, such as Moloney's murine leukemia virus, SV40, vaccinia virus, baculovirus, retroviruses, and plant viruses; and the like.
  • vectors suitable for eukaryotic host cell expression such as plasmid; HAC; YAC; vectors derived from animal viruses, such as Moloney's murine leukemia virus, SV40, vaccinia virus, baculovirus, retroviruses, and plant viruses; and the like.
  • an expression vector comprising an expression cassette disclosed herein can be used to producing the hyperglycosylated polypeptide variants of a parent polypeptide in a host cell.
  • An expression vector comprising the expression cassette can be introduced into a host cell, particularly a eukaryotic cell that is capable of glycosylating proteins.
  • a non-limiting example of methods for producing a hyperglycosylated polypeptide variant of a parent polypeptide can include culturing a eukaryotic host cell, where the host cell comprises a subject recombinant expression vector, under conditions that favor production of the hyperglycosylated polypeptide variant; and isolating the hyperglycosylated polypeptide variant from the culture.
  • the hyperglycosylated polypeptide variant can be isolated and purified to greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%, purity.
  • the expression vectors disclosed herein can include additional sequences that make the vectors suitable for replication and integration in eukaryotes.
  • the expression vectors disclosed herein can include a shuttle element that make the vectors suitable for replication and integration in both prokaryotes and eukaryotes.
  • the expression vectors can include transcription and translation initiation sequences, such as promoters and enhances; and transcription and translation terminators, such as polyadenylation signals.
  • a prokaryotic expression system such as a bacterial expression system
  • a bacterial expression system can be used to express the hyperglycosylated variants of a parent polypeptide as disclosed herein.
  • an expression cassette disclosed herein can be inserted into a bacterial expression vector.
  • bacterial expression vectors include, but are not limited to, the pET series of E. coli expression vectors (see Studier et al., Methods in Enzymol. 185:60-89, 1990).
  • a eukaryotic expression system can be used to express the hyperglycosylated variants of a parent polypeptide as disclosed herein.
  • eukaryotic expression systems include, but are not limited to, yeast expression systems, mammalian expression systems, insect expression systems and plant expression systems.
  • an expression cassette disclosed herein can be inserted into a yeast expression vector.
  • yeast expression vectors containing constitutive or inducible promoters can be used as disclosed in U.S. Pat. No. 5,932,447.
  • yeast expression vectors which promote integration of foreign DNA sequences into the yeast chromosome can be used.
  • an expression cassette disclosed herein can be inserted into a mammalian expression vector.
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+), pGL3, pZeoSV2(+), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen (Carlsbad, CA); pCI which is available from Promega (Madison, WI); pMbac, pPbac, pBK-RSV and pBK-CMV, which are available from Agilent Technologies (La Jolla, CA); pTRES which is available from Clontech (Mountain View, CA); and their derivatives.
  • an expression cassette disclosed herein can be inserted into a plant expression vector. In yet other embodiments, an expression cassette disclosed herein can be inserted into an insect expression vector. [0169] In some embodiment, the expression vectors disclosed herein can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
  • IRS internal ribosome entry site
  • an expression cassette disclosed herein can be inserted into a viral vector, such as an expression vectors comprising regulatory elements from eukaryotic viruses, such as retroviruses.
  • eukaryotic virus examples include, but are not limited to, vectors derived from Simian virus 40 (SV40), for example pSVT7 and pMT2; vectors derived from bovine papilloma virus (BPV), for example pBV-lMTHA; and vectors derived from Epstein Bar virus (EBV), for example pHEBO and p205.
  • Additional exemplary expression vectors that are derived from eukaryotic virus include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vectors allowing expression of proteins under the direction of SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters that are effective for protein expression in eukaryotic cells.
  • Introduction of expression vectors into a host cell may use any convenient method, such as calcium-precipitated DNA, electroporation, fusion, stable or transient transfection, lipofection, infection with viral vectors, biolistics, etc.
  • any convenient method such as calcium-precipitated DNA, electroporation, fusion, stable or transient transfection, lipofection, infection with viral vectors, biolistics, etc.
  • Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gil
  • the recombinant expression vectors can include expression cassettes and/or regulatory sequences ("control sequences” or “control regions") that can effect the expression of a desired polynucleotide to which they are operably linked.
  • control sequences or “control regions”
  • Various regulatory sequences can be used, including, but not limited to, promoter sequences and enhancer sequences.
  • the expression vectors can also have restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding a desired protein or other protein.
  • the signal peptides can be cleaved from the precursor proteins resulting in mature proteins.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can be synthesized in an expression host cell.
  • Various expression host cells for example, prokarytotic and eukaryotic cells, can be used to express the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein.
  • An example of a suitable expression host cell is a eukaryotic cell.
  • Examples of a eukaryotic cell include, but are not limited to; a yeast cell, such as a cell from S.
  • the protein product may include post-translational modification.
  • microorganisms such as bacteria
  • host cells containing expression vectors encoding the hyperglycosylated polypeptide variants of a parent polypeptide can be cultured under effective conditions, which allows for the expression of high amounts of the hyperglycosylated polypeptide variants.
  • Factors that can impact effective culture conditions include, but are not limited to, media, bioreactor, temperature, pH and oxygen content.
  • an effective medium refers to any medium in which a cell is cultured to produce the the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein.
  • a medium can include an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • host cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, micro titer dishes and petri plates. Culturing conditions can be adjusted based on expertise of one of ordinary skill in the art. [0176] The hyperglycosylated polypeptide variant of a parent polypeptide may be isolated and purified in accordance with conventional methods known to those skilled in the art.
  • a lysate may be prepared of the expression host cells and the lysate may be purified using HPLC, hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, ultrafiltration, gel electrophoresis, affinity chromatography, and/or other purification techniques.
  • HPLC hydrophobic interaction chromatography
  • anion exchange chromatography anion exchange chromatography
  • cation exchange chromatography size exclusion chromatography
  • ultrafiltration gel electrophoresis
  • affinity chromatography and/or other purification techniques.
  • Any known assay can be used to determine whether a glycosylated polypeptide, for example, a hyperglycosylated polypeptide variant of a parent polypeptide, exhibits at least one desired pharmacologic activity of the parent polypeptide.
  • useful assays for particular parent polypeptides include, but are not limited to, Testa et al., Assays for hematopoietic growth factors. In: Balkwill F R (edition) Cytokines A practical Approach, pages 229-244; IRL Press Oxford 11021); Kitamura et al., J. Cell. Physiol.
  • compositions including pharmaceutical compositions, which can include a therapeutically effective amount of one or more hyperglycosylated polypeptide variant of a parent polypeptide disclosed herein.
  • the compositions can include one or more hyperglycosylated polypeptide variants of a parent polypeptide described herein and a pharmaceutically acceptable excipient and/or carrier.
  • physiologically acceptable and “pharmaceutically acceptable” refer to a carrier, diluent or excipient that does not abrogate the biological activity and properties of the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein.
  • a “carrier” refers to a compound that facilitates the incorporation of a compound, such as a hyperglycosylated polypeptide variant of a parent polypeptide, into cells or tissues.
  • a compound such as a hyperglycosylated polypeptide variant of a parent polypeptide
  • DMSO dimethyl sulfoxide
  • a "diluent” refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable.
  • a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation.
  • a common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.
  • an “excipient” refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition.
  • a “diluent” is a type of excipient.
  • a therapeutically effective amount refers to an amount of an active compound, or pharmaceutical agent, that elicits the biological or medicinal response indicated.
  • a therapeutically effective amount of a hyperglycosylated polypeptide variant of a parent polypeptide can be the amount need to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • the biological or medicinal response may occur in a tissue, system, animal or human, and includes alleviation of the symptoms of the disease being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art in light of the detailed disclosure provided herein.
  • the therapeutically effective amount of the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein required will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration.
  • the therapeutically effective amount can also depend on factors as weight, diet, concurrent medication; and other factors which those skilled in the medical arts will recognize.
  • compositions disclosed herein may be manufactured in a manner that is itself known, such as by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.
  • the pharmaceutical compositions can be obtained by reacting the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • Pharmaceutical compositions will generally be tailored to the specific intended route of administration. Additionally, the active ingredients are contained in an amount therapeutically effective to achieve its intended purpose.
  • the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharmaceutically compatible counterions.
  • suitable additional components of a pharmaceutical composition include, but are not limited to, salts, buffers, solubilizers, stabilizers, detergents, protease-inhibiting agents, and the like.
  • Suitable routes of administration may, for example, include oral, rectal, topical transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, intraocular injections or as an aerosol inhalant.
  • one or more hyperglycosylated polypeptide variants of a parent polypeptide are formulated into a preparation suitable for oral administration.
  • the hyperglycosylated polypeptide variant can be formulated alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives, and/or flavoring agents.
  • one or more hyperglycosylated polypeptide variants of a parent polypeptide are formulated into a preparation suitable for injection.
  • the hyperglycosylated polypeptide variant(s) of a parent polypeptide can be by dissolved, suspended or emulsified in an aqueous solvent (for example, saline, and the like) or a nonaqueous solvent (for example, vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol); and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • an aqueous solvent for example, saline, and the like
  • a nonaqueous solvent for example, vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
  • compositions described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or carriers, diluents, excipients or combinations thereof. Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the hyperglycosylated polypeptide variants of a parent polypeptide described herein are known to those skilled in the art.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • Compositions that include a hyperglycosylated polypeptide variant of a parent polypeptide disclosed herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • Some embodiments disclosed herein relate to a method of treating and/or ameliorating a disease or condition that can include administering to a subject a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and/or therapy.
  • Treatment covers any treatment of a disease in a subject, for example, in a human.
  • Treatment includes, but is not limited to: (a) increasing survival time; (b) decreasing the risk of death due to the disease; (c) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (d) inhibiting the disease, that is, arresting its development (for example, reducing the rate of disease progression); and (e) relieving the disease, that is, causing regression of the disease.
  • treatment may include acts that may worsen the patient's overall feeling of well- being or appearance.
  • a "subject” refers to an animal that is the object of treatment, observation or experiment.
  • Animal includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles, and in particular, mammals.
  • “Mammal” includes, without limitation, mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates; such as monkeys, chimpanzees and apes, and, in particular, humans.
  • Some embodiments disclosed herein relate to a method of ameliorating and/or treating fibrotic disorders that can include administering to a subject suffering from fibrotic disorders a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein.
  • the method can further include administering one or more additional anti-fibrotic agents.
  • Additional anti-fibrotic agents include, but are not limited to, SAPK inhibitors (such as pirfenidone or pirfenidone analogs), TNF antagonists (such as etanercept, infliximab, or adalimumab), TGF- ⁇ antagonists (such as GLEEVEC), endothelin receptor antagonists (such as TRACLEER), and the like.
  • SAPK inhibitors such as pirfenidone or pirfenidone analogs
  • TNF antagonists such as etanercept, infliximab, or adalimumab
  • TGF- ⁇ antagonists such as GLEEVEC
  • endothelin receptor antagonists such as TRACLEER
  • Fibrosis is generally characterized by the pathologic or excessive accumulation of collagenous connective tissue.
  • the fibrotic disorders can be those diseases or conditions affecting the lung such as idiopathic pulmonary fibrosis, pulmonary fibrosis from a known etiology, liver fibrosis or cirrhosis, cardiac fibrosis, and renal fibrosis.
  • Additional fibrotic disorders include, but are not limited to, collagen disease, interstitial lung disease, human fibrotic lung disease (such as obliterative bronchiolitis, idiopathic pulmonary fibrosis, pulmonary fibrosis from a known etiology, tumor stroma in lung disease, systemic sclerosis affecting the lungs, Hermansky-Pudlak syndrome, coal worker's pneumoconiosis, asbestosis, silicosis, chronic pulmonary hypertension, AIDS- associated pulmonary hypertension, sarcoidosis, and the like), fibrotic vascular disease, arterial sclerosis, atherosclerosis, varicose veins, coronary infarcts, cerebral infarcts, myocardial fibrosis, musculoskeletal fibrosis, post-surgical adhesions, human kidney disease (such as nephritic syndrome, Alport's syndrome, HIV-associated nephropathy, polycystic kidney disease, Fab
  • Some embodiments disclosed herein relate to a method of ameliorating and/or treating cancer that can include administering to a subject suffering from cancer a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein.
  • the method can further include administering one or more additional anti-cancer agents.
  • additional anti-cancer agents include, but are not limited to, chemotherapeutic agents, radiation agents, bone marrow, biological response modifier, agents that act to reduce cellular proliferation, antimetabolite agents, microtubule affecting agents, hormone modulators, antibodies, and anti-angiogenic agents.
  • the cancer can be a carcinoma. In other embodiments, the cancer can be a sarcoma. In still other embodiments, the cancer can be a tumor, such as a solid tumor. In yet other embodiments, the cancer can be leukemia. In yet still other embodiments, the cancer can be lymphoma.
  • carcinomas include, but are not limited to, esophageal carcinoma; hepatocellular carcinoma; basal cell carcinoma, squamous cell carcinoma (various tissues) ; bladder carcinoma, including transitional cell carcinoma; bronchogenic carcinoma; colon carcinoma; colorectal carcinoma; gastric carcinoma; lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung; adrenocortical carcinoma; thyroid carcinoma; pancreatic carcinoma; breast carcinoma; ovarian carcinoma; prostate carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinoma; cystadenocarcinoma; medullary carcinoma; renal cell carcinoma; ductal carcinoma in situ or bile duct carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; Wilm's tumor; cervical carcinoma; uterine carcinoma; testicular carcinoma; osteogenic carcinoma; epithelieal carcinoma; nasopharyngeal carcinoma; etc.
  • sarcomas include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.
  • solid tumors include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
  • leukemias include, but are not limited to, chronic myeloproliferative syndromes; acute myelogenous leukemias; chronic lymphocytic leukemias, including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and acute lymphoblastic leukemias.
  • lymphomas include, but are not limited to, B-cell lymphomas, such as Burkitt's lymphoma; Hodgkin's lymphoma; and the like.
  • Some embodiments disclosed herein relate to a method of inhibiting the growth of a tumor that can include administering to a subject having the tumor a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein.
  • Some embodiments disclosed herein relate to a method of ameliorating and/or treating viral infection that can include administering to a subject suffering from viral infection a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein.
  • the method can further include administering one or more additional anti-viral agents.
  • anti-viral agents include, but are not limited to nucleoside analogs (for example, ribavirin, viramidine and levovirin) and HCV NS3 inhibitors.
  • the viral infection can be caused by a virus selected from an adenovirus, an Alphaviridae, an Arbovirus, an Astrovirus, a Bunyaviridae, a Coronaviridae, a Filoviridae, a Flaviviridae, a Hepadnaviridae, a Herpesviridae, an Alphaherpesvirinae, a Betaherpesvirinae, a Gammaherpesvirinae, a Norwalk Virus, an Astroviridae, a Caliciviridae, an Orthomyxoviridae, a Paramyxoviridae, a Paramyxoviruses, a Rubulavirus, a Morbilli virus, a Papovaviridae, a Parvoviridae, a Picornaviridae, an Aphthoviridae, a Cardioviridae, an Enteroviridae, a Cox
  • Some embodiments provides a method of reducing the risk of viral infection for a subject who has been exposed to a virus (for example, a subject who has come into contact with another subject infected with a virus).
  • the method can include administering to the subject who has been exposed to a virus a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptide variants of a parent polypeptide described herein.
  • the method can further include administering one or more additional anti-viral agents.
  • a hyperglycosylated polypeptide variant of a parent polypeptide can be exhibit one or more of the following activities: antiproliferative activity, anti-viral activity, and anti-fibrotic activity. Whether a hyperglycosylated polypeptide variant of a parent polypeptide exhibits anti-viral activity can be determined using any known assay, including for example, an in vitro cell-based inhibition of viral replication assay described in Patick et al. Antimicrob. Agents Chemother., 1999, 43:2444-2450. Whether a hyperglycosylated polypeptide variant of a parent polypeptide exhibits anti-proliferative activity can be determined using any known assay, including for example, an in vitro cell- based inhibition of proliferation assay.
  • the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, and animal species treated, the particular hyperglycosylated polypeptide variant of a parent polypeptide employed, and the specific use for which these hyperglycosylated polypeptide variants of a parent polypeptide are employed.
  • the determination of effective dosage levels can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved.
  • acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.
  • the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein may be administered orally or via injection at a dose of between 0.01 mg and 3000 mg of each ingredient, preferably between 1 mg and 700 mg, for example, 5 to 200 mg.
  • the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein can be administered orally or via injection at a dose of between about 0.001 mg and about 30 mg of each ingredient, or between about 0.001 mg and about 25 mg, between about 0.001 mg and about 20 mg, between about 0.001 mg and about 15 mg, between about 0.001 mg and about 10 mg, between about 0.005 mg and about 5 mg, between about 0.005 mg and about 4 mg, between about 0.005 mg and about 3 mg, between about 0.005 mg and about 2 mg, between about 0.005 mg and about 1 mg, preferably between 0.01 mg and 1 mg, for example, between about 0.05 to about 0.5 mg, between about 0.01 to about 0.3 mg, or between about 0.01 to about 0.1 mg.
  • human dosages for the hyperglycosylated polypeptide variants of a parent polypeptide have been established for at least some condition, those same dosages, or dosages that are between about 0.1% and 500%, more preferably between about 25% and 250% of the established human dosage can be used.
  • a suitable human dosage can be inferred from ED 50 or ID 50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
  • dosages may be calculated as the free base.
  • dosages may be administered in amounts that exceed, or even far exceed, the above-stated, preferred dosage range in order to effectively and aggressively treat particularly aggressive diseases or infections.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each hyperglycosylated polypeptide variant of a parent polypeptide, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
  • Dosage intervals can also be determined using MEC value.
  • Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
  • the effective local concentration of the drug may not be related to plasma concentration.
  • the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and/or dose frequency may also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
  • the hyperglycosylated polypeptide variants of a parent polypeptide can be administered less frequently at substantially the same amount as compared to the parent polypeptide to achieve substantially similar or the same therapeutic results.
  • the hyperglycosylated polypeptide variants of a parent polypeptide can be administered orally, once every three months, once every two months, once every month, twice per month, three times per month, once every week, once every five days, once every three days, once every two days, once per day, twice per day, or three times per day substantially continuously or continuously, for the desired duration of treatment.
  • the hyperglycosylated polypeptide variants of a parent polypeptide can be administered via injection, once every three months, once every two months, once every month, twice per month, three times per month, once every week, once every five days, once every three days, once every two days, once per day, twice per day, or three times per day substantially continuously or continuously, for the desired duration of treatment.
  • a therapeutic amount of the hyperglycosylated polypeptide variants of a parent polypeptide can be administered to a subject at various points of time for a substantial continuous or continuous period of time, for example, for a week or more, or for a month or more, or for a year or more.
  • the hyperglycosylated polypeptide variants of a parent polypeptide is administered for a period of time, which time period can be, for example, at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 54 weeks, at least about 58 weeks, at least about 62 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 74 weeks, at least about 78 weeks, at least about 82 weeks, at least about 86 weeks, at least about 90 weeks, at least about 94 weeks, at least about 98 weeks, at least about 102 weeks, at least about 106 weeks, or longer.
  • the hyperglycosylated polypeptide variants of a parent polypeptide can be administered once every two weeks for about
  • the hyperglycosylated polypeptide variants of a parent polypeptide can be administered at a dosing interval of about every 3 days to about every 7 days, about every 5 days to every 9 days, about every 7 days to every 11 days, about every 9 days to every 13 days, about every 11 days to every 15 days, about every 13 days to every 17 days, about every 15 days to every 19 days, about every 17 days to every 21 days, about every 19 days to every 23 days, about every 21 days to every 25 days, about every 23 days to every 27 days, about every 25 days to every 29 days, about every 27 days to every 31 days, about every 29 days to every 33 days, about every 31 days to every 35 days, about every 33 days to every 37 days, about every 35 days to every 39 days, about every 37 days to every 41 days, about every 39 days to every 43 days, about every 41 days to every 45 days, about every 43 days to every 47 days, or about every 45 days to every 49 days.
  • dosage levels In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear.
  • the dosage may range broadly, depending upon the desired effects and the therapeutic indication. Alternatively, dosages may be based and calculated upon the surface area of the subject, as understood by those of skill in the art.
  • Hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein can be evaluated for efficacy and toxicity using known methods.
  • the toxicology of a particular hyperglycosylated polypeptide variant of a parent polypeptide, or of a subset of the hyperglycosylated polypeptide variants of a parent polypeptide, sharing certain chemical moieties may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line.
  • a cell line such as a mammalian, and preferably human, cell line.
  • the results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans.
  • the toxicity of particular hyperglycosylated polypeptide variants of a parent polypeptide in an animal model may be determined using known methods.
  • the efficacy of a particular hyperglycosylated polypeptide variant of a parent polypeptide may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. Recognized in vitro models exist for nearly every class of condition, including but not limited to cancer, cardiovascular disease, and various immune dysfunction.
  • acceptable animal models may be used to establish efficacy of chemicals to treat such conditions.
  • the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, and regime.
  • Human clinical trials can also be used to determine the efficacy of a hyperglycosylated polypeptide variant of a parent polypeptide in humans.
  • the expression vector or expression cassette containing sequence encoding a biologically-active peptide, may be expressed in a mammal.
  • the expression vector or cassette may advantageously be introduced in various ways to secure expression thereof, such as transfection of the mammal with a viral vector containing the expression vector or cassette; introduction of the expression vector or cassette through naked DNA injection; introduction through electroporation; ex vivo transfection of cells that are then reintroduced into the mammal; and the like.
  • the expression vector or cassette is under the control of an inducible promoter. In this way, when the mammal is in need of therapy using the biologically-active peptide, systemic or localized delivery of the inducer can effect transcription and translation of the relevant DNA and result in delivery of therapeutic polypeptide to the mammal.
  • the parent polypeptide can be human growth hormone 1 (hGH-1), human insulin-like growth factor 1A (hIGF- ⁇ ), human granulocyte colony-stimulating factor (hG-CSF), human erythropoietin (hEPO), human insulin, an Fab fragment of an antibody specific for human TNF-oc receptor, and an scFv fragment of an antibody specific for human TNF-oc receptor.
  • hGH-1 human growth hormone 1
  • hIGF- ⁇ human insulin-like growth factor 1A
  • hG-CSF human granulocyte colony-stimulating factor
  • hEPO human erythropoietin
  • DNA constructs encoding hyperglycosylated variants of hGH-1 that have peptide extensions with additional glycosylation sites at an amino-terminal and/or a carboxy- terminal region are generated by site-directed mutagenesis using standard DNA techniques known in the art.
  • a DNA sequence encoding a peptide extension is inserted immediately upstream of the amino-terminus of the parent hGH-1 excluding any signal peptide and/or downstream of carboxy-terminus the parent hGH-1.
  • the sequences of the non-limiting examples of hyperglycosylated variants of hGH-1 are given in SEQ ID NOs: 2-7.
  • Figures 7A-I show the structure and sequence of the expression cassette in expression vectors pC6-l- hGHl, pN6-l-hGHl, pN6-2-hGHl, pNlO-l-hGHl, P N10-C4-l-hGHl, P N10-C6-l-hGHl, pN14-l-hGHl, pN14(2)-l-hGHl, and pN14(3)-l-hGHl.
  • sequences of expression vectors pC6-l-hGHl, pN6-l-hGHl, pN6-2-hGHl, pNlO-l-hGHl, pN10-C4-l-hGHl, pNlO- C6-l-hGHl, P N14-l-hGHl, pN14(2)-l-hGHl, and pN14(3)-l-hGHl are given in SEQ ID NOs: 170-178.
  • HEK293 cells are tranfected with the DNA constructs encoding hyperglycosylated variants of hGH-1.
  • Transient expression, isolation of stable expression clones, and large-scale production of hyperglycosylated variants of hGH-1 proteins are performed using standard protein expression techniques known in the art.
  • Sequences of the non-limiting examples of hyperglycosylated variants of hIGF- ⁇ are given in SEQ ID NOs: 9-14.
  • Figure 8 shows the structure and sequence of the expression cassette in expression vector pNlO-1-hIGFlA. The sequence of pNlO-1-hIGFlA is given in SEQ ID NO: 179. Sequences of the non-limiting examples of hyperglycosylated variants of hG-CSF are given in SEQ ID NO: 16-21.
  • Figure 9 shows the structure and sequence of the expression cassette in expression vector pN 10-1 -hG-CSF. The sequence of pNlO-l-h G-CSF is given in SEQ ID NO: 180.
  • Sequences of the non-limiting examples of hyperglycosylated variants of hEPO are given in SEQ ID NOs: 23-28.
  • Figure 10 shows the structure and sequence of the expression cassette in expression vector pNlO-l-hEPO. The sequence of pNlO-l-hEPO is given in SEQ ID NO: 181. Sequences of the non-limiting examples of hyperglycosylated variants of human insulin are given in SEQ ID NOs: 30-35.
  • Figure 11 shows the structure and sequence of the expression cassette in expression vector pN 10-1 -Insulin. The sequence of pN 10-1 -Insulin is given in SEQ ID NO: 182.
  • Sequences of the non-limiting examples of hyperglycosylated variants of the heavy chain of an Fab fragment of an antibody specific for human HER-2 receptor are given in SEQ ID NOs: 37-42. Sequences of the non-limiting examples of hyperglycosylated variants of the light chain of an Fab fragment of an antibody specific for human HER-2 receptor are given in SEQ ID NOs: 44-49. Sequences of the non-limiting examples of hyperglycosylated variants of the heavy chain of an Fab fragment of an antibody specific for human TNF-oc are given in SEQ ID NOs: 51-56.
  • Sequences of the non-limiting examples of hyperglycosylated variants of the light chain of an Fab fragment of an antibody specific for human TNF-oc are given in SEQ ID NOs: 58-63. Sequences of the non-limiting examples of hyperglycosylated variants of the heavy chain of an Fab fragment of an antibody specific for human VEGF are given in SEQ ID NOs: 65-70. Sequences of the non-limiting examples of hyperglycosylated variants of the light chain of an Fab fragment of an antibody specific for human VEGF are given in SEQ ID NOs: 72-77.
  • Sequences of the non-limiting examples of hyperglycosylated variants of human TNF-oc soluble receptor Rl are given in SEQ ID NOs: 79-84. Sequences of the non-limiting examples of hyperglycosylated variants of human TNF-oc soluble receptor R2 are given in SEQ ID NOs: 86-91. Sequences of the non-limiting examples of hyperglycosylated variants of various fragments of human VEGF soluble receptor Rl are given in SEQ ID NOs: 93-98, 100-105, 107-112, 114-119, and 121-126.
  • Expression vector pC6-l-hGHl ( Figure 7 A) that contains an expression cassette for expressing hyperglycosylated variants of hGHl was generated.
  • a CMV promoter is operably linked with a nucleotide sequence encoding the signal peptide of IFN ⁇ and an extension sequence encoding a peptide extension that includes six glycosylation sites.
  • a gene encoding hGHl is inserted between the nucleotide sequence encoding the signal peptide of IFN ⁇ and the extension sequence, and a bovine growth hormone (BGH) polyadenylation signal sequence is located downstream of the extension sequence.
  • BGH bovine growth hormone
  • Expression vector pN6-l-hGHl ( Figure 7B) that contains an expression cassette for expressing hyperglycosylated variants of hGHl was also generated.
  • a CMV promoter is operably linked with a nucleotide sequence encoding the signal peptide of IFN ⁇ and an extension sequence encoding a peptide extension that includes six glycosylation sites.
  • the extension sequence is located immediately downstream of the signal peptide of IFN ⁇ and a gene encoding hGHl is inserted immediately downstream of the extension sequence.
  • Expression vector pN6-2-hGHl ( Figure 7C) was generated similarly as expression vector pN6-l-hGHl except that an artificial signal peptide is used instead of the signal peptide of IFN ⁇ .
  • Expression vector pN 10-1 -hGHl ( Figure 7D) was generated similarly as expression vector pN6-l-hGHl except that the peptide extension in pNlO-1- hGHl includes ten glycosylation sites.
  • Expression vectors pN14-l-hGHl ( Figure 7G), pN 14(2)- 1 -hGHl ( Figure 7H) and pN14(3)-l-hGHl ( Figure 71) were also generated similarly as expression vector pN6-l-hGHl except that the peptide extensions in pN 14-1 -hGHl, pN 14(2)- 1 -hGHl and pN14(3)-l-hGHl include fourteen glycosylation sites.
  • Expression vector pN10-C4-l-hGHl ( Figure 7E) that contains an expression cassette for expressing hyperglycosylated variants of hGHl was generated.
  • a CMV promoter is operably linked with a nucleotide sequence encoding the signal peptide of IFN ⁇ ;
  • a first extension sequence encoding a first peptide extension that includes ten glycosylation sites is located immediately downstream of the signal peptide of IFN ⁇ ;
  • a second extension sequence encoding a second peptide extension includes four glycosylation sites.
  • a gene encoding hGHl is located between the first and second extension sequences.
  • Expression vector pN10-C6-l-hGHl ( Figure 7F) is generated similarly as the expression vector pN10-C4-l-hGHl except that the second peptide extension in pNlO- C6-l-hGHl has six glycosylation sites.
  • sequences of expression vectors pC6-l-hGHl, pN6-l-hGHl, pN6-2- hGHl, pNlO-l-hGHl, pN10-C4-l-hGHl, pN10-C6-l-hGHl, P N14-l-hGHl, pN14(2)-l- hGHl, and pN14(3)-l-hGHl are given in SEQ ID NOs: 170-178.
  • Expression vector pNlO-1-hIGFlA ( Figure 8) that contains an expression cassette for expressing a hyperglycosylated variant of human insulin-like growth factor 1A (hIGF- ⁇ ) was generated.
  • a CMV promoter is operably linked with a nucleotide sequence encoding the signal peptide of IFN ⁇ and an extension sequence encoding a peptide extension includes ten glycosylation sites.
  • the extension sequence is located immediately downstream of the signal peptide of IFN ⁇ , a gene encoding hIGF- ⁇ is inserted immediately downstream of the extension sequence, and a BGH polyadenylation signal sequence is located downstream of the extension sequence.
  • the sequence of pNlO-1-hIGFlA is given in SEQ ID NO: 179.
  • Expression vector pNlO-l-hG-CSF ( Figure 9) that contains an expression cassette for expressing a hyperglycosylated variant of human granulocyte colony-stimulating factor (hG-CSF) was generated similarly as pNlO-1-hIGFlA except that the gene encoding hG-CSF is inserted immediately downstream of the extension sequence.
  • the sequence of pNlO-l-h G-CSF is given in SEQ ID NO: 180.
  • Expression vector pNlO-l-hEPO ( Figure 10) that contains an expression cassette for expressing a hyperglycosylated variant of human Erythropoietin (hEPO) was generated similarly as pNlO-1-hIGFlA except that the gene encoding hEPO is inserted immediately downstream of the extension sequence.
  • the sequence of pN 10-1 -hEPO is given in SEQ ID NO: 181.
  • Expression vector pN 10-1 -Insulin ( Figure 11) that contains an expression cassette for expressing a hyperglycosylated variant of human insulin was generated as pNlO- 1-hIGFlA except that the gene encoding human insulin is inserted immediately downstream of the extension sequence.
  • the sequence of pN 10-1 -Insulin is given in SEQ ID NO: 182.
  • This example illustrates the construction of hyperglycosylated variants of interferon alfacon-1 (CIFN) with various amino-terminal peptide extension: (1) a hyperglycosylated variant of the parent CIFN with an N-terminal peptide extension VNITG and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-Nl-31-102-138”); (2) a hyperglycosylated variant of the parent CIFN with an N- terminal peptide extension (VNITG) 2 and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-N2-31-102-138”); (3) a hyperglycosylated variant of the parent CIFN with an N-terminal peptide extension (VNITG) 3 and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-N3-31-102-138”; and (4) a hyperglycosyl
  • N-terminal or C-terminal peptide extensions can be generated through conventional techniques known in the art, such as polymerase chain reaction (PCR)-based mutagenesis. In some experiments, the shorter extension was generated first and the longer extension was generated based on the shorter extension in a step-wise fashion.
  • PCR polymerase chain reaction
  • the primers used in the PCR reactions to generate DNA sequences encoding CIFN-Nl-31-102-138 (SEQ ID NO:329), CIFN-N2-31-102-138 (SEQ ID NO:330), CIFN-N3-31-102-138 (SEQ ID NO: 331), and CIFN-N4-31-102-138 (SEQ ID NO: 332) are listed in Table 2.
  • step 1 of the PCR reaction a 506bp fragment 1 was generated by using the gene encoding CIFN-31-102- 138 (which is a variant of the parent CIFN with additional glycosylation sites at amino acid positions 31, 102 and 138) as a template, and CIFN-N1-F and ECOR I-R as primers.
  • step 2 a 506bp fragment 1 was generated by using the gene encoding CIFN-31-102- 138 (which is a variant of the parent CIFN with additional glycosylation sites at amino acid positions 31, 102 and 138) as a template, and CIFN-N1-F and ECOR I-R as primers.
  • a 95bp fragment 2 was generated by using the gene encoding CIFN-31-102-138 as a template, and CIFN-N1-R and HIND III-F as primers.
  • step 2 of the PCR reaction fragments 1 and 2 were used as templates to generate a 601bp fragment 3 using ECOR I-R and HIND III-F as primers. Fragment 3 was digested by Hind III and EcoR I, and then cloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA).
  • DNA sequences encoding CIFN-Nl-31-102-138 were generated similarly in which a DNA sequence encoding CIFN-Nl-31-102-138 was used as a template, and CIFN-N2-F and CIFN-N2-R were used as primers in step 1 of the PCR reaction.
  • a similar 2-step PCR was used in which a DNA sequence encoding CIFN-N2-31-102- 138 was used as a template, and CIFN- N3-F and CIFN-N3-R were used as primers in step 1 of the PCR reaction.
  • pcDNA3.1 vectors containing DNA sequences encoding each hyperglycosylated variant of CIFN were transfected into mammalian cell lines, such as HEK293 cells, CHO cells and Cos-7 cells, for protein expression.
  • DNA encoding various hyperglycosylated variants of CIFN with an N-terminal or a C-terminal peptide extension, such as those from Example 4, were synthesized and transfected into mammalian cell line CHO to express hyperglycosylated variants of CIFN protein.
  • the media containing the secreted hyperglycosylated variants of CIFN proteins were collected and analyzed with protein gel electrophoresis and western blot analysis using the polyclonal antibodies targeting the parent CIFN.
  • N-terminal peptide extensions on a CIFN variant increased the molecular weight of the CIFN variant
  • CIFN-Nl-31-102-138 Four hyperglycosylated variants of CIFN: CIFN-Nl-31-102-138, CIFN- N2-31-102-138, CIFN-N3-31-102-138, and CIFN-N4-31-102-138, were produced as described in Example 4.
  • CIFN-31-102-138 which is a variant of the parent CIFN having a sequence identical to the four hyperglycosylated variants of the parent CIFN except for the absence of a N-terminal peptide extension, was used as a control in the western blot analysis shown in Figure 12A.
  • CIFN-N4-31-102-138 which had 4 glycosylation sites in the N-terminal peptide extension had the highest molecular weight among the four hyperglycosylated variants of CIFN; CIFN-N3-31-102-138 which had 3 glycosylation sites in the N-terminal peptide extension had the second highest molecular weight among the four variants; CIFN-N2-31-102-138 which had 2 glycosylation sites in the N-terminal peptide extension had the third highest molecular weight among the four variants; and CIFN-Nl-31-102-138 which had 1 glycosylation site in the N-terminal peptide extension had the lowest molecular weight among the four variants. Therefore, adding a motif with one or more glycosylation sites, such as VNITG motif, increases the molecular weight of the starting interferon.
  • CIFN- 102-138-C2 is a hyperglycosylated variant of CIFN with a C-terminal peptide extension (VNITG) 2 and additional glycosylation sites at amino acid positions 102 and 138.
  • CIFN-102-138 is a variant of CIFN with additional glycosylation sites at amino acid positisions 102 and 138 of the CIFN.
  • a DNA sequence encoding CIFN- 102-138-C2 was generated by the PCR- based mutagenesis method as described in Example 4, and transfected into CHO cells for the expression of CIFN- 102-138-C2 protein.
  • Western blot analysis of Figure 12B showed that CIFN- 102-138-C2 had an increased molecular weight compared to CIFN-102-138.
  • the C-terminal peptide extension (VNITG) 2 that contains 2 N-linked glycosylated sites increased the molecular weight of the starting CIFN-102-138 interferon.
  • the DNA inserts were then double digested with NotI and Swal and cloned into the cloning vector.
  • the four variants are: (1) CIFN-N 14(3)- 102 (SEQ ID NO: 334) having the N-terminal peptide extension with 14 glycosylation sites and an additional glysoylation site at amino acid position 102; (2) CIFN-N14(3)-102-138 (SEQ ID NO:336) having the N-terminal peptide extension with 14 glycosylation sites and two additional glysoylation sites at amino acid positions 102 and 138; (3) CIFN-N14(3)-108-138 (SEQ ID NO:337) having the N-terminal peptide extension with 14 glycosylation sites and two additional glysoylation sites at amino acid positions 108 and 138; and (4) CIFN-N14(3)- 108 (SEQ ID NO:335) having the N-terminal peptide extension with 14 glycosylation sites and an additional glysoy
  • CIFN- Nl 1-31-102-138 had an N-terminal peptide extension with 11 glycosylation sites
  • CIFN-N8- 31-102-138 had an N-terminal peptide extension with 8 glycosylation sites
  • CIFN-N6-31- 102-138 had an N-terminal peptide extension with 6 glycosylation sites
  • CIFN-N4-31- 102-138 had an N-terminal peptide extension with 4 glycosylation sites.
  • all 4 hyperglycosylated variants of the parent CIFN had an increased molecular weight compared to the starting CIFN.
  • N-terminal peptide extensions containing various numbers of glycosylation sites increased the molecular weight of the starting CIFN.
  • CIFN-N11-31-102-138 which had 11 glycosylation sites in the N-terminal peptide extension had the highest molecular weight among the four hyperglycosylated variants of CIFN; CIFN-N8-31-102-138 which had 8 glycosylation sites in the N-terminal peptide extension had the second highest molecular weight among the four variants; CIFN-N6-31-102-138 which had 6 glycosylation sites in the N-terminal peptide extension had the third highest molecular weight among the four variants; and CIFN-N4-31-102-138 which had 4 glycosylation site in the N-terminal peptide extension had the lowest molecular weight among the four variants.
  • N-terminal peptide extension with various numbers of glycosylation sites such as an N-terminal peptide extension with 11, 8, 6 or 4 glycosylation sites, to the CIFN variant CIFN-31-102- 138 increased the molecular weight of CIFN.
  • the proteins of CIFN-N11-31-102-138, CIFN-N8-31- 102-138 and CIFN-N6-31-102-138 were digested with (+) or without (-) glycosidase PNGase F.
  • the digested and undigested proteins were then examined by western blot analysis using the CIFN antibody.
  • Figure 12E when the carbohydrates were removed from the proteins by the treatment of glycosidase PNGase F, the molecular weight of all three hyperglycosylated variants of CIFN decreased to a level that is similar to the molecular weight of the parent CIFN.
  • the interferon specific activity of the parent CIFN and hyperglycosylated variants CIFN-N11-31-102-138, CIFN-N8-31-102- 138, CIFN-N6-31-102- 138, and CIFN-N4-31-102-138 were measured in an IFN gene reporter assay using iLite human interferon alpha kit (PBL).
  • PBL iLite human interferon alpha kit
  • all 4 hyperglycosylated variants of CIFN had the interferon specific activity comparable to that of the parent CIFN. This demonstrates that that biological potencies of these hyperglycosylated variants of CIFN are similar to that of the parent CIFN.
  • Table 3 IFN specific activities of CIFN and hyperglycosylated variants of CIFN
  • HCV replicon cells were plated in 96 well plates and incubated over night. The interferons were serially diluted and added to the replicon cells the next day. After 3day incubation, the cells were lysed and the luciferase activity was assayed with Bright-glo reagent (Promega). The dose response curves were ploted, and EC50 values were calculated. As shown in Figure 13A-C, the HCV replicon activity of all four hyperglycosylated variants were comparable to that of the parent CIFN. This demonstrates that these hyperglycosylated variants of CIFN are as biologically potent as the parent CIFN.
  • test interferons and standard were serially diluted in serum free DMEM media and then added to the plated cells.
  • the wells designated for positive control (cells without virus) and negative control (cells with virus but no drug protection) were not dosed.
  • encephalomyocarditis virus (EMCV) at the MOI of 0.001 was added to the cells, except for the positive controls.
  • the plate was then incubated at 37°C, 5% C0 2 and observed daily for the cytopathic effects (CPE) in the cells.
  • CPE cytopathic effects
  • the culture media was removed from the wells and Hucker' s Crystal Violet Solution was added to the cells and further incubated for 5 minutes at room temperature. The stained plate was them washed to remove the background stain, air dried and scanned. The blue dye were eluted with 33% acetic acid and quantified using ELISA reader. Interferon specific activities of the test interferons were calculated by comparing the 50% CPE of test interferons and the standard.
  • IFNB interferon beta
  • DNA encloding hyperglycosylated variants of the parent IFNB with an N-terminal peptide extension having 10 glycosylation sites and various signal peptides were constructed using the expression cassette systems disclosed herein and transfected into mammalian cell line CHO to express hyperglycosylated variants of IFNB protein by the techniques similar to what was described in Examples 4 and 5.
  • EPO-N10 had the signal peptide from EPO (SEQ ID NO: 165)
  • CD33-N10 had the signal peptide from CD33 (SEQ ID NO:164)
  • IFNB-N10 had the signal peptide from IFNB (SEQ ID NO: 168)
  • DS-N10 had an artificial signal peptide (SEQ ID NO: 163).
  • media containing the secreted hyperglycosylated variants of IFNB proteins were collected. The extent of changes in molecular weight of each protein was determined by the mobility shift of the protein in SDS gel electrophoresis using the polyclonal antibodies targeting the parent IFNB.
  • IFNB interferon beta
  • IFNB-N10 (SEQ ID NO:338) is a hyperglycosylated variant of IFNB with an N-terminal peptide extension (VNITG)io.
  • IFNB-N8 (SEQ ID NO:339) is a hyperglycosylated variant of IFNB with an N-terminal peptide extension (VNITG) 8 .
  • IFNB-N6 (SEQ ID NO: 340) is a hyperglycosylated variant of IFNB with an N-terminal peptide extension (VNITG)e.
  • IFNB-N4 (SEQ ID NO:341) is a hyperglycosylated variant of IFNB with an N- terminal peptide extension (VNITG) 4 .
  • Each VNITG motif contains an N-linked glycosylation site.
  • DNA sequences encoding IFNB-N10, IFNB-N8, IFNB-N6, and IFNB-N4 were generated by the PCR-based mutagenesis method as described in Example 4, and transfected into CHO cells for protein expression.
  • Western blot analysis of Figure 15 A showed that all four hyperglycosylated variants of IFNB with an N-terminal peptide extension had increased molecular weights compared to the parent IFNB.
  • the additions of N-terminal peptide extensions containing various numbers of VNITG motifs increased the molecular weight of IFNB compared to the parent IFNB.
  • Figure 15A shows that IFNB-N10 which had 10 glycosylation sites in the N-terminal peptide extension had the highest molecular weight among the four variants; IFNB-N8 which had 8 glycosylation sites in the N-terminal peptide extension had the second highest molecular weight among the four variants; IFNB-N6 which had 6 glycosylation sites in the N-terminal peptide extension had the third highest molecular weight among the four variants; and IFNB-N4 which had 4 glycosylation sites in the N-terminal peptide extension had the lowest molecular weight among the four variants. Therefore, the addition of an N- terminal peptide extension with various numbers of glycosylation sites to the parent IFNB increased the molecular weight of the parent IFNB.
  • Carbohydrates contribute to most of the molecular weight increase
  • IFNB-NIO, IFNB-N8 and IFNB-N6 were digested with (+) or without (-) glycosidase PNGase F. The digested and undigested proteins were then examined with western blot analysis using the IFNB antibody.
  • IFNal IFN alpha 1
  • N-terminal peptide extension with 10 glycosylation sites was introduced to human interferon alpha 1 (IFNal) using an expression vector with an N- terminal peptide extension with 10 glycosylation sites and methods similarly to what was described in Example 5 to generate a hyperglycosylated variant IFNal-N10 (SEQ ID NO: 342).
  • the extent of glycosylation of IFNal and IFNal-N10 was examined by the mobility shift of the proteins in SDS gel electrophoresis.
  • hyperglycosylated variant IFNal-N10 had an increased molecular weight compared to the parent IFNal.
  • the N-terminal peptide extension containing 10 glycosylation sites increased the molecular weight of the parent IFNal.
  • IFNG-N5 SEQ ID NO: 343
  • VNITG N-terminal peptide extension
  • IFNG-N10 SEQ ID NO: 3444
  • VNITG N-terminal peptide extension
  • Each VNITG motif contains an N- linked glycosylation site.
  • DNA sequences encoding IFNG-N5 and IFNG-N10 were generated by the PCR-based mutagenesis method as described in Example 1, and transfected into CHO cells for protein expression, respectively.
  • Western blot analysis of Figure 16B showed that both of the hyperglycosylated variants of IFNG with an N-terminal peptide extension had an increased molecular weight compared to the parent IFNG.
  • additions of N-terminal peptide extensions containing various numbers of VNITG motif increased the molecular weight of the starting IFNG.
  • Figure 16B also showed that IFNG-N10 which had 10 glycosylation sites in the N-terminal peptide extension had a higher molecular weight than IFNG-N5 which had 5 glycosylation sites in the N-terminal peptide extension. Therefore, the addition of a peptide extension with more glycosylation sites to the parent IFNG resulted in higher increase in the molecular weight of the parent IFNG. 3.
  • N-terminal peptide extension with 6 glycosylation sites was introduced to human interferon ⁇ , ⁇ 2 and ⁇ 3 using an expression vector containing an N-terminal peptide extension with 6 glycosylation sites and methods similarly to what was described in Example 5 to generate hyperglycosylated variants IFN ⁇ 1- ⁇ 6 (SEQ ID NO:347), IFN ⁇ 2- ⁇ 6 (SEQ ID NO:348) and IFN ⁇ 3- ⁇ 6 (SEQ ID NO:349).
  • the extent of glycosylation of each protein was examined by the mobility shift of the protein in SDS gel electrophoresis.
  • N-terminal peptide extensions with 6 glycosylation sites was introduced to human growth hormone 1 (hGHl) using a expression cassette with an N- terminal peptide with 6 glycosylation sites by methods similar to what was described in Example 5 to generate hyperglycosylated variants hGHl-N6 (SEQ ID NO:345).
  • the extent of glycosylation of hGHl, hGHl-N6 and Norditropin ® hGH was examined by the mobility shift of the proteins in SDS gel electrophoresis. As shown in Figure 17, hGHl-N6 had an increased molecular weight compared to the parent hGHl and Norditropin hGH. Therefore, the N-terminal peptide extension containing 6 glycosylation sites increased the molecular weight of the parent hGH.
  • mN14-CIFN-108 A mouse homolog of N14-CIFN-108 (hereafter "mN14-CIFN-108", SEQ ID NO:346) was generated by techniques similar to what was described in Example 5. When expressed, mN14-CIFN-108 was glycosylated well with a molecular weight close to lOOkD and was active in EMCV/L929 CPE assay.
  • Table 5 also supports the results shown in Figure 18A.
  • the serum half-life (Ti /2 ) of mN14-CIFN-108 is about 40 times higher than that of the unglycosylated mIFNal control. This increase in serum half-life indicates that mN14-CIFN-108 stayed in the subject for a markedly longer period of time compared to the mIFNal control.
  • mN14-CIFN-108 shows about 33 fold of improvement in the AUC t and AUQ nf value (AUC means "area under the curve") as compared to the unglycosylated mIFNal control.
  • the AUC value is a measure of drug exposure in a subject.
  • an interferon with a higher AUC value requires less frequent dosing to a subject to achieve approximately the same results as compared to an interferon with a lower AUC value.
  • the data provided in Table 5 indicate that a subject receiving mN14-CIFN-108 can be dosed less frequently compared to a subject receiving the same amount of the unglycosylated mIFNal control. For example, a subject could be dosed approximately twice a month with the mN14-CIFN-108 compared to the daily dosing needed with the unglycosylated mIFN al control to achieve similar clinical results. Table 5. Improved pharmacokinetics profiles of mN14-CIFN-108 compared to the unglycosylated mIFN al control
  • beta2-microglobulin protein level and induction of OASl mRNA in liver are two commonly used biomarkers for the efficacy of interferon treatment.
  • the beta2-microglobulin protein level in the subject administered with mN14- CIFN-108 were well above 1 fold increase over baseline level with the same period of time and were well over 0.5 fold increase until 100 hours after initial dosing.
  • the induction of OASl mRNA in liver dropped significantly to less than 0.1 fold increase over baseline in subjects administered with the unglycosylated mIFN al. In comparison, the induction stayed well above 0.5 fold increase for 100 hours after initial dosing in the subjects administered with mN14-CIFN-108.

Abstract

Disclosed herein are hyperglycosylated polypeptide variants, pharmaceutical compositions comprising the hyperglycosylated polypeptide variants described herein, and method of ameliorating and/or treating diseases and/or conditions with the hyperglycosylated polypeptide variants described herein.

Description

HYPERGLYCOSYLATED POLYPEPTIDE VARIANTS AND METHODS OF USE
RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S. Provisional Application Nos. 61/287,908, entitled "HYPERGLYCOSYLATED POLYPEPTIDE VARIANTS AND METHODS OF USE" filed December 18, 2009 and 61/355,478, entitled "HYPERGLYCOSYLATED POLYPEPTIDE VARIANTS AND METHODS OF USE" filed June 16, 2010, both of which are herein expressly incorporated by reference in their entireties, including any drawings.
REFERENCE TO SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SEQLISTING.TXT, created December 16, 2010, which is 692 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
BACKGROUND
Field
[0003] The present application relates to the fields of chemistry, biochemistry and medicine. More particularly, disclosed herein are hyperglycosylated polypeptide variants, pharmaceutical compositions that include one or more hyperglycosylated polypeptide variants, and methods of treating diseases and/or conditions with one or more hyperglycosylated polypeptide variants.
Description
[0004] Polypeptides are used for a wide range of applications, including industrial uses, and human and veterinary therapy. General recognized drawbacks to the use of many polypeptides include immunogenicity, susceptibility to proteolytic degradation by enzymes produced by the host, suboptimal pharmacokinetic properties such as stability and/or serum half-life, and the like. SUMMARY
[0005] Some embodiments disclosed herein relate to a hyperglycosylated polypeptide variant of a parent polypeptide, wherein the hyperglycosylated polypeptide variant can be the parent polypeptide that has been modified to include a peptide extension inserted at a terminal region, where the peptide extension can be a peptide of 1-200 consecutive amino acids and can include at least two glycosylation sites, where the terminal region can be selected from the group consisting of an amino-terminal region that consists of the first 15 amino acids at the amino-terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide and a carboxy-terminal region that consists of the last 15 amino acids at the carboxy-terminus of the parent polypeptide. In some embodiments, the peptide extension can include at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites. In some embodiments, the parent polypeptide has been further modified to include at least one or more additional glycosylation sites through at least one amino acid substitution or at least one combination of amino acid substitutions in the amino acid sequence of the parent polypeptide.
[0006] Some embodiments disclosed herein relate to a pharmaceutical composition that can include one or more hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein, and a pharmaceutically acceptable carrier, excipient, or combination thereof.
[0007] Some embodiments disclosed herein relate to a method of ameliorating and/or treating a fibrotic disorder. Other embodiments disclosed herein relate to a method of ameliorating and/or treating cancer. Still other embodiments disclosed herein relate to a method of inhibiting the growth of a tumor. Yet other embodiments disclosed herein relate to a method of ameliorating and/or treating a viral infection.
[0008] Some embodiments disclosed herein relate to an expression cassette for expression a hyperglycosylated polypeptide, comprising a promoter operably linked to a nucleotide sequence encoding a signal peptide, a first extension sequence encoding a first peptide extension, and a gene encoding a biologically-active polypeptide, where the first peptide extension can be a peptide of 1-200 consecutive amino acids including at least two glycosylation sites. In some embodiments, the expression cassette can further include a second extension sequence encoding a second peptide extension. It is also contemplated that the expression cassette can be provided without a gene encoding a biologically-active polypeptide, but instead having a restriction site allowing for insertion of any such desired gene. The expression cassette can be in the form of a linear or circular DNA, for example, and may advantageously be in the form of a plasmid.
[0009] Some embodiments disclosed herein relate to an expression vector comprising one or more expression cassettes disclosed herein. Some embodiments disclosed herein relate to a recombinant host cell comprising one or more expression vectors and/or cassette disclosed herein.
[0010] Some embodiments disclosed herein relate to a method for producing a hyperglycosylated polypeptide variant of a parent polypeptide discosed herein.
[0011] Some embodiments disclosed herein relate to a hyperglycosylated polypeptide, where the hyperglycosylated polypeptide comprises a biologically-active polypepide linked to a peptide extension, wherein the peptide extension is a peptide of 1 -200 consecutive amino acids and comprises at least two glycosylation sites, where the biologically-active polypeptide optionally comprises one or more glycosylation sites.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 shows the amino acid sequence of human growth hormone 1 (hGH-1, SEQ ID NO: 1), wherein the signal peptide is shown in bold letters.
[0013] Figure 2 shows the amino acid sequence of human insulin-like growth factor 1A (hIGF-ΙΑ, SEQ ID NO: 8), wherein the signal peptide is shown in bold letters.
[0014] Figure 3 shows the amino acid sequence of human granulocyte colony- stimulating factor (hG-CSF, SEQ ID NO: 15), wherein the signal peptide is shown in bold letters.
[0015] Figure 4 shows the amino acid sequence of human Erythropoietin (hEPO, SEQ ID NO: 22), wherein the signal peptide is shown in bold letters.
[0016] Figure 5 shows the amino acid sequence of human insulin (SEQ ID NO: 29), wherein the signal peptide is shown in bold letters. [0017] Figure 6 are schematic diagrams showing some embodiments of the expression cassette for expressing a hyperglycosylated polypeptide.
[0018] Figures 7A-I show the structure and sequence of the expression cassette in expression vectors pC6-l-hGHl, pN6-l-hGHl, pN6-2-hGHl, pNlO-l-hGHl, pN10-C4-l- hGHl, pN10-C6-l-hGHl, PN14-l-hGHl, PN14(2)-l-hGHl, and PN14(3)-l-hGHl, respectively. The main cassette cloning sites are the upstream Not I site and downstream Pme I or EcoR I site.
[0019] Figure 8 shows the structure and sequence of the expression cassette in expression vector pNlO-1-hIGFlA. The main cassette cloning sites are the upstream Not I site and downstream EcoR I site.
[0020] Figure 9 shows the structure and sequence of the expression cassette in expression vector pNlO-l-hG-CSF. The main cassette cloning sites are the upstream Not I site and downstream EcoR I site.
[0021] Figure 10 shows the structure and sequence of the expression cassette in expression vector pNlO-l-hEPO. The main cassette cloning sites are the upstream Not I site and downstream EcoR I site.
[0022] Figure 11 shows the structure and sequence of the expression cassette in expression vector pN 10-1 -Insulin. The main cassette cloning sites are the upstream Not I site and downstream EcoR I site.
[0023] Figure 12 A-E shows the western blot of various hyperlgyocylated variants of the parent interferon alfacon-1 (CIFN). The interferons shown in Figure 12A are CIFN-31-102-108, CIFN-Nl-31-102-108, CIFN-N2-31-102- 108, CIFN-N3-31-102-108, and CIFN-N4-31-102- 108. The interferons shown in Figure 12B are CIFN-102-138 and CIFN- 102-138-C2. The interferons shown in Figure 12C are CIFN-N 14(3)- 102, CIFN-N 14(3)- 102- 138, CIFN-N14(3)-108-138, and CIFN-N 14(3)- 108. The interferons shown in Figure 12D are CIFN-Nl l-31-102-138, CIFN-N8-31-102- 138, CIFN-N6-31-102- 138, and CIFN-N4-31- 102-138. In Figure 12E, CIFN-N11-31-102-138, CIFN-N11-8-102-138 and CIFN-N6-31- 102-138 were digested with (+) or without (-) glycosidase PNGase F.
[0024] Figure 13 A-D shows the HCV replicon activities and interferon specific activities of CIFN and variouis hyperglycosylated variants of the parent CIFN. Figure 13 A shows the HCV replicon activities of CIFN and CIFN-N14(3)-102. Figure 13B shows the HCV replicon activities of CIFN-N14(3)-108 and CIFN-N14(3)-102-138. Figure 13C shows the HCV replicon activity of CIFN-N14(3)-108-138. Figure 13D shows the interferon specific activities of CIFN, CIFN-N14(3)-102, CIFN-N14(3)-102-138, CIFN-N14(3)-108, and CIFN-N14(3)-108-138.
[0025] Figure 14 shows the western blot of various hyperglycosylated variants of the parent human interferon beta (IFNB) having an N-terminal peptide extension with 10 glycosylation sites. Variant EPO-N10 has the signal peptide from EPO (SEQ ID NO: 165), variant CD33-N10 has the signal peptide from CD33 (SEQ ID NO: 164), variant IFNB-N10 has the signal peptide from IFNB (SEQ ID NO: 168), and variant DS-N10 has an artificial signal peptide (SEQ ID NO: 163).
[0026] Figure 15 shows the western blot of IFNB and various hyperglycosylated variants of the parent IFNB. The interferons shown in Figure 15A are IFNB-N10, IFNB-N8, IFNB-N6, and IFNB-N4. In Figure 15B, IFNB-N10, IFNB-N8 and IFNB-N6 were digested with (+) or without (-) glycosidase PNGase F.
[0027] Figure 16 shows the western blot of hyperglycosylated variants of the parent human interferon alpha 1 (IFN al), the parent human interferon gamma (IFNG) and the parent human interferons lambdal (IFN λΐ), lambda2 (IFN λ2) and lambda3 (IFN λ3). The interferon shown in Figure 16A is IFN Ocl-N10. The interferons shown in Figure 16B are IFNG-N5 and IFNG-N10. The interferons shown in Figure 16C are IFN λ1-Ν16, IFN λ2- N16 and IFN λ3-Ν16.
[0028] Figure 17 shows the western blot of hGHl-N6, a hyperglycosylated variant of the parent human growth horman 1 (hGHl).
[0029] Figure 18A shows a graph illustrating the IFN activity of mN14-CIFN- 108 in mouse plasma over time after a single subcutaneous injection in mice. Figure 18B shows graphs illustrating the beta2-microglobulin protein level, and the induction of OAS1 mRNA in liver in mice over time after a single subcutaneous injection. DETAILED DESCRIPTION
Definitions
[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.
[0031] As used herein, the term "hyperglycosylated polypeptide variant of a parent polypeptide" refers to a polypeptide variant that includes one or more additional glycosylation sites that are not present in a parent polypeptide. In some embodiments, the parent polypeptide has been modified to include a peptide extension inserted at a terminal region, where the peptide extension includes one or more glycosylation sites. In some embodiments, the parent polypeptide has been modified to include (1) one or more additional glycosylation sites in the amino acid sequence of the parent polypeptide, wherein the additional glycosylation site(s) are introduced by an amino acid substitution and/or a combination of amino acid substitutions, and (2) a peptide extension inserted at a terminal region, where the peptide extension includes one or more glycosylation sites. In some embodiments, the parent polypeptide has been modified only by inclusion of the one or more additional glycosylation sites and the peptide extension on either the carboxy or amino terminus of the parent polypeptide. Each added glycosylation site may be an N-linked glycosylation site or an O-linked glycosylation site.
[0032] As used herein, the term "native glycosylation site" refers to a glycosylation site that exists in a first naturally-occurring polypeptide and is introduced into a second naturally-occurring or a non-naturally occurring polypeptide at a homologous amino acid position. A native glycosylation site can exist in one or more naturally-occurring polypeptides, and the native glycosylation site can be glycosylated or non-glycosylated in the first naturally-occurring polypeptide. For example, the glycosylation site N-L-S at the 25th, 26th, 27th amino acids of the sequence of interferon ocl4 can be introduced at amino acid positions 25, 26 and 27 of the sequence of interferon alfacon-1 as a native glycosylation site. [0033] As used herein, the term "non-native glycosylation site" refers to a glycosylation site that does not exist in any naturally-occurring polypeptide, as well as a glycosylation site that exists in a first naturally-occurring polypeptide and is introduced into a second naturally-occurring or a non-naturally occurring polypeptide at a non-homologous amino acid position. For example, the glycosylation site (N-L-S) at amino acid positions 25, 26 and 27 of the sequence of interferon ocl4 can be introduced at amino acid positions 101, 102 and 103 of the sequence of interferon alfacon-1 (D-E-S) as a non-native glycosylation site. As another example, the glycosylation site (N-S-S) at amino acid positions 95, 96 and 97 of the sequence of interferon ocl4 can be introduced to a peptide extension that is inserted at the N-terminal region of human insulin as a non-native glycosylation site.
[0034] As used herein, non-native and native glycosylation sites include N-linked glycosylation sites and O-linked glycosylation sites. N-linked glycosylation sites include, for example, Asn-X-Ser/Thr (N-X-S/T), where the Asparagine (Asn) residue provides a site for N-linked glycosylation and X is any amino acid residue. O-linked glycosylation sites include at least one serine or threonine residue. A number of O-linked glycosylation sites are known in the art and have been described in, for example, Ten Hagen et al., J. Biol. Chem., 1999, 274(39):27867-27874; Hanisch et al., Glycobiology, 2001, 11:731-740; and Ten Hagen et al., Glycobiology, 2003, 13:1R-16R.
[0035] Standard techniques can be used to determine whether a polypeptide comprises N-linked and/or O-linked glycosylation. See, for example, R. Townsend and A. Hotchkiss, eds., Techniques in Glycobiology, Marcel Dekker Inc., 1997; and E. Hounsell, ed. Gly co analy is Protocols (Methods in Molecular Biology, Vol. 76), Humana Press, 1998. For example, the change in electrophoretic mobility of a protein before and after treatment with chemical or enzymatic deglycosylation can be used to determine the glycosylation status of a protein. Enzymatic deglycosylation can be carried out using any of a variety of enzymes, including, but not limited to, peptide-N4-(N-acetyl- -D-glucosaminyl) asparagine amidase (PNGase F), endoglycosidase Fl, endoglycosidase F2, endoglycosidase F3, a(2→3,6,8,9) neuraminidase, and the like. For example, sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the protein, either pre-treated with PNGase F or untreated with PNGaseF, can be conducted. A marked decrease in band width and change in migration position after treatment with PNGaseF is considered diagnostic of N-linked glycosylation. The carbohydrate content of a glycosylated protein can also be detected using lectin analysis of protein blots (for example, proteins separated by SDS-PAGE and transferred to a support, such as a nylon membrane). Lectins, carbohydrate-binding proteins from various plant tissues, have both high affinity and narrow specificity for a wide range of defined sugar epitopes found on glycoprotein glycans. See Cummings, Methods in Enzymol, 1994, 230:66-86. Lectins can be detectably labeled (either directly or indirectly), allowing detection of binding of lectins to carbohydrates on glycosylated proteins. For example, when conjugated with biotin or digoxigenin, a lectin bound to a glycosylated protein can be easily identified on membrane blots through a reaction utilizing avidin or anti-digoxigenin antibodies conjugated with an enzyme such as alkaline phosphatase, β-galactosidase, luciferase, or horse radish peroxidase, to yield a detectable product. Screening with a panel of lectins with well-defined specificity can provide considerable information about a glycoprotein's carbohydrate complement.
[0036] The phrase "increased glycosylation" is used herein to indicate increased levels of attached carbohydrate molecules, normally obtained as a result of increased number or better utilization of glycosylation site(s). The increased glycosylation may be determined by any suitable method known in the art for analyzing attached carbohydrate structures. For example, Western blot can be used to determine the amount of attached carbohydrates in a glycosylated polypeptide.
[0037] An amino acid residue "located close to" a glycosylation site is usually located in position -4, -3, -2, -1, +1, +2, +3, or +4 relative to the amino acid residue of the glycosylation site to which the carbohydrate moiety is attached, in particular in position -2, - 1, +1, +2, such as position -1 or +1, in particular position -1. These positions may be modified to increase glycosylation at the glycosylation site. For example, the modification can be a substitution.
[0038] As used herein, the term "polypeptide" refers to a polymer of amino acids. A polypeptide can be of various lengths. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. A polypeptide can be with or without N- terminal methionine residues. A polypeptide may include post-translational modifications, for example, glycosylations, acetylations, phosphorylations and the like. Examples of "polypeptide" include, but are not limited to, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, non-coded amino acids, etc.), polypeptides with substituted linkages, fusion proteins, as well as polypeptides with other modifications known in the art, both naturally occurring and non-naturally occurring.
[0039] As used herein, the terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to refer to polymeric forms of nucleotides of any length. Thus, oligonucleotides are included within the definition of polynucleotide. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and their analogs. Nucleotides may have any three-dimensional structure, and may perform various functions. The term "polynucleotide" includes single-, double-stranded and triple helical molecules. For example, a polynucleotide can be a double-stranded DNA found, inter alia, in linear DNA molecules (for example, restriction fragments), viruses, plasmids, and chromosomes. "Oligonucleotide" generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as "oligomers" or "oligos," and can be isolated from genes, or chemically synthesized by methods known in the art.
[0040] Non-limiting embodiments of polynucleotides include: genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can also include modified nucleic acid molecules, such as methylated nucleic acid molecules, nucleic acid molecules with modified purine or pyrimidine bases and nucleic acid molecule analogs. Nucleic acids may be naturally occurring DNA or RNA, or may be synthetic analogs of DNA or RNA, such as those known in the art. Synthetic analogs include native structures that have been modified to include alterations in the backbone, sugars and/or heterocyclic bases. Examples of modified backbone include phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters; boranophosphates; achiral phosphate derivatives, such as 3'-0'-5'-S- phosphorothioate, 3'-S-5'-0- phosphorothioate, 3 '-CH2-5' -O-phosphonate and 3'-NH-5'-0 phosphoroamidate; and peptide nucleic acids in which the entire ribose phosphodiester backbone is replaced with a peptide linkage. In some embodiments, modifications of the backbone, sugars and/or heterocyclic bases may increase stability and/or binding affinity.
[0041] "Percent (%) sequence identity" with respect to polynucleotide or polypeptide sequences is defined as the percentage of bases or amino acid residues in a candidate sequence that are identical with the bases or amino acid residues in another sequence, after aligning the two sequences. Gaps can be introduced into the sequence alignment, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not considered as part of the sequence identity. Alignment for purposes of determining percent (%) sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer methods and programs such as BLAST, BLAST-2, ALIGN, FASTA (available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA), or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
[0042] For instance, percent (%) amino acid sequence identity values may be obtained by using the WU-BLAST-2 computer program described in Altschul et al., Methods in Enzymology, 1996, 266:460-480. Many search parameters in the WU-BLAST-2 computer program can be adjusted by those skilled in the art. For example, some of the adjustable parameters can be set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is used, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the amino acid sequence of a first protein of interest and the amino acid sequence of a second protein of interest as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the first protein of interest.
[0043] Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 described in Altschul et al., Nucleic Acids Res., 1997, 25:3389-3402. The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, MD. NCBI-BLAST2 uses several adjustable search parameters. The default values for some of those adjustable search parameters are, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
[0044] In situations where NCBI-BLAST2 is used for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
[0045] 100 times the fraction X/Y
[0046] where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
Hyperglycosylated polypeptide variants of a parent polypeptide
[0047] Some embodiments disclosed herein relate to a hyperglycosylated polypeptide variant of a parent polypeptide that is the parent polypeptide that has been modified to include a peptide extension inserted at a terminal region, where the peptide extension includes one or more glycosylation sites.
Peptide Extension
[0048] As used herein, the term "peptide extension" refers to any polymer of consecutive amino acid residues. A peptide extension can be of various lengths, for example, ranging from a single amino acid residue to a peptide of 500 amino acid residues. In some embodiments, the peptide extension can be at least about 4 amino acids in length, alternatively at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 145 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 155 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 165 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 180 amino acids in length, alternatively at least about 190 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 210 amino acids in length, alternatively at least about 220 amino acids in length, alternatively at least about 230 amino acids in length, alternatively at least about 240 amino acids in length, alternatively at least about 250 amino acids in length, alternatively at least about 260 amino acids in length, alternatively at least about 270 amino acids in length, alternatively at least about 280 amino acids in length, alternatively at least about 290 amino acids in length, alternatively at least about 300 amino acids in length, alternatively at least about 310 amino acids in length, alternatively at least about 320 amino acids in length, alternatively at least about 330 amino acids in length, alternatively at least about 340 amino acids in length, alternatively at least about 350 amino acids in length, alternatively at least about 360 amino acids in length, alternatively at least about 370 amino acids in length, alternatively at least about 380 amino acids in length, alternatively at least about 390 amino acids in length, alternatively at least about 400 amino acids in length, alternatively at least about 450 amino acids in length, alternatively at least about 500 amino acids in length, or more.
[0049] The peptide extension can include any number of glycosylation sites. In some embodiments, the peptide extension can include one glycosylation site. In other embodiments, the peptide extension can include two, three or four glycosylation sites. In still other embodiments, the peptide extension can include five, six, seven, eight, nine, or ten glycosylation sites. In yet other embodiments, the peptide extension can include eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty glycosylation sites. In yet still other embodiments, the peptide extension can include twenty- one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty- eight, twenty-nine, thirty glycosylation sites. In yet still other embodiments, the peptide extension can include thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, or more glycosylation sites. Each glycosylation site that is included in the peptide extension may be an N-linked glycosylation site or an O- linked glycosylation site.
[0050] In some embodiments, the peptide extension can include at least one glycosylation site. In other embodiments, the peptide extension can include at least two, at least three or at least four glycosylation sites. In still other embodiments, the peptide extension can include at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites. In yet other embodiments, the peptide extension can include at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty glycosylation sites. In yet still other embodiments, the peptide extension can include at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, at least twenty-six, at least twenty-seven, at least twenty-eight, at least twenty-nine, at least thirty glycosylation sites. In yet still other embodiments, the peptide extension can include at least thirty-one, at least thirty-two, at least thirty-three, at least thirty-four, at least thirty-five, at least thirty-six, at least thirty-seven, at least thirty-eight, at least thirty-nine, at least forty, or more glycosylation sites.
[0051] A glycosylation site that is included in the peptide extension may be a non- native glycosylation site that is present in the parent polypeptide or a non-native glycosylation site that is not present in the parent polypeptide. For example, when the parent polypeptide is interferon ocl4, the glycosylation site N-L-S at amino acid positions 25, 26 and 27 of the sequence of interferon ocl4 can be introduced to the peptide extension inserted in the parent interferon ocl4 as a non-native glycosylation site. As another example, when the parent polypeptide is human growth hormone I (hGH-1), the glycosylation site N-L-S at amino acid positions 25, 26 and 27 of the sequence of interferon ocl4 can be introduced to the peptide extension inserted in the parent hGH-1 as a non-native glycosylation site.
[0052] The peptide extension can be inserted at an amino-terminal and/or a carboxy-terminal region of the parent polypeptide. In some embodiments, the peptide can be inserted at an amino-terminal region of the parent polypeptide. In other embodiments, the peptide extension can be inserted at a carboxy-terminal region of the parent polypeptide. In still other embodiments, the peptide extension can be inserted at an amino-terminal region and a carboxy-terminal region of the parent polypeptide.
[0053] The terminal region of the parent polypeptide at which the peptide extension is inserted can be of various lengths. In some embodiments, the terminal region where the peptide extension is inserted at can be an amino-terminal region that consists of the first 15 amino acids at the amino terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide. In other embodiments, the terminal region where the peptide extension is inserted at can be an amino terminal region that consists of the first 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) at the amino terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide. In still other embodiments, the peptide extension can be inserted before the first amino acid at the amino terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide. In still other embodiments, the peptide extension is inserted after the fifteenth amino acid at the amino terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide. In yet other embodiments, the peptide extension is not inserted before the first amino acid at the amino-terminus of the parent polypeptide. In yet still other embodiments, the peptide extension includes at least two glycosylation sites and is not inserted before the first amino acid at the amino-terminus of the parent polypeptide. In some embodiments, the peptide extension includes at least four glycosylation sites and is not inserted before the first amino acid at the amino-terminus of the parent polypeptide.
[0054] In some embodiments, when the parent polypeptide is human growth hormone 1 (hGH-1), the peptide extension can be inserted between the 26th and the 27th amino acids of the parent hGH-1. In other embodiments, the peptide extension can be inserted in a position selected from between the 27th and the 28th amino acids, between the 28th and the 29th amino acids, between the 29th and the 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, between the 39th and the 40th amino acids, between the 40th and the 41st amino acids, between the 41st and the 42nd amino acids, between the 42nd and the 43rd amino acids, and between the 43rd and the 44th amino acids of the parent hGH-1.
[0055] In some embodiments, when the parent polypeptide is human insulin-like growth factor 1A (hIGF-ΙΑ), the peptide extension can be located between the 21st and the 22nd amino acids of the parent hIGF-ΙΑ. In other embodiments, the peptide extension can be inserted in a position selected from between the 22nd and the 23rd amino acids, between the 23rd and the 24th amino acids, the 24th and the 25th amino acids, between the 25th and the 26th amino acids, between the 26th and the 27th amino acids, between the 27th and the 28th amino acids, between the 28th and 29th amino acids, between the 29th and the 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, and between the 37th and the 38th amino acids of the parent hIGF-lA.
[0056] In some embodimetns, when the parent polypeptide is human G-CSF (hG- CSF), the peptide extension can be located between the 29th and the 30th amino acids of the parent hG-CSF. In other embodiments, the peptide extension can be inserted in a position selected from between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, between the 39th and the 40th amino acids, between the 40th and the 41st amino acids, between the 41st and the 42nd amino acids, between the 42nd and the 43rd amino acids, between the 43rd and the 44th amino acids, between the 44th and the 45th amino acids, and between the 45th and the 46th amino acids of the parent hG-CSF.
[0057] In some embodiments, when the parent polypeptide is human erythropoietin (hEPO), the peptide extension can be located between the 27th and the 28th amino acids of the parent hEPO. In other embodimetns, the peptide extension can be inserted in a position selected from between the 28th and the 29th amino acids, between the 29th and the 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, between the 39th and the 40th amino acids, between the 40th and the 41st amino acids, between the 41st and the 42nd amino acids, between the 42nd and the 43rd amino acids, between the 43rd and the 44th amino acids, between the 44th and the 45th amino acids, and between the 45th and the 46th amino acids of the parent hEPO.
[0058] In some embodiments, when the parent polypeptide is human insulin, the peptide extension can be located between the 24th and 25th amino acids of the parent human insulin. In other embodiments, the peptide extension can be inserted in a position selected from between the 25th and the 26th amino acids, between the 26th and the 27th amino acids, between the 27th and the 28th amino acids, between the 28th and 29th amino acids, between the 29th and 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, and betweent he 39th and the 40th amino acids of the parent human insulin.
[0059] In some embodiments, the terminal region where the peptide extension is inserted at can be a carboxy-terminal region that consists of the last 15 amino acids at the carboxy- terminus of the parent polypeptide. In other embodiments, the terminal region where the peptide extension is inserted at can be a carboxy-terminal region that consists of the last 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) at the carboxy-terminus of the parent polypeptide. In some embodiments, the peptide extension can be inserted after the last amino acid at the carboxy-terminus of the parent polypeptide. In other embodiments, the peptide extension is not inserted after the last amino acid at the carboxy-terminus of the parent polypeptide. In still other embodiments, the peptide extension includes at least two glycosylation sites and is not inserted after the last amino acid at the carboxy-terminus of the parent polypeptide. In still other embodiments, the peptide extension includes at least four glycosylation sites and is not inserted after the last amino acid at the carboxy-terminus of the parent polypeptide.
Parent polypeptide
[0060] As used herein, a "parent polypeptide" can be any polypeptide, including naturally-occurring and non-naturally occurring polypeptides.
[0061] As used herein, a "naturally occurring polypeptide" refers to a polypeptide that can be found in nature as distinct from being artificially produced by man. For example, a polypeptide that is produced by an organism in nature, where the organism has not been intentionally modified by man is naturally occurring. Examples of organisms that can produce naturally-occurring polypeptides include, but are not limited to, bacteria, such as gram-positive bacteria and gram-negative bacteria; archaea, such as euryachaeota and cre- archaeota; fungus, such as yeasts, molds, mushrooms; plants, such as seed plants, bryophytes, ferns, fern allies, and algae; and animals, including non-mammalian vertebrates such as birds (for example, turkey, chicken, and duck) and fish (for example, zebrafish), and mammalian vertebrates such as human, pig, mouse, dog, cat, horse, rat, cattle, and sheep.
[0062] A naturally-occurring polypeptide can be at least about 50 amino acids in length, at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 180 amino acids in length, alternatively at least about 190 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 210 amino acids in length, alternatively at least about 220 amino acids in length, alternatively at least about 230 amino acids in length, alternatively at least about 240 amino acids in length, alternatively at least about 250 amino acids in length, alternatively at least about 260 amino acids in length, alternatively at least about 270 amino acids in length, alternatively at least about 280 amino acids in length, alternatively at least about 290 amino acids in length, alternatively at least about 300 amino acids in length, alternatively at least about 310 amino acids in length, alternatively at least about 320 amino acids in length, alternatively at least about 330 amino acids in length, alternatively at least about 340 amino acids in length, alternatively at least about 350 amino acids in length, alternatively at least about 360 amino acids in length, alternatively at least about 370 amino acids in length, alternatively at least about 380 amino acids in length, alternatively at least about 390 amino acids in length, alternatively at least about 400 amino acids in length, alternatively at least about 410 amino acids in length, alternatively at least about 420 amino acids in length, alternatively at least about 430 amino acids in length, alternatively at least about 440 amino acids in length, alternatively at least about 450 amino acids in length, alternatively at least about 460 amino acids in length, alternatively at least about 470 amino acids in length, alternatively at least about 480 amino acids in length, alternatively at least about 490 amino acids in length, alternatively at least about 500 amino acids in length, alternatively at least about 500 amino acids in length, alternatively at least about 600 amino acids in length, alternatively at least about 600 amino acids in length, alternatively at least about 700 amino acids in length, alternatively at least about 700 amino acids in length, alternatively at least about 800 amino acids in length, alternatively at least about 800 amino acids in length, alternatively at least about 900 amino acids in length, alternatively at least about 900 amino acids in length, alternatively at least about 1000 amino acids in length, alternatively at least about 1000 amino acids in length, alternatively at least about 1100 amino acids in length or more. In some embodiments, the parent polypeptide can be a naturally-occurring polypeptide.
[0063] As used herein, the term "non-naturally occurring polypeptide" is used interchangeably with the term "synthetic polypeptide," and refers to a polypeptide that cannot be found in nature and is artificially produced by man. Non-naturally occurring polypeptides include, but are not limited to, hybrid polypeptides, consensus polypeptides, fusion polypeptides, recombinant polypeptides, and other non-naturally occurring variants of a naturally-occurring or a non-naturally occurring polypeptide.
[0064] In some embodiments, the parent polypeptide can be a non-naturally occurring polypeptide. In an embodiment, the parent polypeptide can be a hybrid polypeptide. In another embodiment, the parent polypeptide can be a consensus polypeptide. In still another embodiment, the parent polypeptide can be a fusion polypeptide. In yet another embodiment, the parent polypeptide can be a recombinant polypeptide.
[0065] A non-naturally occurring polypeptide can be of various lengths. In some embodiments, a non-naturally occurring polypeptide is at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 145 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 155 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 165 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 180 amino acids in length, alternatively at least about 190 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 210 amino acids in length, alternatively at least about 220 amino acids in length, alternatively at least about 230 amino acids in length, alternatively at least about 240 amino acids in length, alternatively at least about 250 amino acids in length, alternatively at least about 260 amino acids in length, alternatively at least about 270 amino acids in length, alternatively at least about 280 amino acids in length, alternatively at least about 290 amino acids in length, alternatively at least about 300 amino acids in length, alternatively at least about 310 amino acids in length, alternatively at least about 320 amino acids in length, alternatively at least about 330 amino acids in length, alternatively at least about 340 amino acids in length, alternatively at least about 350 amino acids in length, alternatively at least about 360 amino acids in length, alternatively at least about 370 amino acids in length, alternatively at least about 380 amino acids in length, alternatively at least about 390 amino acids in length, alternatively at least about 400 amino acids in length, alternatively at least about 410 amino acids in length, alternatively at least about 420 amino acids in length, alternatively at least about 430 amino acids in length, alternatively at least about 440 amino acids in length, alternatively at least about 450 amino acids in length, alternatively at least about 460 amino acids in length, alternatively at least about 470 amino acids in length, alternatively at least about 480 amino acids in length, alternatively at least about 490 amino acids in length, alternatively at least about 500 amino acids in length, alternatively at least about 550 amino acids in length, alternatively at least about 600 amino acids in length, alternatively at least about 650 amino acids in length, alternatively at least about 700 amino acids in length, alternatively at least about 750 amino acids in length, alternatively at least about 800 amino acids in length, alternatively at least about 850 amino acids in length, alternatively at least about 900 amino acids in length, alternatively at least about 950 amino acids in length, alternatively at least about 1000 amino acids in length, or more.
Hybrid polypeptide
[0066] As used herein, a "hybrid polypeptide" is a hybrid polypeptide having an amino acid sequence comprising discrete sub-sequences corresponding in amino acid identity and number to sub-sequences of more than one naturally-occurring and/or non-naturally occurring polypeptides. Each of the naturally-occurring and/or non-naturally occurring polypeptides from which the discrete sub-sequences are derived from may be the same or different from each other. For example, a hybrid polypeptide can include discrete subsequences from two naturally-occurring and/or non-naturally polypeptides, where the two naturally-occurring and/or non-naturally polypeptides are different from each other. As another example, a hybrid polypeptide can include discrete sub-sequences from three, four, five, six, seven, eight, nine, or ten naturally-occurring and/or non-naturally occurring polypeptides.
[0067] For example, the discrete sub-sequences can be selected from naturally- occurring human growth hormone 1 (hGH-1, SEQ ID NO: 1), naturally-occurring human insulin-like growth factor 1A (hIGF-ΙΑ, SEQ ID NO: 8), naturally-occurring human granulocyte colony-stimulating factor (hG-CSF, SEQ ID NO: 15), naturally-occurring human Erythropoietin (hEPO, SEQ ID NO:22), naturally-occurring human insulin (SEQ ID NO: 29), and the amino acid sequence of the hybrid polypeptide differs from each of the amino acid sequences of naturally-occurring hGH-1, naturally-occurring hIGF-ΙΑ, naturally-occurring hG-CSF, naturally-occurring hEPO, and naturally-occurring human insulin, respectively. As another example, the discrete sub-sequences can be selected from naturally-occurring IFN- a2b, naturally-occurring IFN-al4, naturally-occurring IFN-βΙ, and naturally-occurring IFN- co, and the amino acid sequence of the hybrid interferon differs from each of the amino acid sequences of naturally occurring IFN-a2b, naturally-occurring IFN-al4, naturally-occurring IFN-βΙ, and naturally-occurring IFN-co, respectively. As still another example, the discrete sub-sequences can be selected from naturally-occurring IFN-a2b, naturally-occurring IFN- al4, naturally-occurring IFN-βΙ, Infergen® consensus IFN-a, and naturally-occurring IFN-co, and the amino acid sequence of the hybrid interferon differs from each of the amino acid sequences of naturally-occurring IFN-a2b, naturally-occurring IFN-a 14, naturally-occurring IFN-βΙ, Infergen® consensus IFN-a, and naturally-occurring IFN-co, respectively.
[0068] In some embodiments, a hybrid polypeptide can include, in the order from N-terminus to C-terminus, from about 2 to about 90, for example, from about 2 to about 5, from about 5 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from about 85 to about 90 contiguous amino acids of two, three, four, or five polypeptides selected from hGH-1 (SEQ ID NO: 1), hIGF-ΙΑ (SEQ ID NO: 8), hG-CSF (SEQ ID NO: 15), hEPO (SEQ ID NO: 22), and human insulin (SEQ ID NO: 29).
Consensus polypeptide
[0069] As used herein, a "consensus polypeptide" is a polypeptide having a consensus amino acid sequence that is derived by aligning the amino acid sequences of three or more naturally occurring and/or non-naturally occurring polypeptides, and identifying the amino acids that are shared by at least two of the naturally occurring and/or non-naturally occurring polypeptide sequences. For example, a consensus polypeptide can include a consensus amino acid sequence that is derived by aligning the amino acid sequences of three, four, five, six, seven, eight, nine, or ten naturally occurring and/or non-naturally occurring polypeptides, and identifying amino acids that are shared by at least two, three, five, six, seven, eight, nine, or ten of the naturally occurring and/or non-naturally occurring polypeptide sequences.
[0070] As an example, a consensus polypeptide can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-al4, and naturally-occurring human IFN-βΙ. As another example, a consensus polypeptide can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-al4, and naturally-occurring human IFN- col. As still another example, a consensus polypeptide can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-βΙ, and naturally-occurring human IFN-col. As yet another example, a consensus polypeptide can be a sequence derived from aligning the sequences of naturally occurring human IFN-al4, naturally-occurring human IFN-βΙ, and naturally-occurring human IFN-col. As still yet another example, a consensus polypeptide can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-al4, naturally-occurring human IFN-βΙ, and naturally-occurring human IFN-col. As still yet another example, a consensus polypeptide can be a sequence derived from aligning the sequences of naturally-occurring human IFN-λΙ, naturally-occurring human ΙΡΝ-λ2, and naturally-occurring human ΙΡΝ-λ3.
[0071] An example of consensus polypeptide is Infergen® consensus IFN-a (Three Rivers Pharmaceuticals, Warrendale, PA). In some embodiments, a consensus sequence can be derived by including one or more consensus polypeptides, such as Infergen® consensus IFN-a, in the amino acid sequence alignment.
Fusion polypeptide
[0072] As used herein, a "fusion polypeptide" is a polypeptide comprising a fusion partner, such as one or more heterologous peptides or polypeptides. Suitable fusion partners include, but are not limited to, peptides and polypeptides that confer enhanced stability in vivo (for example, enhanced serum half-life); peptides and polypeptides that can provide ease of purification (for example, (His)n) and the like; peptides and polypeptides that can provide for secretion of the fusion protein from a cell, and the like; peptides and polypeptides that can provide an epitope tag, such as GST, hemagglutinin (for example, CYPYDVPDYA), FLAG (for example, DYKDDDDK), c-myc (for example, CEQKLISEEDL), and the like; peptides and polypeptides that can provide a detectable signal, such as an enzyme that generates a detectable product (for example, β-galactosidase and luciferase), and the like; a peptide or polypeptide that is itself detectable (for example, a green fluorescent protein) and the like; and peptides and polypeptides that can provide for multimerization, such as a multimerization domain (for example, an Fc portion of an immunoglobulin) and the like.
[0073] Various signal peptide sequences can be incorporated into a polypeptide as a fusion partner to provide for secretion of the fusion protein from a cell. In some embodiments, the fusion partner can be a signal peptide that provides for secretion from a mammalian cell. Such signal peptides include, but are not limited to, a signal peptide from human insulin-like growth factor II (hIGF-II). Methods of producing a fusion polypeptide comprising a hIGF-II signal peptide are described in U.S. Patent No. 7,396,811, the contents of which are hereby incorporated by reference in its entirety. In other embodiments, the fusion partner can be a bacterial secretion signal peptide. Such signal peptides include, but are not limited to, the secretion signal of Braun's lipoprotein of E. coli, S. marcescens, E. amylosora, M. morganii, and P. mirabilis; the TraT protein of E. coli and Salmonella; the penicillinase (PenP) protein of B. licheniformis, B. cereus and S. aureus; pullulanase proteins of Klebsiella pneumoniae and Klebsiella aerogenese; E. coli lipoproteins lpp-28, Pal, RplA, RplB, OsmB, NIpB, and Orll7; chitobiase protein of V. harseyi; the -l,4-endoglucanase protein of Pseudomonas solanacearum; the Pal and Pep proteins of H. influenzae; the Oprl protein of P. aeruginosa; the MalX and AmiA proteins of S. pneumoniae; the 34 kda antigen and TpmA protein of Treponema pallidum; the P37 protein of Mycoplasma hyorhinis; the neutral protease of Bacillus amyloliquefaciens; and the 17 kda antigen of Rickettsia rickettsii. In still other embodiments, the fusion partner can be a yeast secretion signal peptide. Secretion signal peptides that can be used in yeast are known in the art. See, for example, U.S. Patent No. 5,712,113, the contents of which are hereby incorporated by reference in its entirety.
[0074] In some embodiments, a fusion polypeptide can further include a protease cleavage site that is positioned between the fusion partner and the remainder of the fusion polypeptide. Examples of proteolytic cleavage sites that can be included in a fusion polypeptide disclosed herein include, but are no limited to, an enterokinase cleavage site: (Asp)4Lys; a factor Xa cleavage site: Ile-Glu-Gly-Arg; a thrombin cleavage site, such as Leu- Val-Pro-Arg-Gly-Ser; a renin cleavage site, such as His-Pro-Phe-His-Leu-Val-Ile-His; a collagenase cleavage site, such as X-Gly-Pro (where X is any amino acid); a trypsin cleavage site, such as Arg-Lys; a viral protease cleavage site, such as a viral 2 A or 3C protease cleavage site, including, but not limited to, a protease 2A cleavage site from a picornavirus described in Sommergruber et al., Virol., 1994, 198:741-745, a Hepatitis A virus 3C cleavage site described in Schultheiss et al., J. Virol., 1995, 69: 1727-1733, human rhinovirus 2A protease cleavage site described in Wang et al., Biochem. Biophys. Res. Comm., 1997, 235:562-566, and a picornavirus 3 protease cleavage site described in Walker et al., Biotechnol., 1994, 12:601-605.
Recombinant polypeptide
[0075] As used herein, a "recombinant polypeptide" is a non-naturally occurring polypeptide that is produced utilizing recombinant techniques. For example, a recombinant polypeptide can be produced by genetic engineering techniques known in the art, such as recursive sequence recombination of nucleic acid segments, diversity generation methods (such as shuffling) of nucleotides, or manipulation of isolated segments of polynucleotides or polypeptides. Various recombinant polypeptides are known in the art, including, but not limited to, recombinant human growth hormone (hGH) such as Genotropin available from Pflizer, New York, NY; recombinant human insulin-like growth factor 1 such as Increlex available from Tercica, Brisbane, CA; recombinant human G-CSF such as Neupogen® available from Amgen Inc., Thousand Oaks, CA; recombinant erythropoietin (EPO) such as Epogen® available from Amgen Inc., Thousand Oaks, CA; and recombinant insulin such as NovoLog® available from Novonordisk Inc., Princeton NJ. [0076] In some embodiments, the parent polypeptide can include one or more modified amino acids. Examples of modified amino acid include, but are not limited to, a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, a carboxylated amino acid, a phosphorylated amino acid (for example, phosphotyrosine, phosphoserine, and phosphothreonine), an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, an amino acid conjugated to an organic derivatizing agent, and the like.
[0077] In some embodiments, the parent polypeptide can be a naturally occurring or non-naturally occurring polypeptide that includes one or more polyethylene glycol (PEG) moieties. The PEG molecule(s) can be conjugated to one or more amino acid side chains of the PEGlyated polypeptide. The PEG can be coupled directly to a polypeptide without a linking group, or through an amino group, a sulfhydryl group, a hydroxyl group, or a carboxyl group. Methods for attaching a PEG to a polypeptide are known in the art, and any known method can be used. See, for example, Park et al, Anticancer Res., 1981, 1:373-376; and U.S. Patent No. 5,985,265. In some embodiments, the PEGylated polypeptide can contain a PEG moiety on only one amino acid. In other embodiments, a PEGylated polypeptide contains two, three, four, five, six, seven, eight, nine or ten PEG moieties on two, three, four, five, six, seven, eight, nine or ten different amino acid residues. In some embodiments, the parent polypeptide can be a PEGylated polypeptide that is PEGylated at or near the amino- terminus, where a peptide extension can be inserted at the carboxy-terminal region of the parent polypeptide. For example, the PEG moiety can be conjugated to the polypeptide at one or more amino acid residues from amino acid 1 through amino acid 4, or from amino acid 5 through amino acid 10. In other embodiments, the parent polypeptide can be a PEGylated polypeptide that is PEGylated at or near the carboxy-terminus, where a peptide extension can be inserted at the amino-terminal region of the parent polypeptide. In an embodiment, the PEGylated polypeptide is PEGylated at one or more amino acid residues at one or more residues from amino acid 100 through amino acid 114. In some embodiments, the addition of one or more pegylation sites can provide PEG-derivatized polypeptide with reduced serum clearance. [0078] In some embodiments, the PEG can be a monomethoxyPEG molecule (mPEG) that reacts with a primary amine group on the subject polypeptide. Methods of modifying polypeptides with monomethoxy PEG via reductive alkylation are known in the art. See, for example, Chamow et al., Bioconj. Chem., 1994, 5: 133-140.
[0079] As discussed herein, various polypeptides, including therapeutic proteins, can be a parent polypeptide. These include a large number of biologically-active polypeptides, and preferably include those that are biologically active in humans and those that are biologically active in veterinary applications, including in mammals, birds, reptiles, amphibians, and fish, and those active in vertebrates in general. A non-limiting list of examples of biologically-active polypeptides that are active in humans and other mammals includes, but is not limited to, naturally-occurring and non-naturally occurring growth hormones (GHs); naturally-occurring and non-naturally occurring insulin-like growth factors (IGFs); naturally-occurring and non-naturally occurring granulocyte colony-stimulating factors (G-CSFs); naturally-occurring and non-naturally occurring erythropoietin (EPO); naturally-occurring and non-naturally occurring insulin; naturally-occuring and non-naturally ouccuring antibodies, such as hybrid antibodies, chimeric antibodies, humanized antibodies, monoclonal antibodies; naturally-occurring and non-naturally occurring antigen binding fragments of an antibody (Fab fragments); naturally-occurring and non-naturally occurring single-chain variable fragments of an antibody (scFV fragments); naturally-occurring and non-naturally occurring blood factors such as β-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; naturally-occurring and non-naturally occurring colony stimulating factors; naturally-occurring and non-naturally occurring growth hormones; naturally-occurring and non-naturally occurring interleukin, such as IL-1, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, etc.; naturally-occurring and non-naturally occurring growth factors; naturally-occurring and non-naturally occurring soluble receptors; naturally-occurring and non-naturally occurring enzymes, such as a-glucosidase, imiglucarase, β-glucocerebrosidase, and alglucerase; naturally-occurring and non-naturally occurring enzyme activators, such as tissue plasminogen activator; naturally-occurring and non-naturally occurring chemokines; naturally-occurring and non-naturally occurring angiogenic agents; naturally-occurring and non-naturally occurring anti-angiogenic agents, such as a soluble VEGF receptor; naturally- occurring and non-naturally occurring protein vaccine; naturally-occurring and non-naturally occurring neuroactive peptides; and other naturally-occurring and non-naturally occurring proteins such as a thrombolytic agents, atrial natriuretic peptide, relaxin, glial fibrillary acidic protein, follicle stimulating hormone (FSH), human alpha- 1 antitrypsin, leukemia inhibitory factor (LIF), transforming growth factors (TGFs), tissue factors, luteinizing hormone, macrophage activating factors, tumor necrosis factor (TNF), neutrophil chemotactic factor (NCF), nerve growth factor, tissue inhibitors of metalloproteinases, vasoactive intestinal peptide, angiogenin, angiotropin, fibrin, hirudin, IL-1 receptor antagonists, and the like. Also suitable for use as a parent polypeptide are fusion proteins comprising all or a portion of any of the aforementioned proteins. Other categories of biologically-active polypeptides are as follows:
Insulin
[0080] In some embodiments, the parent polypeptide can be an insulin. In some embodiments, the parent polypeptide can be human insulin.
[0081] Examples of insulins include, but are not limited to, proinsulin, preproinsuhn, insulin, insulin analogs, and the like. Some examples of insulins are disclosed in U.S. Patent Nos. 5,474,978, 5,514,646, 5,504,188, 5,547,929, 5,650,486, 5,693,609, 5,700,662, 5,747,642, 5,922,675, 5,952,297, 6,034,054, and 6,211,144; and International Patent Publications Nos. WO 00/121197, WO 09/010645 and WO 90/12814, which hereby are incorporated by reference for the limited purpose of their disclosure of insulin. A non- limiting list of non-naturally occurring insulins include synthetic insulins available under the tradenames of NovoLog®, Novolin®, Humulin®, Humalog®, Lantus®, Lente®, and Ultralente®.
[0082] Insulin analogs include, but are not limited to, superactive insulin analogs, monomeric insulins, and hepatospecific insulin analogs. Various examples of insulin analogs include, but are not limited to, insulin analogs available under the tradenames of Humalog®; Humalog® Mix 50/50™; Humalog® Mix 75/25™; Humulin® 50/50; Humulin® 70/30; Humulin® L; Humulin® N; Humulin® R; Humulin® Ultralente; Lantus®; Lente® Iletin® II; Lente® Insulin; Lente® L; Novolin® 70/30; Novolin® L; Novolin® N; Novolin® R; NovoLog™; NPH Iletin® I; NPH-N; Pork NPH Iletin® II; Pork Regular Iletin® II; Regular
(Concentrated) Iletin ® II U-500; Regular Iletin ® I; and Velosulin ® BR Human (Buffered).
[0083] Additional examples of insulin analogs include, but are not limited to, acylated insulin, glycosylated insulin, and the like. Examples of acylated insulin include those disclosed in U.S. Patent No. 5,922,675, for example, insulin derivatized with a C6-C2i fatty acid (such as myristic, pentadecylic, palmitic, heptadecylic, or stearic acid) at an a- or ε- amino acid of glycine, phenylalanine or lysine.
[0084] Amino acid sequences of various insulin polypeptides are publicly available. For example, human insulin can comprise any one of the amino acid sequences as set forth in GenBank under the following accession numbers: P01308, CAA00714; CAA00713; CAA00712; CAA01254; IHISA and IHISB; 1 HIQA and 1 HIQB; IHITA and 1HITB; 1 HLSA and 1HLSB; 1VKTA and 1VKTB; which hereby are incorporated by reference for the limited purpose of their disclosure of amino acid sequences of insulin.
Antibodies
[0085] In some embodiments, the parent polypeptide can be an antibody. Examples of antibodies include, but are not limited to, antibodies of various isotypes (for example, IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM); monoclonal antibodies produced by any means known to those skilled in the art, including an antigen-binding fragment of a monoclonal antibody; humanized antibodies; chimeric antibodies; single-chain antibodies; antibody fragments such as Fv, F(ab')2, Fab', Fab, Facb, scFv and the like; provided that the antibody is capable of binding to antigen. A list of non-limiting examples of antibodies include antibodies that are specific for a cell surface receptor and that function as antagonists to the receptor, including, but not limited to, antibodies to TGF-β receptor; antibodies to TNF-a receptor; antibodies to VEGF receptor (see for example, U.S. Patent Nos. 6,617,160, 6,448,077, and 6,365,157); antibodies to epidermal growth factor receptor; antibodies specific for receptor ligands, including, but not limited to, antibodies to TGF-β, antibodies to TNF-a, antibodies to VEGF, and the like; antibodies specific for a tumor-associated antigen; antibodies specific for CD20; antibodies specific for epidermal growth factor receptor-2 (HER-2); antibodies specific for the receptor binding domain of IgE; antibodies specific for adhesion molecules, such as antibodies specific for a subunit (CD 11 a) of LFA-1; antibodies specific for α4β7; and the like. Examples of recombinant antibodies include, but are not limited to rituximab (a chimeric monoclonal antibody specific for CD20) available under the tradename of Rituxan , infliximab (a monoclonal antibody specific for TNF-oc) available under the tradename of Remicade , trastuzumab (a monoclonal antibody specific for HER- 2/neu receptor) avaible under the tradename of Herceptin®, adalimumab (a human monoclonal antibody specific for TNF-oc) available under the tradename of Humira™, omalizumab (a humanized antibody specific for human immunoglobulin E (IgE)) available under the tradename of Xolair®, tositumomab (a monoclonal antibody specific for CD20) available under the tradename of Bexxar®, efalizumab (a humanized monoclonal antibody specific for CD 11 a) available under the tradename of Raptiva™, cetuximab (a chimeric monoclonal antibody specific for an epidermal growth factor receptor (EGFR)) available under the tradename of Erbitux™, and the like.
[0086] In some embodiments, the parent polypeptide can be an antibody Fab fragment. In an embodiment, the parent polypeptide can be a Fab fragment of an antibody specific for TNF-a. In another embodiment, the parent polypeptide can be a Fab fragment of an antibody specific for HER-2 receptor. In still another embodiment, the parent polypeptide can be a Fab fragment of an antibody specific for VEGF. In other embodiments, the parent polypeptide can be an antibody scFV fragment. In an embodiment, the parent polypeptide can be an scFV fragment of an antibody specific for TNF-a. In another embodiment, the parent polypeptide can be an scFV fragment of an antibody specific for HER-2 receptor. In still another embodiment, the parent polypeptide can be an scFV fragment of an antibody specific for VEGF.
Blood factors
[0087] In some embodiments, the parent polypeptide can be a blood factor. Examples of blood factor include, but are not limited to, tissue plasminogen activators (TPA); β-globin; hemoglobin; coagulation factors such as Factor Vila, Factor VIII, and Factor IX; and the like. The amino acid sequences of various blood factors are publicly available. For example, human TPA can comprise any one of the amino acid sequences as set forth in GenBank under the following accession numbers: P0070, NP_127509, and NP- 000921 ; human Factor Vila can comprise the amino acid sequence set forth in GenBank Accession No. KFHU7; human Factor IX can comprise any one of the amino acid sequences as set forth in GenBank under the following accession numbers P00740 and NP_000124; and human Factor VIII can comprise any one of the amino acid sequences as set forth in GenBank under the following accession numbers: AAH64380, AAH22513, and P00451 ; which hereby are incorporated by reference for the limited purpose of their disclosure of amino acid sequences of blood factors.
[0088] A non-limiting list of examples of non-naturaly occurring blood factors include recombinant human factor Vila available under the tradename of NovoSeven®, recombinant factor VIII available under the tradename of Kogenate , and recombinant human factor IX available under the tradename of BeneFIX .
Colony stimulating factors ( CSFs)
[0089] In some embodiments, the parent polypeptide can be a colony stimulating factor. In an embodiment, the parent polypeptide can be human granulocyte colony stimulating factor (hG-CSF). In another embodiment, the parent polypeptide can be human granulocyte-monocyte colony stimulating factor (hGM-CSF).
[0090] Examples of colony stimulating factors include, but are not limited to, granulocyte colony stimulating factor (G-CSF), such as NEUPOGEN filgrastim and NEULASTA™ pegfilgrastim; granulocyte-monocyte colony stimulating factor (GM-CSF), such as LEUKINE® sargramostim; macrophage colony stimulating factor (M-CSF); megakaryocyte colony stimulating factor (Meg-CSF); IL-3; and the like. The amino acid sequences of various blood factors are publicly available. For example, amino acid sequences of IL-3 are disclosed in U.S. Pat. Nos. 4,877,729 and 4,959,455, and International Patent Publication No. WO 88/00598; amino acid sequences of human G-CSF are disclosed in U.S. Pat. No. 4,810,643; amino acid sequences of fusion proteins comprising IL-3 are disclosed in International Patent Publication Nos. WO 91/02754 and WO 92/04455; amino acid sequences of human G-CSF can be found under GenBank Accession Nos. P09919, NP_757374, P010219, and NP_000750; amino acid sequences of human GM-CSF can be found under GenBank Accession Nos. NP_000749 and P04141 ; amino acid sequences of IL- 3 can be found under GenBank Accession Nos. AAH66272, AAH66273, and AAH66276; and amino acid sequences of M-CSF can be found under GenBank Accession Nos. AAA59572.1, AAB59527.1, AAA59573.1, AAB29303.1, 1HMCB, 1HCMB, and AAA64849.1. All patent references and GenBank records disclosed in this paragraph are hereby incorporated by reference for the limited purpose of their disclosure of amino acid sequences of clony stimulating factors.
Growth hormones
[0091] In some embodiments, the parent polypeptide can be a growth hormone. In some embodiments, the parent polypeptide can be human growth hormone 1 (hGH-1).
[0092] Examples of growth hormones include, but are not limited to, somatotropin; human growth hormones; growth hormone variants disclosed in U.S. Patent Nos. 6,143,523, 6,136,563, 6,022,711, and 5,688,666; fusion proteins comprising a growth hormone, such as those disclosed in U.S. Patent No. 5,889,144; growth hormone fragments that retain growth hormone activity; a growth hormone disclosed in U.S. Patent No. 6,387,879; and the like. Non-limiting examples of growth hormones also include alternative forms of known growth hormones, including naturally-occurring derivatives, variants and metabolic products (for example, degradation products primarily of biosynthetic hGH and engineered variants of hGH produced by recombinant methods (see for example, U.S. Patent No. 6,348,444)). Non-limiting examples of non-naturally occurring human growth hormone include synthetic human growth hormone available under the tradenames of Genotropin , Nutropin®, Norditropin®, Saizen®, Serostim®, and Humatrope®. In some embodiments, hGH-1 can comprise an amino acid sequence as set forth in GenBnk Accession No. P01241. Growth factors
[0093] In some embodiments, the parent polypeptide can be a growth factor. In some embodiments, the parent polypeptide can be a human insulin-like growth factor (hIGF). In some embodiments, the parent polypeptide can be human insulin-like growth factor 1A (hIGF-ΙΑ). In other embodiments, the parent polypeptide can be an erythropoietin (EPO). In some embodiments, the parent polypeptide can be a human erythropoietin (hEPO).
[0094] Examples of growth factors include, but are not limited to, keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), active fragments of a growth factor, fusion proteins comprising a growth factor, bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor- 9 (GDF-9), hepatoma derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-β), and the like.
[0095] Amino acid sequences of various growth factors are publicly available. For example, amino acid sequences of basic FGFs can be found under GenBank Accession Nos. AAB20640, AAA57275, A43498, and AAB20639; amino acid sequences of acidic FGFs can be found under GenBank Accession Nos. AAB29059, CAA46661, and 1605206A; amino acid sequences of human stem cell factor can be found under GenBank Accession Nos. AAH69733, AAH69783, AAH69797, AAD22048.1, AAA85450.1, AAK92486.1, AAK92485.1, 1EXZA, 1EXZB and 1EXZC; amino acid sequences of keratinocyte growth factor are found under GenBank Accession Nos. 035565, AAL05875, and P21781; amino acid sequences of hepatocye growth factor are found under GenBank Accession Nos. AAA64239, AAB20169, and CAA40802; amino acid sequences of human insulin-like growth factor can be found under GenBank Accession Nos. P01343, AAA52538.1, AAA52787.1, BAD92421.1, P01343, P01344.1, NP_001007140.2, and NP_001121070.1. Examples of non-naturally occurring EPO include, but are not limited to synthetic EPO available under the tradenames of Procrit®, Eprex®, Epogen® (epoetin-a), Aranesp® (darbepoetin-a), NeoRecormon®, and Epogin® (epoetin-β). Amino acid sequence of human erythropoietin (hEPO) can be found under GenBank Accession Nos. P01588, CAA26095.1, AAA52400.1, and AAD 13964.1.
Soluble receptors
[0096] In some embodiments, the parent polypeptide can be a soluble receptor. Examples of soluble receptor polypeptides include, but are not limited to, soluble TNF-a receptors; soluble VEGF receptors; soluble interleukin receptors, such as soluble IL-1 receptors and soluble type II IL-1 receptors; soluble γ/δ T cell receptors; ligand-binding fragments of a soluble receptor, and the like. Suitable soluble receptors bind a ligand that, under normal physiological conditions, binds to and activates the corresponding membrane- bound or cell surface receptor. Thus, a suitable soluble receptor can function as a receptor antagonist, by binding the ligand that would ordinarily bind the receptor in its native (for example, membrane-bound) form.
[0097] Amino acid sequences of various soluble receptors are publicly available. For example, amino acid sequences of soluble VEGF receptors can be found under GenBank Accession Nos. AAC50060, NP_002010, P17948, and P35968; soluble VEGF receptors can be found described in U.S. Patent Nos. 6,383,486, 6,375,929, and 6,100,071 ; soluble IL-4 receptors are described in U.S. Pat. No. 5,5102,905; and soluble IL-1 receptors are described in U.S. Patent Publication No. 20040023869; amino acid sequences of soluble TNF-a receptors can be found under GenBank Accession Nos. P19438 and P20333.
Chemokines
[0098] In some embodiments, the parent polypeptide can be a chemokine. Examples of chemokines include, but are not limited to, IP- 10, monokine induced by interferon-gamma (Mig), Groa/IL-8, RANTES, MIP-la, ΜΙΡ-Ιβ, MCP-1, PF-4, and the like. The amino acid sequences of various chemokines are publicly available. For example, amino acid sequences of IP- 10 are disclosed in U.S. Patent Nos. 6,491,906, 5,935,567, 6,153,600, 5,728,377, and 5,1024,292; amino acid sequences of Mig are disclosed in U.S. Patent No. 6,491,906, and Farber, Biochemical and Biophysical Research Communications, 1993, 192(l):223-230; and amino acid sequences of RANTES are disclosed in U.S. Patent Nos. 6,709,649, 6,168,784, and 5,965,697.
Angiogenic agents
[0099] In some embodiments, the parent polypeptide can be an angiogenic agent. Examples of angiogenic polypeptides include, but are not limited to, vascular endothelial growth factors (VEGFs), including VEGF121, VEGF165, VEGF-C, VEGF-2, etc.; transforming growth factor-beta; basic fibroblast growth factor; glioma-derived growth factor; angiogenin; angiogenin-2; and the like. The amino acid sequences of various angiogenic agents are publicly available. For example, amino acid sequences of VEGF polypeptides are disclosed in U.S. Patent Nos. 5,194,596, 5,332,671, 5,240,848, 6,475,796, 6,485,942, and 6,057,428; amino acid sequences of VEGF-2 polypeptides are disclosed in U.S. Patent Nos. 5,726,152 and 6,608,182; amino acid sequences of glioma-derived growth factors having angiogenic activity are disclosed in U.S. Patent Nos. 5,338,840 and 5,532,343; amino acid sequences of angiogenin can be found under GenBank Accession Nos. AAA72611, AAA51678, AAA02369, AAL67710, AAL67711, AAL67712, AAL67713, and AAL67714.
Neuroactive peptides
[0100] In some embodiments, the parent polypeptide can be a neuroactive peptide. Examples of neuroactive polypeptides include, but are not limited to, nerve growth factor (NGF), bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagons, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, and the like.
Additional polypeptides
[0101] Various other polypeptides can be used as a parent polypeptide in the methods and/or compositions disclosed herein. Additional polypeptides include, but are not limited to, thrombolytic agents, atrial natriuretic peptides, glial fibrillary acidic protein, follicle stimulating hormone (FSH), human alpha- 1 antitrypsin, leukemia inhibitory factor (LIF), transforming growth factor (TGF), a luteinizing hormone, a macrophage activating factor, tumor necrosis factor (TNF), neutrophil chemotactic factor (NCF), nerve growth factor (NGF), tissue inhibitor of metalloproteinases; vasoactive intestinal peptide, angiotropin, fibrin, hirudin, relaxin, and the like. The amino acid sequences of various therapeutic proteins are publicly available. For example, amino acid sequences of tissue plasminogen activator can be found under GenBank Accession Nos. P00750, AAA01895, AAA01378, AAB06956, and CAA00642.
[0102] In some embodiments, the parent peptide is not an interferon. In some embodiments, the parent peptide is not a Type I interferon. In other embodiments, the parent peptide is not a Type II interferon. In still other embodiments, the parent peptide is not a Type III interferon. In yet other embodiments, the parent peptide is not a follicle-stimulating hormone (FSH), for example human FSH (hFSH). In yet still other embodiments, the parent peptide is not a cytokine. In yet still other embodiments, the parent peptide is not a growth hormone, for example, human growth hormone. Conversely, in other embodiments, the parent peptide or polypeptide is one of those mentioned in this paragraph.
Hyper glycosylated polypeptide variants
[0103] In some embodiments, a hyperglycosylated polypeptide variant of a parent polypeptide, such as a biologically-active polypeptide, can be the parent polypeptide that has been modified to include a peptide extension inserted at a terminal region, where the peptide extension can include at least one glycosylation site. In some embodiments, the peptide extension can include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites. In some embodiments, the only modifications to the parent polypeptide are addition of the peptide extension to the amino or carboxy terminus and, optionally, addition of one or more glycosylation sites in the parent polypeptide.
[0104] The hyperglycosylated polypeptide can be of various lengths. In some embodiments, a hyperglycosylated polypeptide variant of a parent polypeptide can be at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 145 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 155 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 165 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 175 amino acids in length, alternatively at least about 180 amino acids in length, alternatively at least about 185 amino acids in length, alternatively at least about 190 amino acids in length, alternatively at least about 195 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 205 amino acids in length, alternatively at least about 210 amino acids in length, alternatively at least about 215 amino acids in length, alternatively at least about 220 amino acids in length, alternatively at least about 230 amino acids in length, alternatively at least about 240 amino acids in length, alternatively at least about 250 amino acids in length, alternatively at least about 260 amino acids in length, alternatively at least about 270 amino acids in length, alternatively at least about 280 amino acids in length, alternatively at least about 290 amino acids in length, alternatively at least about 300 amino acids in length, alternatively at least about 310 amino acids in length, alternatively at least about 320 amino acids in length, alternatively at least about 330 amino acids in length, alternatively at least about 340 amino acids in length, alternatively at least about 350 amino acids in length, alternatively at least about 360 amino acids in length, alternatively at least about 370 amino acids in length, alternatively at least about 380 amino acids in length, alternatively at least about 390 amino acids in length, alternatively at least about 400 amino acids in length, alternatively at least about 410 amino acids in length, alternatively at least about 420 amino acids in length, alternatively at least about 430 amino acids in length, alternatively at least about 440 amino acids in length, alternatively at least about 450 amino acids in length, alternatively at least about 460 amino acids in length, alternatively at least about 470 amino acids in length, alternatively at least about 480 amino acids in length, alternatively at least about 490 amino acids in length, alternatively at least about 500 amino acids in length, alternatively at least about 510 amino acids in length, alternatively at least about 520 amino acids in length, alternatively at least about 530 amino acids in length, alternatively at least about 540 amino acids in length, alternatively at least about 550 amino acids in length, alternatively at least about 560 amino acids in length, alternatively at least about 570 amino acids in length, alternatively at least about 580 amino acids in length, alternatively at least about 590 amino acids in length, alternatively at least about 600 amino acids in length, alternatively at least about 700 amino acids in length, alternatively at least about 800 amino acids in length, alternatively at least about 900 amino acids in length, alternatively at least about 1000 amino acids in length, alternatively at least about 1100 amino acids in length, alternatively at least about 1200 amino acids in length, alternatively at least about 1300 amino acids in length, alternatively at least about 1400 amino acids in length, alternatively at least about 1500 amino acids in length or more.
[0105] In some embodiments, the parent polypeptide can be a growth hormone (GH) and the peptide extension can be inserted between the 26th and the 27th amino acids of the parent growth hormone. In some embodiments, the parent polypeptide can be human growth hormone 1 (hGH-1) and the peptide extension can be inserted between the 26th and the 27th amino acids of the parent hGH-1.
[0106] In other embodiments, the parent polypeptide can be an insulin-like growth factor. In some embodiments, the parent polypeptide can be human insulin-like growth factor 1A (hIGF-ΙΑ) and the peptide extension can be located between the 21st and the 22nd amino acids of the parent hIGF-lA.
[0107] In still other embodiments, the parent polypeptide can be a granulocyte colony-stimulating factor (G-CSF). In some embodiments, the parent polypeptide can be human G-CSF and the peptide extension can be located between the 29th and the 30th amino acids of the parent human G-CSF.
[0108] In yet other embodiments, the parent polypeptide can be an erythropoietin. In some embodiments, the parent polypeptide can be human erythropoietin (EPO) and the peptide extension can be located between the 27th and the 28th amino acids of the parent human EPO.
[0109] In yet other embodiments, the parent polypeptide can be an insulin. In some embodiments, the parent polypeptide can be human insulin and the peptide extension can be located between the 24th and the 25th amino acids of the parent human insulin.
[0110] In some embodiments, the peptide extension can include an amino acid motif, where the amino acid motif can include one or more glycosylation sites. In some embodiments, the amino acid motif can be
Figure imgf000038_0001
where B 1 is an amino acid residue; B2 is a Serine (S) or a Threonine (T); and B3 is a sequence of Zl amino acids, where Zl is an integer from 1 to 8 and each amino acid in the sequence B is independently an amino acid residue.
[0111] In some embodiments, B1 can be any amino acid. In other embodiments, B1 can be any amino acid except for Proline (P). In an embodiment, B1 can be a Valine (V). In another embodiment, B1 can be an Isoleucine (I). In still another embodiment, B1 can be a Glycine (G). In yet another embodiment, B1 can be an Alanine (A).
[0112] In some embodiments, B2 can be a Serine (S). In other embodiments, B2 can be a Threonine (T). [0113] In some embodiments, B3 can be any amino acid. In an embodiment, B3 can be a Valine (V). In another embodiment, B can be an Isoleucine (I). In still another embodiment, B 3 can be a Glycine (G). In yet another embodiment, B 3 can be an Alanine (A).
[0114] Non-limiting examples of the amino acid motif ΝΒ 1Έ2 Β 3¾ι include NI[T/S]V, NI[T/S]I, NI[T/S]A, NI[T/S]G, NA[T/S]V, NA[T/S]I, NA[T/S]A, NA[T/S]G, NV[T/S]V, NV[T/S]I, NV[T/S]A, NV[T/S]G, NG[T/S]V, NG[T/S]I, NG[T/S]A, NG[T/S]G, NI[T/S]VNI[T/S]V, NI[T/S]VNA[T/S]G, NI[T/S]VNV[T/S]V, NI[T/S]ANI[T/S]A, NI[T/S]ANI[T/S]G, and NV[T/S]ANG[T/S]I, where [T/S] represents the presence of either a Threonone (T) or a Serine (S) residue. In some embodiments, the amino acid motif
1 2 3
NB B [B ]zi can be NITV. Sequences of some non-limiting examples of the amino acid motif NB1B2[B3]zi are given in SEQ ID NOs: 183-238.
[0115] In some embodiments, the amino acid motif included in the peptide extension can be [B4]z2NB5B6[B7]z3, where B4 is a sequence of Z2 amino acids, Z2 is an integer from 1 to 8, and each amino acid in the sequence B4 is independently an amino acid residue; B5 is an amino acid residue; B6 is a Serine (S) or a Threonine (T); B7 is a sequence of Z3 amino acids, wherein Z3 is an integer from 1 to 8, wherein each amino acid in the η
sequence B is independently an amino acid residue.
[0116] In some embodiments, B4 can be any amino acid. In an embodiment, B4 can be a Valine (V). In another embodiment, B4 can be an Isoleucine (I). In still another embodiment, B4 can be a Glycine (G). In yet another embodiment, B4 can be an Alanine (A).
[0117] In some embodiments, B5 can be any amino acid. In an embodiment, B5 can be any amino acid except for Proline (P). In an embodiment, B5 can be a Valine (V). In another embodiment, B5 can be an Isoleucine (I). In still another embodiment, B5 can be a Glycine (G). In yet another embodiment, B5 can be an Alanine (A).
[0118] In some embodiments, B6 can be a Serine (S). In other embodiments, B6 can be a Threonine (T).
[0119] In some embodiments, B7 can be any amino acid. In an embodiment, B7 can be a Valine (V). In another embodiment, B can be an Isoleucine (I). In still another embodiment, B 7 can be a Glycine (G). In yet another embodiment, B 7 can be an Alanine (A). In yet still another embodiment, B 7 can be GG. In yet still another embodiment, B 7 can be η
GGG. In yet still another embodiment, B can be GR.
[0120] Examples of the amino acid motif [B4]z2NB5B6[B7]z3 include, but are not limited to, INI[T/S]V, INI[T/S]I, VNI[T/S]A, VNI[T/S]G, VNI[T/S]GR, VNI[T/S]GG, VNI[T/S]GGG, INA[T/S]V, INA[T/S]G, GNA[T/S]I, INA[T/S]A, GNI[T/S]G, GNA[T/S]G, INA[T/S]G, ANA[T/S]G, ANV[T/S]V, VNV[T/S]I, VNV[T/S]A, ANI[T/S]V, VNV[T/S]G, ING[T/S]V, ANG[T/S]I, VNG[T/S]A, VNG[T/S]G, SNI[T/S]G, ASNI[T/S]G, VNI[T/S]VNI[T/S]V, ANI[T/S]VNA[T/S]G, INI[T/S]VNV[T/S]V, GNI[T/S]ANI[T/S]A, GNI[T/S]ANI[T/S]G, VNI[T/S]GVNI[T/S]GR, VNI[T/S]GGVNI[T/S]GR, VNI[T/S]GGGVNI[T/S]GR, and GNV[T/S]ANG[T/S]I, where [T/S] represents the presence of either a Threonone (T) or a Serine (S) residue. In some embodiments, the amino acid motif [B4]Z2NB5B6[B7]z3 can be VNITG. In other embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNITGG. In still other embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNITGGG. In yet other embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNISGR. In yet still other embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNITGVNISGR. In some embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNITGG VNIS GR. In other embodiments, the amino acid motif [B4]Z2NB5B6[B7]Z3 can be VNITGGGVNISGR. These various motifs can be repeated in the peptide extension, either by repeating the same glycosylation motive, or by mixing various motifs in the peptide extension. In addition, the motifs can be directly linked to each other, or can be separated by additional amino acids acting as spacers between the motifs. The amino acids used as the spacers can be charged or uncharged, hydrophobic or hydrophilic or neither, and each spacer can be of any length desired, from zero to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more amino acids in length, including any intermediate values not specifically mentioned here. Sequences of some non-limiting examples of the amino acid motif [B4]Z2NB5B6[B7]z3 are given in SEQ ID NOs: 239-328.
[0121] The amino acid motif (or mixture of motifs), such as NB1B2[B3]zi and [B4]z2NB5B6[B7]z3, can be present one or more times in the peptide extension. In some embodiments, the amino acid motif can be present once, twice, three times, or four times in the peptide extension. In still other embodiments, the amino acid motif can be present five times, six times, seven times, eight times, nine times, or ten times in the peptide extension. In yet other embodiments, the amino acid motif can be present eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-one times, twenty-two times, twenty-three times, twenty-four times, twenty-five times, twenty-six times, twenty-seven times, twenty-eight times, twenty-nine times, thirty times, or more in the peptide extension.
[0122] In some embodiments, the amino acid motif can be present at least once, at least twice, at least three times, or at least four times in the peptide extension. In still other embodiments, the amino acid motif can be present at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times in the peptide extension. In yet other embodiments, the amino acid motif can be present at least eleven times, at least twelve times, at least thirteen times, at least fourteen times, at least fifteen times, at least sixteen times, at least seventeen times, at least eighteen times, at least nineteen times, at least twenty times, at least twenty-one times, at least twenty- two times, at least twenty-three times, at least twenty-four times, at least twenty-five times, at least twenty-six times, at least twenty-seven times, at least twenty-eight times, at least twenty-nine times, at least thirty times, or more in the peptide extension.
[0123] In some embodiments, the amino acid motif can include one N-linked glycosylation site. In other embodiments, the amino acid motif can include two, three, four, five, or six N-linked glycosylation sites. In some embodiments, the amino acid motif can include one O-linked glycosylation sites. In other embodiments, the amino acid motif can include two, three, four, five, or six O-linked glycosylation sites. In still other embodiments, the amino acid motif can include one, two, three, four, five, or six N-linked gltycosylation sites and/or one, two, three, four, five, or six O-linked glycosylation sites.
[0124] In some embodiments, the amino acid motif can include at least one N- linked glycosylation site. In other embodiments, the amino acid motif can include at least two, at least three, at least four, at least five, or at least six N-linked glycosylation sites. In some embodiments, the amino acid motif can include at least one O-linked glycosylation sites. In other embodiments, the amino acid motif can include at least two, at least three, at least four, at least five, or at least six O-linked glycosylation sites. In still other embodiments, the amino acid motif can include at least one, at least two, at least three, at least four, at least five, or at least six N-linked gltycosylation sites and/or at least one, at least two, at least three, at least four, at least five, or at least six O-linked glycosylation sites.
[0125] Non-limiting examples of peptide extensions include:
VNITG (SEQ ID NO: l)
VNITGVNITG (SEQ ID NO: 2)
VNITGVNITGVNITG (SEQ ID NO:3)
VNITGVNITGVNITGVNITG (SEQ ID NO: 4)
VNITGVNITGVNITGVNITGVNITG (SEQ ID NO:5)
VNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO:6)
VNITGVNITGVNITG VNITG VNITG VNITGVNITG (SEQ ID NO: 7)
VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 8)
VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 9) VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 10)
VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 11)
VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG
(SEQ ID NO: 12)
VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITGVNITG (SEQ ID NO: 13)
VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITGVNITGVNITGVNITG (SEQ ID NO: 14)
VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 15) VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGV NITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNISGR (SEQ ID NO: 16)
VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITG VNISGR (SEQ ID NO: 17)
VNITGGG (SEQ ID NO: 18) VNITGGGVNITGGG (SEQ ID NO: 19)
VNITGGGVNITGGGVNITGGG (SEQ ID NO: 20)
VNITGGGVNITGGGVNITGGGVNITGGG (SEQ ID NO: 21)
VNITGGGVNITGGGVNITGGGVNITGGGVNITGGG (SEQ ID NO: 22)
VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGG (SEQ ID NO: 23) VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGG (SEQ ID NO: 24)
VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGG
(SEQ ID NO: 25)
VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGV NITGGG (SEQ ID NO: 26)
VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGV NITGGGVNITGGG (SEQ ID NO: 27)
VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGV NITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNISGR (SEQ ID NO: 28)
VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITGVNISGR (SEQ ID NO: 29)
VNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITG G VNITGG VNITGG VNITGG VNIS GR (SEQ ID NO: 30)
[0126] In some embodiments, in addition to the peptide extension that is inserted at a terminal region, the parent polypeptide can be further modified to include at least one additional glycosylation site in the amino acid sequence of the parent polypeptide. Each additional glycosylation site that is introduced in the amino acid sequence of the parent polypeptide can be by at least one amino acid substitution or at least one combination of amino acid substitutions. As such, a hyperglycosylated polypeptide variant of a parent polypeptide can be a parent polypeptide that has been modified to include a peptide extension inserted at a terminal region and at least one additional glycoyslation site introduced to the amino acid sequence of the parent polypeptide.
[0127] In some embodiments, the additional glycosylation sites can be introduced to the parent polypeptide through amino acid substitution(s) located in a region that consists of the amino acid residues after the first 15 amino acids at the amino-terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide and before the last 15 amino acids at the carboxy-terminus of the parent polypeptide. In other embodiments, the additional glycosylation sites can be introduced to the parent polypeptide through amino acid substitution(s) located in a region consisting of the first 15 amino acid residues at the amino- terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide. In still other embodiments, the additional glycosylation sites can be introduced to the parent polypeptide through amino acid substitution(s) located in a region consisting of the last 15 amino acid residues at the carboxy-terminus of the parent polypeptide.
[0128] In some embodiments, the parent polypeptide can be a growth hormone (GH), where the additional glycosylation sites can be introduced to the parent GH through amino acid substitution(s) located at a region consisting of the 15th to 180th amino acids, the 20th to 160th amino acids, the 30th to 140th amino acids, the 40th to 120th amino acids, or the 50th to 110th amino acids of the parent GH, where any signal peptide in the parent GH is excluded. In other embodiments, the parent polypeptide can be an insulin-like growth factor (IGF), where the additional glycosylation site(s) can be introduced to the parent IGF through amino acid substitution(s) located at a region consisting of the 16th to 130th amino acids, the 40th to 110th amino acids, the 50th to 100th amino acids, or the 60th to 80th amino acids of the parent IGF, where any signal peptide in the parent IGF is excluded. In still other embodiments, the parent polypeptide can be a G-CSF, where the additional glycosylation site(s) can be introduced to the parent G-CSF through amino acid substitution(s) located at a region consisting of the 16th to 170th amino acids, the 20th to 150th amino acids, the 25th to 130th amino acids, or the 30th to 110th amino acids in the parent G-CSF, where any signal peptide in the parent G-CSF is excluded. In yet other embodiments, the parent polypeptide can be an erythropoietin (EPO), where the additional glycosylation site(s) can be introduced to the parent EPO through amino acid substitution(s) located at a region consisting of the 16th to 160th amino acids, the 20th to 140th amino acids, the 25th to 120th amino acids, or the 30th to 100th amino acids in the parent EPO, where any signal peptide in the parent EPO is excluded. In yet other embodiments, the parent polypeptide can be an insulin, where the additional glycosylation site(s) can be introduced to the parent insulin through amino acid substitution(s) located at a region consisting of the 16th to 85th amino acids, the 20th to 148th amino acids, the 25th to 75th amino acids, or the 30th to 65th amino acids in the parent insulin, where any signal peptide in the parent insulin is excluded.
[0129] In some embodiments, in addition to the peptide extension that is inserted at a terminal region, the parent polypeptide has been further modified to include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten additional glycosylation sites in the amino acid sequence of the parent polypeptide. Each additional glycosylation site that is introduced to the parent polypeptide may be a native glycosylation site that is not present in the parent polypeptide, or a non- native glycosylation site.
[0130] In some embodiments, the amino acid sequence of the parent polypeptide can be modified to include a native glycosylation site and a non-native glycosylation site In some embodiments, the amino acid sequence of the parent polypeptide can be modified to include at least two, three, four, five, six, seven, eight, nine, or ten native glycosylation sites from the parent polypeptide, and at least two, three, four, five, six, seven, eight, nine, or ten non-native glycosylation sites.
[0131] In some embodiments, the amino acid sequence of the parent polypeptide can be modified to include an O-linked glycosylation site. In other embodiments, the amino acid sequence of the parent polypeptide can be modified to include an N-linked glycosylation. In other embodiments, the amino acid sequence of the parent polypeptide can be modified to include both O-linked and N-linked glycosylation.
[0132] In some embodiments, a hyperglycosylated polypeptide variant of a parent polypeptide can further include at least one native glycosylation site from the parent polypeptide that is glycosylated in the hyperglycosylated polypeptide variant, but is not glycosylated in the parent polypeptide.
[0133] In some embodiments, a hyperglycosylated polypeptide variant of a parent polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-7, 9- 14, 16-21, 23-28, and 30-35. In other embodiments, a hyperglycosylated polypeptide variant of a parent polypeptide is a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 2-7, 9-14, 16-21, 23-28, and 30-35. [0134] In some embodiments, a hyperglycosylated polypeptide variant of a parent polypeptide can have at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a hyperglycosylated polypeptide variant sequence as disclosed herein (for example, SEQ ID NOs: 2-7, 9-14, 16- 21, 23-28, and 30-35), or any fragment of a hyperglycosylated polypeptide variant polypeptide sequence as disclosed herein.
[0135] In some embodiments, the glycosylation of hyperglycosylated polypeptide variants of a parent polypeptide is altered compared to that of the parent polypeptide. In some embodiments, the glycosylation pattern of hyperglycosylated polypeptide variants of a parent polypeptide is altered compared to that of the parent polypeptide. In some embodiments, the hyperglycosylated polypeptide variants of a parent polypeptide exhibit increased amount of glycosylation compared to the parent polypeptide.
Functional features of glycosylated polypeptides
[0136] In some embodiments, a hyperglycosylated polypeptide variant of a parent polypeptide can exhibit one or more of the following properties: increased serum half-life; reduced immunogenicity in vivo; increased functional in vivo half-life; increased stability; reduced degradation by gastrointestinal tract conditions; and improved water solubility. In an embodiment, a hyperglycosylated polypeptide variant of a parent polypeptide can have an increased serum half-life compared to a naturally occurring polypeptide or compared to the parent polypeptide under substantial similar or the same conditions. In another embodiment, a hyperglycosylated polypeptide variant of a parent polypeptide can have an increased AUC compared to a naturally occurring polypeptide or compared to the parent polypeptide under substantial similar or the same conditions. The term "serum half-life" is used interchangeably herein with the terms "plasma half-life," and "circulating half-life."
[0137] In some embodiments, the hyperglycosylated polypeptide variants of a parent polypeptide can have a serum half-life that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9- fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40- fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80- fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 300- fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, or more, greater than the serum half-life of the parent polypeptide under substantial similar or the same conditions. In some embodiments, the extent of the increase in serum half-life of a hyperglycosylated polypeptide variant of a parent polypeptide is determined by comparing the serum half-life of the hyperglycosylated polypeptide variant of the parent polypeptide to the serum half-life of the parent polypeptide in human blood or human serum in vivo.
[0138] In some embodiments, the hyperglycosylated polypeptide variants of a parent polypeptide can have an AUC that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5- fold, at least 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least 9.5-fold, at least 10-fold, or more, greater than the AUC of the parent polypeptide when administered under substantially similar or the same conditions.
[0139] The serum half-life of a hyperglycosylated polypeptide variant of a parent polypeptide can be readily determined using conventional methods. For example, a hyperglycosylated polypeptide variant of a parent polypeptide can be detectably labeled, and be administered to a subject (for example, an experimental non-human animal, or a human subject), and, at various time points following administration of the hyperglycosylated polypeptide variant, a blood sample is drawn and the amount of detectably labeled hyperglycosylated polypeptide variant in the blood sample can be determined.
[0140] In some embodiments, the hyperglycosylated polypeptide variants of a parent polypeptide can be detected in the serum of the subject after at least about 3 days, at least about 5 days, at least about 7 days, at least about 9 days, at least about 11 days, at least about 13 days, at least about 15 days, at least about 17 days, at least about 19 days, at least about 21 days, at least about 23 days, at least about 25 days, at least about 27 days, at least about 29 days, at least about 31 days, at least about 33 days, at least about 35 days, at least about 37 days, at least about 39 days, at least about 41 days, at least about 43 days, at least about 45 days, at least about 47 days, at least about 49 days, at least about 51 days, or longer after administration.
[0141] The hyperglycosylated polypeptide variants disclosed herein can have various molecular weight. In some embodiments, the molecular weight of the hyperglycosylated polypeptide variants is at least 70 kD, at least 75 kD, at least 80 kD, at least 85 kD, at least 90 kD, at least 95 kD, at least 100 kD, at least 105 kD, at least 110 kD, at least 115 kD, at least 120 kD, at least 125 kD, or at least 130 kD. In some embodiments, the molecular weight of the hyperglycosylated polypeptide variants is in the range of about 70 kD to about 200 kD. In other embodiments, the molecular weight of the hyperglycosylated polypeptide variants is in the range of about 70 kD to about 150 kD. In still other embodiments, the molecular weight of the hyperglycosylated polypeptide variants is in the range of about 70 kD to about 100 kD. In yet still other embodiments, the molecular weight of the hyperglycosylated polypeptide variants is in the range of about 80 kD to about 100 kD. Preparation of a hyperglycosylated polypeptide variant of a parent polypeptide
[0142] A hyperglycosylated polypeptide variant of a parent polypeptide can be prepared using conventional techniques, including chemical synthesis methods, production by standard recombinant techniques, and combinations thereof. For example, the hyperglycosylated polypeptide variant can be synthesized using an automated solid-phase tert-butyloxycarbonyl and benzyl protection strategy. A hyperglycosylated polypeptide variant of a parent polypeptide can also be synthesized by native chemical ligation using standard methods of chemical synthesis. The purity of synthesized polypeptides may be assessed by reverse-phase high performance liquid chromatography (HPLC) and isoelectric focusing. The primary structures of the ligands may be verified by Edman sequencing methods. Examples of techniques and methods for generating hyperglycosylated polypeptide variants of a parent polypeptide have been described in U.S. Patent Publication No. 2006- 0182716 and U.S. Patent No. 7,597,884, the contents of which are hereby incorporated by reference in their entirety. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
[0143] As used herein, the term "host cell" includes an individual cell or cell culture, which can be or has been a recipient of any recombinant vector(s), or synthetic or exogenous polynucleotide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a synthetic or exogenous polynucleotide. The term "recombinant host cell" refers to a host cell that includes one or more recombinant vectors. In some embodiments, a host cell can be a prokaryotic cell. In other embodiments, a host cell can be a eukaryotic cell. Non-limiting examples of recombinant vectors include propagation vectors and expression vectors.
[0144] The term "construct," as used herein, refers to a recombinant nucleic acid that has been generated for the purpose of the expression of a specific nucleotide sequence(s), or is to be used in the construction of other recombinant nucleotide sequences. An example of a construct is a recombinant DNA.
[0145] As used herein, the term "vector" refers to a polynucleotide construct, typically a plasmid or a virus, used to transmit genetic material to a host cell. In some embodiments, a vector can be an agent such as a plasmid, for example, a circular plasmid. A vector as used herein can be composed of either DNA or RNA. In some embodiments, a vector is composed of DNA.
[0146] As used herein, the term "expression vector" refers to a polynucleotide construct that can express a gene in a host cell. Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and a gene is said to be "operably linked to" the promoter.
[0147] The terms "DNA regulatory sequences," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a coding sequence and/or production of an encoded polypeptide in a host cell.
[0148] As used herein the term "promoter" is a nucleotide sequence that directs the transcription of a gene. Typically, a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of the gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals (including humans). A promoter can be inducible, repressible, and/or constitutive.
[0149] As used herein, the term "enhancer" refers to a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
[0150] The term "transformation" as used herein refers to a permanent or transient genetic change induced in a cell following introduction of exogenous nucleic acid to the cell. Genetic modification can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.
[0151] The term "operably linked," as used herein, refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter effects transcription or expression of the coding sequence.
[0152] Various chemical synthesis methods can be used to obtain hyperglycosylated polypeptide variants of a parent polypeptide described herein. For example, a hyperglycosylated polypeptide variant can be prepared by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide variant. The codons can be selected such that they are favored in the host cell in which the recombinant polypeptide will be produced. For example, several small oligonucleotides coding for portions of the hyperglycosylated polypeptide variant may be synthesized and assembled by PCR techniques, ligation, ligation chain reaction (LCR), or any other method or procedure known to one skilled in the art. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly. Once assembled, the nucleotide sequence encoding the hyperglycosylated polypeptide variant can be inserted into a recombinant propagation vector to produce a sufficient amount of the polynucleotide encoding the amino acid sequence of a hyperglycosylated polypeptide variant. Alternatively, once assembled, the nucleotide sequence encoding the hyperglycosylated polypeptide variant can be inserted into a recombinant expression vector for production of the hyperglycosylated polypeptide variant in a host cell.
[0153] In some embodiments, the polynucleotide encoding the amino acid sequence of a hyperglycosylated polypeptide variant of a parent polypeptide can be generated such that at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or more, of the codons are codons that are preferred in human sequences. A non-limiting list of example of codons that are preferred in human is shown in Table 1. Table 1 : Codon Usage in Human. Molecular Cloning: A
Laboratory Manual. Sambrook J. and Russell D. W.
Third Edition ©2001 by Cold Spring Harbor Press.
Alanine 6.99 GCU (28.0) GCC (41.6)
GCA (20.0) GCG (10.3)
Arginine 5.28 CGU (8.9) CGC (21.4)
CGA (5.4) CGG (10.4)
AGA (9.9) AGG (l l. l)
Asparagine 3.92 AAU (42.3) AAC (57.7)
Aspartic Acid 5.07 GAU (42.8) GAC (57.2)
Cysteine 2.44 UGU (40.6) UGC (59.4)
Glutamic Acid 6.82 GAA (39.2) GAG (60.7)
Glutamine 4.47 CAA (24.8) CAG (75.2)
Glycine 7.10 GGU (15.8) GGC (35.8)
GGA (24.1) GGG (24.3)
Histidine 2.35 CAU (39.6) CAC (60.4)
Isoleucine 4.50 AUU (33.1) AUC (54.0)
AUA (12.9)
Leucine 9.56 UUA (5.5) UUG (11.5)
CUU (11.1) CUC (20.8)
CUA (6.5) CUG (44.5)
Lysine 5.71 AAA (38.9) AAG (61.1)
Methionine 2.23 AUG (100)
Phenylalanine 3.84 UUU (41.1) UUC (58.2)
Proline 5.67 CCU (27.3) CCC (35.2)
CCA (25.7) CCG (11.6)
Serine 7.25 UCU (18.3) UCC (23.7)
UCA (12.9) UCG (5.9)
AGU (13.2) AGC (25.9)
Threonine 5.68 ACU (22.4) ACC (40.5)
ACA (25.4) ACG ( 11.8)
Tryptophan 1.38 UGG ( IOO)
Tyrosine 3.13 UAU (40.0) UAC (60.0)
Valine 6.35 GUU (16.4) GUC (25.7)
GUA (9.3) GUG (48.7)
[0154] The polypeptide-encoding nucleic acid molecules can be propagated by placing a nucleotide sequence that encodes a hyperglycosylated polypeptide variant of a parent polypeptide in a recombinant propagation vector. Various viral and non-viral vectors, including plasmids, bacteriophages (for example, lambda, PI, Ml 3, etc.), cosmids, fosmids, PI -derived artificial chromosomes (PAC), bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), animal viruses, plant virus, or Human Artificial Chromosomes (HAC) may be used as propagation vectors. The choice of vectors will depend on the type of cell in which propagation is desired and the purpose of propagation, and is within the knowledge of one skilled in the art. Propagation vectors that are useful for amplifying and making large amounts of the desired DNA sequence can be used.
Expression Cassette
[0155] In some embodiments, an expression cassette for expressing a hyperglycosylated polypeptide variant can include a promoter operably linked to a nucleotide sequence encoding a signal peptide, a first extension sequence under the control of the promoter encoding a first peptide extension, and a restriction site allowing for insertion of a gene encoding a biologically-active polypeptide, where the first peptide extension can be a peptide of 1-200 consecutive amino acids and include at least two glycosylation sites. In some embodiments, upon insertion of a gene encoding a biologically-active polypeptide at the restriction site, the expression cassette directs expression of a fusion protein comprosiing the biologically-active polypeptide linked to the first peptide extension. In some embodiments, the restriction site can be either immediately upstream or downstream of the first extension sequence encoding the first peptide extension. In some embodiments, the first peptide extension can be a peptide of 1-180, 1-150, 10-140, 20-130, 30-120, 40-110, 50-100, 60-90, 70-80 consecutive amino acids. In some embodiments, the first peptide extension can include at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty glycosylation sites.
[0156] In some embodiments, the expression cassette can further include a gene encoding a biologically-active polypeptide inserted at the restriction site. In some embodiments, the first extension sequence is located between the gene encoding the biologically-active polypeptide and the nucleotide sequence encoding the signal peptide. In some embodiments, the gene encoding the biologically-active polypeptide is located between the nucleotide sequence encoding the signal peptide and the first extension sequence.
[0157] In some embodiments, the expression cassette can further include a second extension sequence encoding a second peptide extension. In some embodiments, the gene encoding the biologically-active polypeptide is located between the first extension sequence and the second extension sequence. In some embodiments, the second peptide extension can include at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty glycosylation sites. The expression cassette can be in the form of a linear or circular DNA, for example, and may be in the form of a plasmid.
[0158] In some embodiments, the expression cassette can further include a second extension sequence encoding a second peptide extension. In some embodiments, the gene encoding the parent polypeptide is located between the first extension sequence and the second extension sequence. In some embodiments, the second peptide extension can include at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites.
[0159] In Figure 6, schematic diagrams of some embodiments of the expression cassette for expressing a hyperglycosylated polypeptide are shown. Figure 6A shows an expression cassette comprising a promoter operably linked with a nucleotide encoding a signal peptide, a first extension sequence under control of the promoter encoding a first peptide extension, and a gene encoding a biologically-active polypeptide inserted at the restriction site that is downstream of the first extension sequence. Figure 6B shows an expression cassette comprising a promoter operably linked with a nucleotide encoding a signal peptide, a first extension sequence under control of the promoter encoding a first peptide extension, a second extension sequence encoding a second peptide extension, and a gene encoding a biologically-active polypeptide inserted at the restriction site that is downstream of the first extension sequence and upstream of the second extension sequence. Figure 6C shows an expression cassette comprising a promoter operably linked with a nucleotide encoding a signal peptide, a first extension sequence under control of the promoter encoding a first peptide extension, and a gene encoding a biologically-active polypeptide inserted at the restriction site that is upstream of the first extension sequence.
[0160] Various promoters can be used to drive expression of the hyperglycosylated variants of a parent polypeptide in the expression cassettes disclosed herein. Examples of promoters, include, but are not limited to, viral promoters, plant promoters and mammalian promoters. Examples of viral promoters include, but are not limited to Cytomegalovirus (CMV) promoter, Simian virus 40 (SV40) promoter, the 35S RNA and 19S RNA promoters of cauliflower mosaic virs (CaMV) described in Brisson et al., Nature 1984, 310:511-514, and the coat protein promoter to tobacco mosaic virus (TMV) described in Takamatsu et al., EMBO J. 1987, 6:307-311. Examples of plant promoters include, but are not limited to, heat shock promoters, such as soybean hspl7.5-E or hspl7.3- B described in Gurley et al., Mol. Cell. Biol. 1986, 6:559-565. Examples of mammalian promoters include, but are not limited to, human elongation factor loc-subunit (EFl-l oc) promoter, human ubiquitin C (Ubc) promoter, and murine phosphoglycerate kinase- 1 (PGK) promoter.
[0161] Various signal peptides can be used in the expression cassettes and/or expression vectors disclosed herein. Examples of signal peptides include, but are not limited to, the endogenous signal peptide for interferons, including the signal peptide of type I, II and III interferons; and the endogenous signal pepide for any known cytokine, such as the signal peptide of erythropoietin (EPO), TGF-βΙ, TNF, ILl-oc, and ILl-β. Nucleotide sequences of the non-limiting examples of signal peptides are given in SEQ ID NOs: 162-169. For example, SEQ ID NO: 162 shows the sequence of signal peptide of human insulin, SEQ ID NO: 163 shows the sequence of an artificial signal peptide, SEQ ID NO: 164 shows the sequence of signal peptide of human CD33, SEQ ID NO: 165 shows the sequence of signal peptide of human EPO, SEQ ID NO: 166 shows the sequence of signal peptide of human G- CSF, SEQ ID NO: 167 shows the sequence of signal peptide of human growth hormone 1, SEQ ID NO: 168 shows the sequence of signal peptide of human interferon beta, and SEQ ID NO: 169 shows the sequence of signal peptide of human insulin like growth factor 1A.
[0162] In some embodiments, an artificial signal peptide can be used in an expression cassette and/or expression vector to facilitate the secretion of the biologically active polypeptide expressed from the expression cassette and/or expression vector. In some embodiments, the signal polypeptide for a protein that is different from the biologically active polypeptide can be used in an expression cassette and/or expression vector to facilitate the secretion of the biologically active polypeptide expressed from the expression cassette and/or expression vector. In some embodiments, the native signal polypeptide for a biologically active polypeptide can be used in an expression cassette and/or expression vector to facilitate the secretion of the biologically active polypeptide. In some instances, a biologically active polypeptide can utilize an artificial signal peptide or a signal peptide from another molecule and get effectively secreted out of the host cells. In some instances, a biologically active polypeptide prefers its own native signal peptide for efficient secretion out of the host cells. In some embodiments, the biologically active polypeptide can be human growth hormone, interferon lambda 1, interferon lambda 2, or interferon lambda 3.
[0163] Expression cassettes disclosed herein can include a transcription initiation region and/or a transcriptional termination region. Examples of transcription termination region include, but are not limited to, the Bovine growth hormone (BGH) polyA, SV40 polyA, and thymidine kinase (TK) polyA sites. After introduction of the expression cassette and/or expression vector, the cells containing the expression cassette and/or vector may be selected by means of a selectable marker. Examples of selectable markers include, but are not limited to, antibiotic selection markers such as Neomycine resistance gene, kanamycin resistance gene, gentamycin resistance gene, and Zeocin resistance gene. Expression cassettes may be introduced into a variety of vectors suitable for eukaryotic host cell expression, such as plasmid; HAC; YAC; vectors derived from animal viruses, such as Moloney's murine leukemia virus, SV40, vaccinia virus, baculovirus, retroviruses, and plant viruses; and the like.
[0164] In some embodiments, an expression vector comprising an expression cassette disclosed herein can be used to producing the hyperglycosylated polypeptide variants of a parent polypeptide in a host cell. An expression vector comprising the expression cassette can be introduced into a host cell, particularly a eukaryotic cell that is capable of glycosylating proteins. A non-limiting example of methods for producing a hyperglycosylated polypeptide variant of a parent polypeptide can include culturing a eukaryotic host cell, where the host cell comprises a subject recombinant expression vector, under conditions that favor production of the hyperglycosylated polypeptide variant; and isolating the hyperglycosylated polypeptide variant from the culture. In some embodiments, the hyperglycosylated polypeptide variant can be isolated and purified to greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%, purity.
[0165] In some embodiments, the expression vectors disclosed herein can include additional sequences that make the vectors suitable for replication and integration in eukaryotes. In other embodiments, the expression vectors disclosed herein can include a shuttle element that make the vectors suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, the expression vectors can include transcription and translation initiation sequences, such as promoters and enhances; and transcription and translation terminators, such as polyadenylation signals.
[0166] In some embodiments, a prokaryotic expression system, such as a bacterial expression system, can be used to express the hyperglycosylated variants of a parent polypeptide as disclosed herein. For example, an expression cassette disclosed herein can be inserted into a bacterial expression vector. Examples of bacterial expression vectors include, but are not limited to, the pET series of E. coli expression vectors (see Studier et al., Methods in Enzymol. 185:60-89, 1990).
[0167] In some embodiments, a eukaryotic expression system can be used to express the hyperglycosylated variants of a parent polypeptide as disclosed herein. Examples of eukaryotic expression systems include, but are not limited to, yeast expression systems, mammalian expression systems, insect expression systems and plant expression systems.
[0168] In some embodiments, an expression cassette disclosed herein can be inserted into a yeast expression vector. For example, yeast expression vectors containing constitutive or inducible promoters can be used as disclosed in U.S. Pat. No. 5,932,447. As another example, yeast expression vectors which promote integration of foreign DNA sequences into the yeast chromosome can be used. In other embodiments, an expression cassette disclosed herein can be inserted into a mammalian expression vector. Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+), pGL3, pZeoSV2(+), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen (Carlsbad, CA); pCI which is available from Promega (Madison, WI); pMbac, pPbac, pBK-RSV and pBK-CMV, which are available from Agilent Technologies (La Jolla, CA); pTRES which is available from Clontech (Mountain View, CA); and their derivatives. In still other embodiments, an expression cassette disclosed herein can be inserted into a plant expression vector. In yet other embodiments, an expression cassette disclosed herein can be inserted into an insect expression vector. [0169] In some embodiment, the expression vectors disclosed herein can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
[0170] In some embodiments, an expression cassette disclosed herein can be inserted into a viral vector, such as an expression vectors comprising regulatory elements from eukaryotic viruses, such as retroviruses. Examples of expression vectors derived from eukaryotic virus include, but are not limited to, vectors derived from Simian virus 40 (SV40), for example pSVT7 and pMT2; vectors derived from bovine papilloma virus (BPV), for example pBV-lMTHA; and vectors derived from Epstein Bar virus (EBV), for example pHEBO and p205. Additional exemplary expression vectors that are derived from eukaryotic virus include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vectors allowing expression of proteins under the direction of SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters that are effective for protein expression in eukaryotic cells.
[0171] Introduction of expression vectors into a host cell may use any convenient method, such as calcium-precipitated DNA, electroporation, fusion, stable or transient transfection, lipofection, infection with viral vectors, biolistics, etc. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986].
[0172] The recombinant expression vectors can include expression cassettes and/or regulatory sequences ("control sequences" or "control regions") that can effect the expression of a desired polynucleotide to which they are operably linked. Various regulatory sequences can be used, including, but not limited to, promoter sequences and enhancer sequences. The expression vectors can also have restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding a desired protein or other protein.
[0173] In some embodiments, following protein expression and/or secretion, the signal peptides can be cleaved from the precursor proteins resulting in mature proteins.
[0174] As discussed previously, a hyperglycosylated polypeptide variant of a parent polypeptide can be synthesized in an expression host cell. Various expression host cells, for example, prokarytotic and eukaryotic cells, can be used to express the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein. An example of a suitable expression host cell is a eukaryotic cell. Examples of a eukaryotic cell include, but are not limited to; a yeast cell, such as a cell from S. cerevisiae; an insect cell in combination with baculovirus vectors; a plan cell in combination with recombinant virus expression vectors (for example, cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or recombinant plasmid expression vectors, such as Ti plasmid; a mammalian cell, such as COS 7 cell, CHO cell, and HEK293 cell; and the like. When a gene is expressed in eukaryotic cells, the protein product may include post-translational modification. In some embodiments, microorganisms, such as bacteria, can be transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding a hyperglycosylated polypeptide variant of a parent polypeptide.
[0175] In some embodiments, host cells containing expression vectors encoding the hyperglycosylated polypeptide variants of a parent polypeptide can be cultured under effective conditions, which allows for the expression of high amounts of the hyperglycosylated polypeptide variants. Factors that can impact effective culture conditions include, but are not limited to, media, bioreactor, temperature, pH and oxygen content. In some embodiments, an effective medium refers to any medium in which a cell is cultured to produce the the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein. In some embodiments, a medium can include an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, host cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, micro titer dishes and petri plates. Culturing conditions can be adjusted based on expertise of one of ordinary skill in the art. [0176] The hyperglycosylated polypeptide variant of a parent polypeptide may be isolated and purified in accordance with conventional methods known to those skilled in the art. For example, a lysate may be prepared of the expression host cells and the lysate may be purified using HPLC, hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, ultrafiltration, gel electrophoresis, affinity chromatography, and/or other purification techniques.
[0177] Any known assay can be used to determine whether a glycosylated polypeptide, for example, a hyperglycosylated polypeptide variant of a parent polypeptide, exhibits at least one desired pharmacologic activity of the parent polypeptide. Examples of useful assays for particular parent polypeptides include, but are not limited to, Testa et al., Assays for hematopoietic growth factors. In: Balkwill F R (edition) Cytokines A practical Approach, pages 229-244; IRL Press Oxford 11021); Kitamura et al., J. Cell. Physiol. 1989, 140:323; Hirudin, Blood Coagul Fibrinolysis, 1996, 7(2):259-261 ; Rubinstein et al., J. Virol., 1981, 37(2):755-758; Gao et al., Mol Cell Biol., 1999, 19(11):7305-7313; Czarniecki et al., J. Virol., 1984, 49:490; Shirafuji et al., Exp. Hematol., 1989, 17: 116; Weinstein et al, Proc. Natl. Acad. Sci. (1986) 83:5010-5014; Steppan et al., Nature, 2001, 409(6818):307-312; Moura et al., J Clin Endocrinol Metab, 2000, 85(l l):4274-4279; Bristow, Horm Res, 1999, 51 Suppl 1:7-12; Van Wijk et al., Thromb. Res., 1981, 22:681-686; Belaaouaj et al., J. Biol. Chem., 2000, 275:27123-27128; Diaz-Collier et al., Thromb. Haemost., 1994, 71:339-346. Pharmaceutical Compositions
[0178] Some embodiments disclosed herein relate to compositions, including pharmaceutical compositions, which can include a therapeutically effective amount of one or more hyperglycosylated polypeptide variant of a parent polypeptide disclosed herein. In some embodiments, the compositions can include one or more hyperglycosylated polypeptide variants of a parent polypeptide described herein and a pharmaceutically acceptable excipient and/or carrier.
[0179] The terms "physiologically acceptable" and "pharmaceutically acceptable" refer to a carrier, diluent or excipient that does not abrogate the biological activity and properties of the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein.
[0180] As used herein, a "carrier" refers to a compound that facilitates the incorporation of a compound, such as a hyperglycosylated polypeptide variant of a parent polypeptide, into cells or tissues. For example, without limitation, dimethyl sulfoxide (DMSO) is a commonly utilized carrier that facilitates the uptake of many organic compounds into cells or tissues of a subject.
[0181] As used herein, a "diluent" refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.
[0182] As used herein, an "excipient" refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition. For example, a "diluent" is a type of excipient.
[0183] As used herein, the term "therapeutically effective amount" refers to an amount of an active compound, or pharmaceutical agent, that elicits the biological or medicinal response indicated. For example, a therapeutically effective amount of a hyperglycosylated polypeptide variant of a parent polypeptide can be the amount need to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. The biological or medicinal response may occur in a tissue, system, animal or human, and includes alleviation of the symptoms of the disease being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art in light of the detailed disclosure provided herein. The therapeutically effective amount of the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein required will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The therapeutically effective amount can also depend on factors as weight, diet, concurrent medication; and other factors which those skilled in the medical arts will recognize.
[0184] The pharmaceutical compositions disclosed herein may be manufactured in a manner that is itself known, such as by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes. For example, the pharmaceutical compositions can be obtained by reacting the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutical compositions will generally be tailored to the specific intended route of administration. Additionally, the active ingredients are contained in an amount therapeutically effective to achieve its intended purpose.
[0185] The compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharmaceutically compatible counterions. Suitable additional components of a pharmaceutical composition include, but are not limited to, salts, buffers, solubilizers, stabilizers, detergents, protease-inhibiting agents, and the like.
[0186] Suitable routes of administration may, for example, include oral, rectal, topical transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, intraocular injections or as an aerosol inhalant.
[0187] In some embodiments, one or more hyperglycosylated polypeptide variants of a parent polypeptide are formulated into a preparation suitable for oral administration. For oral preparations, the hyperglycosylated polypeptide variant can be formulated alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives, and/or flavoring agents. [0188] In other embodiments, one or more hyperglycosylated polypeptide variants of a parent polypeptide are formulated into a preparation suitable for injection. For preparations suitable for injection, the hyperglycosylated polypeptide variant(s) of a parent polypeptide can be by dissolved, suspended or emulsified in an aqueous solvent (for example, saline, and the like) or a nonaqueous solvent (for example, vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol); and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
[0189] The pharmaceutical compositions described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or carriers, diluents, excipients or combinations thereof. Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the hyperglycosylated polypeptide variants of a parent polypeptide described herein are known to those skilled in the art.
[0190] One may also administer the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein in a local rather than systemic manner, for example, via injection of the hyperglycosylated polypeptide variants of a parent polypeptide directly into the infected area, often in a depot or sustained release formulation. Furthermore, one may administer the hyperglycosylated polypeptide variants of a parent polypeptide in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposomes can be targeted to and taken up selectively by the organ.
[0191] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions that include a hyperglycosylated polypeptide variant of a parent polypeptide disclosed herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
Methods of Use
[0192] Some embodiments disclosed herein relate to a method of treating and/or ameliorating a disease or condition that can include administering to a subject a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein.
[0193] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and/or therapy. "Treatment," as used herein, covers any treatment of a disease in a subject, for example, in a human. "Treatment," as used herein, includes, but is not limited to: (a) increasing survival time; (b) decreasing the risk of death due to the disease; (c) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (d) inhibiting the disease, that is, arresting its development (for example, reducing the rate of disease progression); and (e) relieving the disease, that is, causing regression of the disease. Furthermore, treatment may include acts that may worsen the patient's overall feeling of well- being or appearance.
[0194] As used herein, a "subject" refers to an animal that is the object of treatment, observation or experiment. "Animal" includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles, and in particular, mammals. "Mammal" includes, without limitation, mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates; such as monkeys, chimpanzees and apes, and, in particular, humans. [0195] Some embodiments disclosed herein relate to a method of ameliorating and/or treating fibrotic disorders that can include administering to a subject suffering from fibrotic disorders a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein. In some embodiments, the method can further include administering one or more additional anti-fibrotic agents. Additional anti-fibrotic agents include, but are not limited to, SAPK inhibitors (such as pirfenidone or pirfenidone analogs), TNF antagonists (such as etanercept, infliximab, or adalimumab), TGF-β antagonists (such as GLEEVEC), endothelin receptor antagonists (such as TRACLEER), and the like.
[0196] Fibrosis is generally characterized by the pathologic or excessive accumulation of collagenous connective tissue. In some embodiments, the fibrotic disorders can be those diseases or conditions affecting the lung such as idiopathic pulmonary fibrosis, pulmonary fibrosis from a known etiology, liver fibrosis or cirrhosis, cardiac fibrosis, and renal fibrosis. Additional fibrotic disorders include, but are not limited to, collagen disease, interstitial lung disease, human fibrotic lung disease (such as obliterative bronchiolitis, idiopathic pulmonary fibrosis, pulmonary fibrosis from a known etiology, tumor stroma in lung disease, systemic sclerosis affecting the lungs, Hermansky-Pudlak syndrome, coal worker's pneumoconiosis, asbestosis, silicosis, chronic pulmonary hypertension, AIDS- associated pulmonary hypertension, sarcoidosis, and the like), fibrotic vascular disease, arterial sclerosis, atherosclerosis, varicose veins, coronary infarcts, cerebral infarcts, myocardial fibrosis, musculoskeletal fibrosis, post-surgical adhesions, human kidney disease (such as nephritic syndrome, Alport's syndrome, HIV-associated nephropathy, polycystic kidney disease, Fabry's disease, diabetic nephropathy, chronic glomerulonephritis, nephritis associated with systemic lupus, and the like), cutis keloid formation, progressive systemic sclerosis (PSS), primary sclerosing cholangitis (PSC), liver fibrosis, liver cirrhosis, renal fibrosis, pulmonary fibrosis, cystic fibrosis, chronic graft versus host disease, scleroderma (local and systemic), Grave's opthalmopathy, diabetic retinopathy, glaucoma, Peyronie's disease, penis fibrosis, urethrostenosis after the test using a cystoscope, inner accretion after surgery, scarring, myelofibrosis, idiopathic retroperitoneal fibrosis, peritoneal fibrosis from a known etiology, drug-induced ergotism, fibrosis incident to benign or malignant cancer, fibrosis incident to microbial infection (such as viral, bacterial, parasitic and fungal infection), Alzheimer's disease, fibrosis incident to inflammatory bowel disease (including stricture formation in Crohn's disease and microscopic colitis), fibrosis induced by chemical or environmental insult (such as cancer chemotherapy, pesticides, radiation, and the like), and the like.
[0197] Some embodiments disclosed herein relate to a method of ameliorating and/or treating cancer that can include administering to a subject suffering from cancer a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein. In some embodiments, the method can further include administering one or more additional anti-cancer agents. Examples of additional anti-cancer agents include, but are not limited to, chemotherapeutic agents, radiation agents, bone marrow, biological response modifier, agents that act to reduce cellular proliferation, antimetabolite agents, microtubule affecting agents, hormone modulators, antibodies, and anti-angiogenic agents.
[0198] In some embodiments, the cancer can be a carcinoma. In other embodiments, the cancer can be a sarcoma. In still other embodiments, the cancer can be a tumor, such as a solid tumor. In yet other embodiments, the cancer can be leukemia. In yet still other embodiments, the cancer can be lymphoma.
[0199] Examples of carcinomas include, but are not limited to, esophageal carcinoma; hepatocellular carcinoma; basal cell carcinoma, squamous cell carcinoma (various tissues) ; bladder carcinoma, including transitional cell carcinoma; bronchogenic carcinoma; colon carcinoma; colorectal carcinoma; gastric carcinoma; lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung; adrenocortical carcinoma; thyroid carcinoma; pancreatic carcinoma; breast carcinoma; ovarian carcinoma; prostate carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinoma; cystadenocarcinoma; medullary carcinoma; renal cell carcinoma; ductal carcinoma in situ or bile duct carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; Wilm's tumor; cervical carcinoma; uterine carcinoma; testicular carcinoma; osteogenic carcinoma; epithelieal carcinoma; nasopharyngeal carcinoma; etc.
[0200] Examples of sarcomas include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.
[0201] Examples of solid tumors include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
[0202] Examples of leukemias include, but are not limited to, chronic myeloproliferative syndromes; acute myelogenous leukemias; chronic lymphocytic leukemias, including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and acute lymphoblastic leukemias.
[0203] Examples of lymphomas include, but are not limited to, B-cell lymphomas, such as Burkitt's lymphoma; Hodgkin's lymphoma; and the like.
[0204] Some embodiments disclosed herein relate to a method of inhibiting the growth of a tumor that can include administering to a subject having the tumor a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein.
[0205] Some embodiments disclosed herein relate to a method of ameliorating and/or treating viral infection that can include administering to a subject suffering from viral infection a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein. In some embodiments, the method can further include administering one or more additional anti-viral agents. Examples of anti-viral agents include, but are not limited to nucleoside analogs (for example, ribavirin, viramidine and levovirin) and HCV NS3 inhibitors.
[0206] In some embodiments, the viral infection can be caused by a virus selected from an adenovirus, an Alphaviridae, an Arbovirus, an Astrovirus, a Bunyaviridae, a Coronaviridae, a Filoviridae, a Flaviviridae, a Hepadnaviridae, a Herpesviridae, an Alphaherpesvirinae, a Betaherpesvirinae, a Gammaherpesvirinae, a Norwalk Virus, an Astroviridae, a Caliciviridae, an Orthomyxoviridae, a Paramyxoviridae, a Paramyxoviruses, a Rubulavirus, a Morbilli virus, a Papovaviridae, a Parvoviridae, a Picornaviridae, an Aphthoviridae, a Cardioviridae, an Enteroviridae, a Coxsackie virus, a Polio Virus, a Rhinoviridae, a Phycodnaviridae, a Poxviridae, a Reoviridae, a Rotavirus, a Retroviridae, an A-Type Retrovirus, an Immunodeficiency Virus, a Leukemia Viruses, an Avian Sarcoma Viruses, a Rhabdoviruses, a Rubiviridae and/or a Togaviridae. In some embodiments, the viral infection can be a hepatitis C viral infection. In other embodiments, the viral infection can be an HIV infection.
[0207] Some embodiments provides a method of reducing the risk of viral infection for a subject who has been exposed to a virus (for example, a subject who has come into contact with another subject infected with a virus). The method can include administering to the subject who has been exposed to a virus a therapeutically effective amount of one or more hyperglycosylated polypeptides variants of a parent polypeptide described herein, or a pharmaceutical composition that includes one or more hyperglycosylated polypeptide variants of a parent polypeptide described herein. In some embodiments, the method can further include administering one or more additional anti-viral agents.
[0208] In some embodiments, a hyperglycosylated polypeptide variant of a parent polypeptide can be exhibit one or more of the following activities: antiproliferative activity, anti-viral activity, and anti-fibrotic activity. Whether a hyperglycosylated polypeptide variant of a parent polypeptide exhibits anti-viral activity can be determined using any known assay, including for example, an in vitro cell-based inhibition of viral replication assay described in Patick et al. Antimicrob. Agents Chemother., 1999, 43:2444-2450. Whether a hyperglycosylated polypeptide variant of a parent polypeptide exhibits anti-proliferative activity can be determined using any known assay, including for example, an in vitro cell- based inhibition of proliferation assay.
[0209] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, and animal species treated, the particular hyperglycosylated polypeptide variant of a parent polypeptide employed, and the specific use for which these hyperglycosylated polypeptide variants of a parent polypeptide are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.
[0210] Although the exact dosage will be determined on a drug-by-drug basis, in most cases, some generalizations regarding the dosage can be made. In some embodiments, the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein may be administered orally or via injection at a dose of between 0.01 mg and 3000 mg of each ingredient, preferably between 1 mg and 700 mg, for example, 5 to 200 mg. In some embodiments, the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein can be administered orally or via injection at a dose of between about 0.001 mg and about 30 mg of each ingredient, or between about 0.001 mg and about 25 mg, between about 0.001 mg and about 20 mg, between about 0.001 mg and about 15 mg, between about 0.001 mg and about 10 mg, between about 0.005 mg and about 5 mg, between about 0.005 mg and about 4 mg, between about 0.005 mg and about 3 mg, between about 0.005 mg and about 2 mg, between about 0.005 mg and about 1 mg, preferably between 0.01 mg and 1 mg, for example, between about 0.05 to about 0.5 mg, between about 0.01 to about 0.3 mg, or between about 0.01 to about 0.1 mg.
[0211] In instances where human dosages for the hyperglycosylated polypeptide variants of a parent polypeptide have been established for at least some condition, those same dosages, or dosages that are between about 0.1% and 500%, more preferably between about 25% and 250% of the established human dosage can be used. Where no human dosage is established, as will be the case for newly-discovered pharmaceutical compositions, a suitable human dosage can be inferred from ED50 or ID50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
[0212] In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free base. As will be understood by those of skill in the art, in certain situations it may be necessary to administer the hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein in amounts that exceed, or even far exceed, the above-stated, preferred dosage range in order to effectively and aggressively treat particularly aggressive diseases or infections.
[0213] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each hyperglycosylated polypeptide variant of a parent polypeptide, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
[0214] Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
[0215] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and/or dose frequency may also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
[0216] In some embodiments, the hyperglycosylated polypeptide variants of a parent polypeptide can be administered less frequently at substantially the same amount as compared to the parent polypeptide to achieve substantially similar or the same therapeutic results. In an embodiment, the hyperglycosylated polypeptide variants of a parent polypeptide can be administered orally, once every three months, once every two months, once every month, twice per month, three times per month, once every week, once every five days, once every three days, once every two days, once per day, twice per day, or three times per day substantially continuously or continuously, for the desired duration of treatment. In another embodiment, the hyperglycosylated polypeptide variants of a parent polypeptide can be administered via injection, once every three months, once every two months, once every month, twice per month, three times per month, once every week, once every five days, once every three days, once every two days, once per day, twice per day, or three times per day substantially continuously or continuously, for the desired duration of treatment.
[0217] A therapeutic amount of the hyperglycosylated polypeptide variants of a parent polypeptide can be administered to a subject at various points of time for a substantial continuous or continuous period of time, for example, for a week or more, or for a month or more, or for a year or more. In some embodiments, the hyperglycosylated polypeptide variants of a parent polypeptide is administered for a period of time, which time period can be, for example, at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 54 weeks, at least about 58 weeks, at least about 62 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 74 weeks, at least about 78 weeks, at least about 82 weeks, at least about 86 weeks, at least about 90 weeks, at least about 94 weeks, at least about 98 weeks, at least about 102 weeks, at least about 106 weeks, or longer. For example, the hyperglycosylated polypeptide variants of a parent polypeptide can be administered once every two weeks for about six months, once every month for about a year, or twice every month for about two years.
[0218] In other embodiments, the hyperglycosylated polypeptide variants of a parent polypeptide can be administered at a dosing interval of about every 3 days to about every 7 days, about every 5 days to every 9 days, about every 7 days to every 11 days, about every 9 days to every 13 days, about every 11 days to every 15 days, about every 13 days to every 17 days, about every 15 days to every 19 days, about every 17 days to every 21 days, about every 19 days to every 23 days, about every 21 days to every 25 days, about every 23 days to every 27 days, about every 25 days to every 29 days, about every 27 days to every 31 days, about every 29 days to every 33 days, about every 31 days to every 35 days, about every 33 days to every 37 days, about every 35 days to every 39 days, about every 37 days to every 41 days, about every 39 days to every 43 days, about every 41 days to every 45 days, about every 43 days to every 47 days, or about every 45 days to every 49 days.
[0219] In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear. The dosage may range broadly, depending upon the desired effects and the therapeutic indication. Alternatively, dosages may be based and calculated upon the surface area of the subject, as understood by those of skill in the art.
[0220] Hyperglycosylated polypeptide variants of a parent polypeptide disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular hyperglycosylated polypeptide variant of a parent polypeptide, or of a subset of the hyperglycosylated polypeptide variants of a parent polypeptide, sharing certain chemical moieties, may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular hyperglycosylated polypeptide variants of a parent polypeptide in an animal model, such as mice, rats, rabbits, or monkeys, may be determined using known methods. The efficacy of a particular hyperglycosylated polypeptide variant of a parent polypeptide may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. Recognized in vitro models exist for nearly every class of condition, including but not limited to cancer, cardiovascular disease, and various immune dysfunction. Similarly, acceptable animal models may be used to establish efficacy of chemicals to treat such conditions. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, and regime. Human clinical trials can also be used to determine the efficacy of a hyperglycosylated polypeptide variant of a parent polypeptide in humans.
[0221] In certain embodiments, the expression vector or expression cassette, containing sequence encoding a biologically-active peptide, may be expressed in a mammal. The expression vector or cassette may advantageously be introduced in various ways to secure expression thereof, such as transfection of the mammal with a viral vector containing the expression vector or cassette; introduction of the expression vector or cassette through naked DNA injection; introduction through electroporation; ex vivo transfection of cells that are then reintroduced into the mammal; and the like. In one preferred embodiment, the expression vector or cassette is under the control of an inducible promoter. In this way, when the mammal is in need of therapy using the biologically-active peptide, systemic or localized delivery of the inducer can effect transcription and translation of the relevant DNA and result in delivery of therapeutic polypeptide to the mammal.
EXAMPLES
[0222] Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.
Example 1
Construction of hyperglycosylated peptide variants of a parent polypeptide
[0223] This example illustrates the construction of hyperglycosylated variants of a parent polypeptide with various amino-terminal and/or carboxy-terminal peptide extensions. The parent polypeptide can be human growth hormone 1 (hGH-1), human insulin-like growth factor 1A (hIGF-ΙΑ), human granulocyte colony-stimulating factor (hG-CSF), human erythropoietin (hEPO), human insulin, an Fab fragment of an antibody specific for human TNF-oc receptor, and an scFv fragment of an antibody specific for human TNF-oc receptor. [0224] DNA constructs encoding hyperglycosylated variants of hGH-1 that have peptide extensions with additional glycosylation sites at an amino-terminal and/or a carboxy- terminal region are generated by site-directed mutagenesis using standard DNA techniques known in the art. A DNA sequence encoding a peptide extension is inserted immediately upstream of the amino-terminus of the parent hGH-1 excluding any signal peptide and/or downstream of carboxy-terminus the parent hGH-1. The sequences of the non-limiting examples of hyperglycosylated variants of hGH-1 are given in SEQ ID NOs: 2-7. Figures 7A-I show the structure and sequence of the expression cassette in expression vectors pC6-l- hGHl, pN6-l-hGHl, pN6-2-hGHl, pNlO-l-hGHl, PN10-C4-l-hGHl, PN10-C6-l-hGHl, pN14-l-hGHl, pN14(2)-l-hGHl, and pN14(3)-l-hGHl. The sequences of expression vectors pC6-l-hGHl, pN6-l-hGHl, pN6-2-hGHl, pNlO-l-hGHl, pN10-C4-l-hGHl, pNlO- C6-l-hGHl, PN14-l-hGHl, pN14(2)-l-hGHl, and pN14(3)-l-hGHl are given in SEQ ID NOs: 170-178.
[0225] For expression of the hyperglycosylated variants of hGH-1, HEK293 cells are tranfected with the DNA constructs encoding hyperglycosylated variants of hGH-1. Transient expression, isolation of stable expression clones, and large-scale production of hyperglycosylated variants of hGH-1 proteins are performed using standard protein expression techniques known in the art.
[0226] Similarly, hyperglycosylated variants of the parent polypeptides hIGF-lA, hG-CSF, hEPO, human insulin, Fab fragments of an antibody specific for human HER-2 receptor, Fab fragments of an antibody specific for human TNF-oc, Fab fragments of an antibody specific for human VEGF-A, human TNF-oc soluble receptors Rl and R2, fragments of human VEGF soluble receptor Rl (human VEGF Rl-III, Rl-IV, Rl-V, Rl-VI, and Rl- VII), and fragments of human VEGF soluble receptor R2 (human VEGF R2-VI and R2-VII) are generated. Sequences of the non-limiting examples of hyperglycosylated variants of hIGF-ΙΑ are given in SEQ ID NOs: 9-14. Figure 8 shows the structure and sequence of the expression cassette in expression vector pNlO-1-hIGFlA. The sequence of pNlO-1-hIGFlA is given in SEQ ID NO: 179. Sequences of the non-limiting examples of hyperglycosylated variants of hG-CSF are given in SEQ ID NO: 16-21. Figure 9 shows the structure and sequence of the expression cassette in expression vector pN 10-1 -hG-CSF. The sequence of pNlO-l-h G-CSF is given in SEQ ID NO: 180. Sequences of the non-limiting examples of hyperglycosylated variants of hEPO are given in SEQ ID NOs: 23-28. Figure 10 shows the structure and sequence of the expression cassette in expression vector pNlO-l-hEPO. The sequence of pNlO-l-hEPO is given in SEQ ID NO: 181. Sequences of the non-limiting examples of hyperglycosylated variants of human insulin are given in SEQ ID NOs: 30-35. Figure 11 shows the structure and sequence of the expression cassette in expression vector pN 10-1 -Insulin. The sequence of pN 10-1 -Insulin is given in SEQ ID NO: 182. Sequences of the non-limiting examples of hyperglycosylated variants of the heavy chain of an Fab fragment of an antibody specific for human HER-2 receptor are given in SEQ ID NOs: 37-42. Sequences of the non-limiting examples of hyperglycosylated variants of the light chain of an Fab fragment of an antibody specific for human HER-2 receptor are given in SEQ ID NOs: 44-49. Sequences of the non-limiting examples of hyperglycosylated variants of the heavy chain of an Fab fragment of an antibody specific for human TNF-oc are given in SEQ ID NOs: 51-56. Sequences of the non-limiting examples of hyperglycosylated variants of the light chain of an Fab fragment of an antibody specific for human TNF-oc are given in SEQ ID NOs: 58-63. Sequences of the non-limiting examples of hyperglycosylated variants of the heavy chain of an Fab fragment of an antibody specific for human VEGF are given in SEQ ID NOs: 65-70. Sequences of the non-limiting examples of hyperglycosylated variants of the light chain of an Fab fragment of an antibody specific for human VEGF are given in SEQ ID NOs: 72-77. Sequences of the non-limiting examples of hyperglycosylated variants of human TNF-oc soluble receptor Rl are given in SEQ ID NOs: 79-84. Sequences of the non-limiting examples of hyperglycosylated variants of human TNF-oc soluble receptor R2 are given in SEQ ID NOs: 86-91. Sequences of the non-limiting examples of hyperglycosylated variants of various fragments of human VEGF soluble receptor Rl are given in SEQ ID NOs: 93-98, 100-105, 107-112, 114-119, and 121-126. Sequences of the non-limiting examples of hyperglycosylated variants of various fragments of human VEGF soluble receptor R2 are given in SEQ ID NOs: 128-133, 135-140, 142-147, 148-154, and 156-161. Example 2
Expression Cassette for Expressing Hyperglycosylated Variants of
Human Growth Hormone 1 (hGHl)
[0227] Expression vector pC6-l-hGHl (Figure 7 A) that contains an expression cassette for expressing hyperglycosylated variants of hGHl was generated. In pC6-l-hGHl, a CMV promoter is operably linked with a nucleotide sequence encoding the signal peptide of IFN β and an extension sequence encoding a peptide extension that includes six glycosylation sites. In pC6-l-hGHl, a gene encoding hGHl is inserted between the nucleotide sequence encoding the signal peptide of IFN β and the extension sequence, and a bovine growth hormone (BGH) polyadenylation signal sequence is located downstream of the extension sequence.
[0228] Expression vector pN6-l-hGHl (Figure 7B) that contains an expression cassette for expressing hyperglycosylated variants of hGHl was also generated. In pN6-l- hGHl, a CMV promoter is operably linked with a nucleotide sequence encoding the signal peptide of IFN β and an extension sequence encoding a peptide extension that includes six glycosylation sites. In pN6-l-hGHl, the extension sequence is located immediately downstream of the signal peptide of IFN β and a gene encoding hGHl is inserted immediately downstream of the extension sequence.
[0229] Expression vector pN6-2-hGHl (Figure 7C) was generated similarly as expression vector pN6-l-hGHl except that an artificial signal peptide is used instead of the signal peptide of IFN β. Expression vector pN 10-1 -hGHl (Figure 7D) was generated similarly as expression vector pN6-l-hGHl except that the peptide extension in pNlO-1- hGHl includes ten glycosylation sites. Expression vectors pN14-l-hGHl (Figure 7G), pN 14(2)- 1 -hGHl (Figure 7H) and pN14(3)-l-hGHl (Figure 71) were also generated similarly as expression vector pN6-l-hGHl except that the peptide extensions in pN 14-1 -hGHl, pN 14(2)- 1 -hGHl and pN14(3)-l-hGHl include fourteen glycosylation sites.
[0230] Expression vector pN10-C4-l-hGHl (Figure 7E) that contains an expression cassette for expressing hyperglycosylated variants of hGHl was generated. In pN10-C4-l-hGHl, a CMV promoter is operably linked with a nucleotide sequence encoding the signal peptide of IFN β; a first extension sequence encoding a first peptide extension that includes ten glycosylation sites is located immediately downstream of the signal peptide of IFN β; and a second extension sequence encoding a second peptide extension includes four glycosylation sites. In pN10-C4-l-hGHl, a gene encoding hGHl is located between the first and second extension sequences.
[0231] Expression vector pN10-C6-l-hGHl (Figure 7F) is generated similarly as the expression vector pN10-C4-l-hGHl except that the second peptide extension in pNlO- C6-l-hGHl has six glycosylation sites.
[0232] The sequences of expression vectors pC6-l-hGHl, pN6-l-hGHl, pN6-2- hGHl, pNlO-l-hGHl, pN10-C4-l-hGHl, pN10-C6-l-hGHl, PN14-l-hGHl, pN14(2)-l- hGHl, and pN14(3)-l-hGHl are given in SEQ ID NOs: 170-178.
Example 3
Expression Cassettes for Expressing Hyperglycosylated Variants of
Human IGF-1A, G-CSF, EPO, and Insulin
[0233] Expression vector pNlO-1-hIGFlA (Figure 8) that contains an expression cassette for expressing a hyperglycosylated variant of human insulin-like growth factor 1A (hIGF-ΙΑ) was generated. In pNlO-1-hIGFlA, a CMV promoter is operably linked with a nucleotide sequence encoding the signal peptide of IFN β and an extension sequence encoding a peptide extension includes ten glycosylation sites. Also, in pNlO-1-hIGFlA, the extension sequence is located immediately downstream of the signal peptide of IFN β, a gene encoding hIGF-ΙΑ is inserted immediately downstream of the extension sequence, and a BGH polyadenylation signal sequence is located downstream of the extension sequence. The sequence of pNlO-1-hIGFlA is given in SEQ ID NO: 179.
[0234] Expression vector pNlO-l-hG-CSF (Figure 9) that contains an expression cassette for expressing a hyperglycosylated variant of human granulocyte colony-stimulating factor (hG-CSF) was generated similarly as pNlO-1-hIGFlA except that the gene encoding hG-CSF is inserted immediately downstream of the extension sequence. The sequence of pNlO-l-h G-CSF is given in SEQ ID NO: 180.
[0235] Expression vector pNlO-l-hEPO (Figure 10) that contains an expression cassette for expressing a hyperglycosylated variant of human Erythropoietin (hEPO) was generated similarly as pNlO-1-hIGFlA except that the gene encoding hEPO is inserted immediately downstream of the extension sequence. The sequence of pN 10-1 -hEPO is given in SEQ ID NO: 181.
[0236] Expression vector pN 10-1 -Insulin (Figure 11) that contains an expression cassette for expressing a hyperglycosylated variant of human insulin was generated as pNlO- 1-hIGFlA except that the gene encoding human insulin is inserted immediately downstream of the extension sequence. The sequence of pN 10-1 -Insulin is given in SEQ ID NO: 182.
Example 4
Construction of hyperglycosylated variants of interferon alfacon-1 (CIFN)
[0237] This example illustrates the construction of hyperglycosylated variants of interferon alfacon-1 (CIFN) with various amino-terminal peptide extension: (1) a hyperglycosylated variant of the parent CIFN with an N-terminal peptide extension VNITG and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-Nl-31-102-138"); (2) a hyperglycosylated variant of the parent CIFN with an N- terminal peptide extension (VNITG)2 and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-N2-31-102-138"); (3) a hyperglycosylated variant of the parent CIFN with an N-terminal peptide extension (VNITG)3 and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-N3-31-102-138"; and (4) a hyperglycosylated variant of the parent CIFN with an N-terminal peptide extension (VNITG)4 and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-N4-31-102-138"). Each VNITG motif contains an N-linked glycosylation site.
[0238] N-terminal or C-terminal peptide extensions can be generated through conventional techniques known in the art, such as polymerase chain reaction (PCR)-based mutagenesis. In some experiments, the shorter extension was generated first and the longer extension was generated based on the shorter extension in a step-wise fashion.
[0239] The primers used in the PCR reactions to generate DNA sequences encoding CIFN-Nl-31-102-138 (SEQ ID NO:329), CIFN-N2-31-102-138 (SEQ ID NO:330), CIFN-N3-31-102-138 (SEQ ID NO: 331), and CIFN-N4-31-102-138 (SEQ ID NO: 332) are listed in Table 2.
Table 2. Sequences of primers for generating of hyperglycosylated variants CIFN-N1- 31-102-138, CIFN-N2-31-102-138, CIFN-N3-31-102-138, and CIFN-N4-31-102-138
Figure imgf000079_0001
[0240] To generate DNA sequences encoding CIFN-Nl-31-102-138, a 2-step PCR was used. In step 1 of the PCR reaction, a 506bp fragment 1 was generated by using the gene encoding CIFN-31-102- 138 (which is a variant of the parent CIFN with additional glycosylation sites at amino acid positions 31, 102 and 138) as a template, and CIFN-N1-F and ECOR I-R as primers. A 95bp fragment 2 was generated by using the gene encoding CIFN-31-102-138 as a template, and CIFN-N1-R and HIND III-F as primers. In step 2 of the PCR reaction, fragments 1 and 2 were used as templates to generate a 601bp fragment 3 using ECOR I-R and HIND III-F as primers. Fragment 3 was digested by Hind III and EcoR I, and then cloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA).
[0241] DNA sequences encoding CIFN-Nl-31-102-138 were generated similarly in which a DNA sequence encoding CIFN-Nl-31-102-138 was used as a template, and CIFN-N2-F and CIFN-N2-R were used as primers in step 1 of the PCR reaction. To synthesize DNA sequences encoding CIFN-N3-31-102-138, a similar 2-step PCR was used in which a DNA sequence encoding CIFN-N2-31-102- 138 was used as a template, and CIFN- N3-F and CIFN-N3-R were used as primers in step 1 of the PCR reaction. To synthesize DNA sequences encoding CIFN-N4-31-102- 138, a similar 2-step PCR was used except that DNA sequence encoding CIFN-N3-31-102-138 was used as a template, and CIFN-N4-F and CIFN-N4-R were used as primers in step 1 of the PCR reaction.
[0242] pcDNA3.1 vectors containing DNA sequences encoding each hyperglycosylated variant of CIFN were transfected into mammalian cell lines, such as HEK293 cells, CHO cells and Cos-7 cells, for protein expression.
Example 5
Increased glycosylation and maintained biological activity in hyperglycosylated variants of interferon alfacon-1 (CIFN)
[0243] To analyze the effects of an N-terminal or a C-terminal peptide extension on the molecular weight of the parent interferon, DNA encoding various hyperglycosylated variants of CIFN with an N-terminal or a C-terminal peptide extension, such as those from Example 4, were synthesized and transfected into mammalian cell line CHO to express hyperglycosylated variants of CIFN protein. Five days after transfection, the media containing the secreted hyperglycosylated variants of CIFN proteins were collected and analyzed with protein gel electrophoresis and western blot analysis using the polyclonal antibodies targeting the parent CIFN.
1. N-terminal peptide extensions on a CIFN variant (CIFN-31-102-138) increased the molecular weight of the CIFN variant
[0244] Four hyperglycosylated variants of CIFN: CIFN-Nl-31-102-138, CIFN- N2-31-102-138, CIFN-N3-31-102-138, and CIFN-N4-31-102-138, were produced as described in Example 4. CIFN-31-102-138, which is a variant of the parent CIFN having a sequence identical to the four hyperglycosylated variants of the parent CIFN except for the absence of a N-terminal peptide extension, was used as a control in the western blot analysis shown in Figure 12A. As shown in Figure 12A, all 4 hyperglycosylated variants of the parent CIFN had an increased molecular weight compared to the control CIFN-31-102-138. Thus, N-terminal peptide extensions containing various numbers of the VNITG motif increased the molecular weight of the starting CIFN-31-102-138 interferon. [0245] Also shown in Figure 12A, CIFN-N4-31-102-138 which had 4 glycosylation sites in the N-terminal peptide extension had the highest molecular weight among the four hyperglycosylated variants of CIFN; CIFN-N3-31-102-138 which had 3 glycosylation sites in the N-terminal peptide extension had the second highest molecular weight among the four variants; CIFN-N2-31-102-138 which had 2 glycosylation sites in the N-terminal peptide extension had the third highest molecular weight among the four variants; and CIFN-Nl-31-102-138 which had 1 glycosylation site in the N-terminal peptide extension had the lowest molecular weight among the four variants. Therefore, adding a motif with one or more glycosylation sites, such as VNITG motif, increases the molecular weight of the starting interferon.
2. A C-terminal peptide extension on CIFN variant (CIFN-102-138) increased the molecular weight of CIFN
[0246] CIFN- 102-138-C2 (SEQ ID NO:333) is a hyperglycosylated variant of CIFN with a C-terminal peptide extension (VNITG)2 and additional glycosylation sites at amino acid positions 102 and 138. CIFN-102-138 is a variant of CIFN with additional glycosylation sites at amino acid positisions 102 and 138 of the CIFN.
[0247] A DNA sequence encoding CIFN- 102-138-C2 was generated by the PCR- based mutagenesis method as described in Example 4, and transfected into CHO cells for the expression of CIFN- 102-138-C2 protein. Western blot analysis of Figure 12B showed that CIFN- 102-138-C2 had an increased molecular weight compared to CIFN-102-138. Thus, the C-terminal peptide extension (VNITG)2 that contains 2 N-linked glycosylated sites increased the molecular weight of the starting CIFN-102-138 interferon.
3. Glycosylation of variants of CIFN havins a N-terminal peptide extension with 14 slycosylation sites, and l or 2 internal slycosylation sites
[0248] Four hyperglycosylated variants of CIFN having a N-terminal peptide extension with 14 glycosylation sites and 1 or 2 internal glycosylation sites were produced using the expression cassette system in vector pN14(3)-l-hGHl (Figure 71). Vector pN14(3)- 1-hGHl was double digested with restriction enzymes Notl and Swal to remove the DNA insert containing hGHl gene. The remaining portion of the vector was used as a cloning vector. DNA inserts were generated by PCR method using primers containing Notl or Swal site at the 5 ' end and templates containing the CIFN sequence or sequences of CIFN variants with internal glycosylation site(s). The DNA inserts were then double digested with NotI and Swal and cloned into the cloning vector. The four variants are: (1) CIFN-N 14(3)- 102 (SEQ ID NO: 334) having the N-terminal peptide extension with 14 glycosylation sites and an additional glysoylation site at amino acid position 102; (2) CIFN-N14(3)-102-138 (SEQ ID NO:336) having the N-terminal peptide extension with 14 glycosylation sites and two additional glysoylation sites at amino acid positions 102 and 138; (3) CIFN-N14(3)-108-138 (SEQ ID NO:337) having the N-terminal peptide extension with 14 glycosylation sites and two additional glysoylation sites at amino acid positions 108 and 138; and (4) CIFN-N14(3)- 108 (SEQ ID NO:335) having the N-terminal peptide extension with 14 glycosylation sites and an additional glysoylation site at amino acid position 108.
[0249] As shown in Figure 12C, all 4 hyperglycosylated variants of the parent CIFN had a significantly increased molecular weight compared to the parent CIFN. Thus, the addition of internal glycosylation sites and an N-terminal peptide extension containing additional glycosylation sites to CIFN increased the molecular weight of the starting CIFN. 4. Glycosylation and IFN specific activity of variant of CIFN having additional sly cosy lation sites at amino acid positions 31, 102 and 138, and various N-terminal peptide extensions
[0250] Four hyperglycosylated variants of CIFN: CIFN-N11-31-102-138, CIFN- N8-31-102-138, CIFN-N6-31-102-138, and CIFN-N4-31-102- 138 were produced similarly by the techniques described above using the expression cassette systems disclosed herein. Other than the additional glycosylation sites at aminio acid positions 31, 102 and 138, CIFN- Nl 1-31-102-138 had an N-terminal peptide extension with 11 glycosylation sites, CIFN-N8- 31-102-138 had an N-terminal peptide extension with 8 glycosylation sites, CIFN-N6-31- 102-138 had an N-terminal peptide extension with 6 glycosylation sites, and CIFN-N4-31- 102-138 had an N-terminal peptide extension with 4 glycosylation sites. As shown in Figure 12D, all 4 hyperglycosylated variants of the parent CIFN had an increased molecular weight compared to the starting CIFN. Thus, N-terminal peptide extensions containing various numbers of glycosylation sites increased the molecular weight of the starting CIFN.
[0251] Also shown in Figure 12D, CIFN-N11-31-102-138 which had 11 glycosylation sites in the N-terminal peptide extension had the highest molecular weight among the four hyperglycosylated variants of CIFN; CIFN-N8-31-102-138 which had 8 glycosylation sites in the N-terminal peptide extension had the second highest molecular weight among the four variants; CIFN-N6-31-102-138 which had 6 glycosylation sites in the N-terminal peptide extension had the third highest molecular weight among the four variants; and CIFN-N4-31-102-138 which had 4 glycosylation site in the N-terminal peptide extension had the lowest molecular weight among the four variants. Therefore, adding an N-terminal peptide extension with various numbers of glycosylation sites, such as an N-terminal peptide extension with 11, 8, 6 or 4 glycosylation sites, to the CIFN variant CIFN-31-102- 138 increased the molecular weight of CIFN.
[0252] To identify the source of the molecular weight increase in the hyperglycosylated variants of CIFN, the proteins of CIFN-N11-31-102-138, CIFN-N8-31- 102-138 and CIFN-N6-31-102-138 were digested with (+) or without (-) glycosidase PNGase F. The digested and undigested proteins were then examined by western blot analysis using the CIFN antibody. As shown in Figure 12E, when the carbohydrates were removed from the proteins by the treatment of glycosidase PNGase F, the molecular weight of all three hyperglycosylated variants of CIFN decreased to a level that is similar to the molecular weight of the parent CIFN. This indicates that the increase in the molecular weight of the hyperglycosylated variants of CIFN were largely caused by the addition of carbohydrates, and thus demonstrates that adding an N-terminal peptide extension with various numbers of glycosylation sites and a number of internal glycosylation sites increased the molecular weight of the hyperglycosylated variants of CIFN compared to the parent CIFN.
[0253] Further, the interferon specific activity of the parent CIFN and hyperglycosylated variants CIFN-N11-31-102-138, CIFN-N8-31-102- 138, CIFN-N6-31-102- 138, and CIFN-N4-31-102-138 were measured in an IFN gene reporter assay using iLite human interferon alpha kit (PBL). As shown in Table 3, all 4 hyperglycosylated variants of CIFN had the interferon specific activity comparable to that of the parent CIFN. This demonstrates that that biological potencies of these hyperglycosylated variants of CIFN are similar to that of the parent CIFN. Table 3. IFN specific activities of CIFN and hyperglycosylated variants of CIFN
Figure imgf000084_0001
Example 6
Biological activities of hyperglycosylated variants of CIFN
[0254] Four hyperglycosylated variants of CIFN: CIFN-N14(3)-102, CIFN- N14(3)-108, CIFN-N14(3)-102-138 and CIFN-N14(3)-108-138 were produced similarly by the techniques described in Example 5. These hyperglycosylated variants were assayed for HCV replicon activity and interferon specific activity.
1. HCV replicon activity
[0255] HCV replicon cells were plated in 96 well plates and incubated over night. The interferons were serially diluted and added to the replicon cells the next day. After 3day incubation, the cells were lysed and the luciferase activity was assayed with Bright-glo reagent (Promega). The dose response curves were ploted, and EC50 values were calculated. As shown in Figure 13A-C, the HCV replicon activity of all four hyperglycosylated variants were comparable to that of the parent CIFN. This demonstrates that these hyperglycosylated variants of CIFN are as biologically potent as the parent CIFN.
2. Interferon specific activity
[0256] The day before the experiment, the A549 cells were plated at 30,000 cell/well in 96 well plates. On the day of the experiment, test interferons and standard (CIFN) were serially diluted in serum free DMEM media and then added to the plated cells. The wells designated for positive control (cells without virus) and negative control (cells with virus but no drug protection) were not dosed. After 24 hours preincubation with the test interferons or the standard, encephalomyocarditis virus (EMCV) at the MOI of 0.001 was added to the cells, except for the positive controls. The plate was then incubated at 37°C, 5% C02 and observed daily for the cytopathic effects (CPE) in the cells. When the negative controls achieved full or near full CPE, the culture media was removed from the wells and Hucker' s Crystal Violet Solution was added to the cells and further incubated for 5 minutes at room temperature. The stained plate was them washed to remove the background stain, air dried and scanned. The blue dye were eluted with 33% acetic acid and quantified using ELISA reader. Interferon specific activities of the test interferons were calculated by comparing the 50% CPE of test interferons and the standard.
[0257] All four hyperglycosylated variants of CIFN showed comparable interferon specific activity to the parent CIFN (Figure 13D), demonstrating that these hyperglycosylated variants of CIFN are as biologically potent as the parent CIFN.
Example 7
Increased glycosylation in hyperglycosylated variants of interferon beta (IFNB)
[0258] To analyze the effects of different signal peptides on the secretion of target molecules in the expression cassette systems disclosed herein, DNA encloding hyperglycosylated variants of the parent IFNB with an N-terminal peptide extension having 10 glycosylation sites and various signal peptides were constructed using the expression cassette systems disclosed herein and transfected into mammalian cell line CHO to express hyperglycosylated variants of IFNB protein by the techniques similar to what was described in Examples 4 and 5. Other than the N-terminal peptide extension, EPO-N10 had the signal peptide from EPO (SEQ ID NO: 165), CD33-N10 had the signal peptide from CD33 (SEQ ID NO:164), IFNB-N10 had the signal peptide from IFNB (SEQ ID NO: 168), and DS-N10 had an artificial signal peptide (SEQ ID NO: 163). Several days after transfection, the media containing the secreted hyperglycosylated variants of IFNB proteins were collected. The extent of changes in molecular weight of each protein was determined by the mobility shift of the protein in SDS gel electrophoresis using the polyclonal antibodies targeting the parent IFNB.
[0259] As shown in Figure 14, all hyperglycosylated variants of IFNB with the N- terminal peptide extension with 10 glycosylation sites have increased molecular weight regardless of the signal peptide used. This indicates that the expression cassette systems disclosed herein can accommodate signal peptides from a wide range of secreted proteins and are applicable to express hybrid molecules with a foreign signal peptide.
Example 8
Increased glycosylation in hyperglycosylated variants of interferon beta (IFNB)
1. N-terminal peptide extensions on IFNB significantly increased the molecular weight of IFNB
[0260] Various N-terminal peptide extensions were added to human interferon beta (IFNB). IFNB-N10 (SEQ ID NO:338) is a hyperglycosylated variant of IFNB with an N-terminal peptide extension (VNITG)io. IFNB-N8 (SEQ ID NO:339) is a hyperglycosylated variant of IFNB with an N-terminal peptide extension (VNITG)8. IFNB-N6 (SEQ ID NO: 340) is a hyperglycosylated variant of IFNB with an N-terminal peptide extension (VNITG)e. IFNB-N4 (SEQ ID NO:341) is a hyperglycosylated variant of IFNB with an N- terminal peptide extension (VNITG)4. Each VNITG motif contains an N-linked glycosylation site.
[0261] DNA sequences encoding IFNB-N10, IFNB-N8, IFNB-N6, and IFNB-N4 were generated by the PCR-based mutagenesis method as described in Example 4, and transfected into CHO cells for protein expression. Western blot analysis of Figure 15 A showed that all four hyperglycosylated variants of IFNB with an N-terminal peptide extension had increased molecular weights compared to the parent IFNB. Thus, the additions of N-terminal peptide extensions containing various numbers of VNITG motifs increased the molecular weight of IFNB compared to the parent IFNB.
[0262] Figure 15A shows that IFNB-N10 which had 10 glycosylation sites in the N-terminal peptide extension had the highest molecular weight among the four variants; IFNB-N8 which had 8 glycosylation sites in the N-terminal peptide extension had the second highest molecular weight among the four variants; IFNB-N6 which had 6 glycosylation sites in the N-terminal peptide extension had the third highest molecular weight among the four variants; and IFNB-N4 which had 4 glycosylation sites in the N-terminal peptide extension had the lowest molecular weight among the four variants. Therefore, the addition of an N- terminal peptide extension with various numbers of glycosylation sites to the parent IFNB increased the molecular weight of the parent IFNB.
2. Carbohydrates contribute to most of the molecular weight increase
[0263] To identify the source of the molecular weight increase in the hyperglycosylated variants of IFNB, the proteins of IFNB-NIO, IFNB-N8 and IFNB-N6 were digested with (+) or without (-) glycosidase PNGase F. The digested and undigested proteins were then examined with western blot analysis using the IFNB antibody.
[0264] As shown in Figure 15B, when the carbohydrates were removed from the proteins by the treatment of glycosidase PNGase F, the molecular weight of IFNB-N10, IFNB-N8 and IFNB-N6 decreased to a level similar to the molecular weight of the parent IFNB. These results indicated that the increase in the molecular weight of the hyperglycosylated variants of IFNB were largely contributed by the addition of carbohydrates from the VNITG motif in the N-terminal extension.
3. Interferon specific activity of hyperglycosylated variants of IFNB
[0265] Interferon specific activities of the parent IFNB and hyperglycosylated variants IFNB-N6 and IFNB-N10 were measured similarly by the EMCV/A549 CPE assay described in Example 6. As shown in Tables 4, hyperglycosylated variants IFNB-N6 and IFNB-N10 have an interferon specific activity comparable to that of the parent IFNB.
Table 4. IFNB specific activity of IFNB, IFNB-N6 and IFNB-N10
Figure imgf000087_0001
Example 9
Increased glycosylation in hyperglycosylated variants of type I, II and III interferons 1. An N-terminal peptide extension to IFN alpha 1 (IFNal ), a type I interferon, increased the molecular weight of IFNal
[0266] An N-terminal peptide extension with 10 glycosylation sites was introduced to human interferon alpha 1 (IFNal) using an expression vector with an N- terminal peptide extension with 10 glycosylation sites and methods similarly to what was described in Example 5 to generate a hyperglycosylated variant IFNal-N10 (SEQ ID NO: 342). The extent of glycosylation of IFNal and IFNal-N10 was examined by the mobility shift of the proteins in SDS gel electrophoresis.
[0267] As shown in Figure 16A, hyperglycosylated variant IFNal-N10 had an increased molecular weight compared to the parent IFNal. Thus, the N-terminal peptide extension containing 10 glycosylation sites increased the molecular weight of the parent IFNal.
2. N-terminal peptide extensions on human interferon gamma (IFNG) increased the molecular weight of!FNG
[0268] Two N-terminal peptide extensions having different number of VNITG motif were added to human interferon gamma (IFNG), a type II interferon, respectively. IFNG-N5 (SEQ ID NO: 343) is a hyperglycosylated variant of IFNG with an N-terminal peptide extension (VNITG)5. IFNG-N10 (SEQ ID NO: 344) is a hyperglycosylated variant of IFNG with an N-terminal peptide extension (VNITG) io. Each VNITG motif contains an N- linked glycosylation site.
[0269] DNA sequences encoding IFNG-N5 and IFNG-N10 were generated by the PCR-based mutagenesis method as described in Example 1, and transfected into CHO cells for protein expression, respectively. Western blot analysis of Figure 16B showed that both of the hyperglycosylated variants of IFNG with an N-terminal peptide extension had an increased molecular weight compared to the parent IFNG. Thus, additions of N-terminal peptide extensions containing various numbers of VNITG motif increased the molecular weight of the starting IFNG.
[0270] Figure 16B also showed that IFNG-N10 which had 10 glycosylation sites in the N-terminal peptide extension had a higher molecular weight than IFNG-N5 which had 5 glycosylation sites in the N-terminal peptide extension. Therefore, the addition of a peptide extension with more glycosylation sites to the parent IFNG resulted in higher increase in the molecular weight of the parent IFNG. 3. An N-terminal peptide extension to type III interferons, increased the molecular weight of the type III interferons
[0271] An N-terminal peptide extension with 6 glycosylation sites was introduced to human interferon λΐ, λ2 and λ3 using an expression vector containing an N-terminal peptide extension with 6 glycosylation sites and methods similarly to what was described in Example 5 to generate hyperglycosylated variants IFN λ1-Ν6 (SEQ ID NO:347), IFN λ2-Ν6 (SEQ ID NO:348) and IFN λ3-Ν6 (SEQ ID NO:349). The extent of glycosylation of each protein was examined by the mobility shift of the protein in SDS gel electrophoresis.
[0272] As shown in Figure 16C, all hyperglycosylated variants had an increased molecular weight compared to its parent IFN λ. Thus, N-terminal peptide extensions containing glycosylation sites increased the molecular weight of the parent IFN λ.
Example 10
Increased glycosylation in hyperglycosylated variants human growth hormone (hGH)
[0273] An N-terminal peptide extensions with 6 glycosylation sites was introduced to human growth hormone 1 (hGHl) using a expression cassette with an N- terminal peptide with 6 glycosylation sites by methods similar to what was described in Example 5 to generate hyperglycosylated variants hGHl-N6 (SEQ ID NO:345). The extent of glycosylation of hGHl, hGHl-N6 and Norditropin® hGH was examined by the mobility shift of the proteins in SDS gel electrophoresis. As shown in Figure 17, hGHl-N6 had an increased molecular weight compared to the parent hGHl and Norditropin hGH. Therefore, the N-terminal peptide extension containing 6 glycosylation sites increased the molecular weight of the parent hGH.
Example 11
Pharmacokinetic (PK) and pharmacodynamic (PD) studies of
a mouse homolog of N14-CIFN-108
[0274] A mouse homolog of N14-CIFN-108 (hereafter "mN14-CIFN-108", SEQ ID NO:346) was generated by techniques similar to what was described in Example 5. When expressed, mN14-CIFN-108 was glycosylated well with a molecular weight close to lOOkD and was active in EMCV/L929 CPE assay.
[0275] Both mN14-CIFN-108 and unglycosylated mouse IFNal (mIFNal) were administered to mice as a single dose (1000 IU/g) subcutaneously. Animals (N=3) were sacrificed at pre-dose and post-dose for unglycosylated mouse IFNal at 0.5, 1 , 3, 5, 12, 24, 48, 96, and 168 hours, and mN14-CIFN-108 at 12, 24, 48, 96, and 168 hours. Mouse plasma and liver were harvested and analyzed for PK and PD markers. Mouse IFNal in plasma was quantified by sandwich ELISA and then converted to international units (IU)/ml based on protein specific activity. Plasma β-2 microglobulin was measured with competitive ELISA and liver OAS 1 mRNA with Real-Time PCR normalized against GAPDH.
[0276] As shown in Figure 18 A, the activity of the unglycosylated mIFNal control in the subject droped to a baseline level (about 0.5 IU/ml) within approximately the first 10 hours post initial dosing. Comparatively, the activity of mN14-CIFN-108 were well above 50 IU/ml during the same period of time, and continued to be well above 10 IU/ml during the first 100 hours post initial dosing. This demonstrated that mN14-CIFN-108 stayed in the subject for a markedly longer period of time compared to the mIFNal control.
[0277] Table 5 also supports the results shown in Figure 18A. As shown by the data provided in Table 5, the serum half-life (Ti/2) of mN14-CIFN-108 is about 40 times higher than that of the unglycosylated mIFNal control. This increase in serum half-life indicates that mN14-CIFN-108 stayed in the subject for a markedly longer period of time compared to the mIFNal control. Moreover, mN14-CIFN-108 shows about 33 fold of improvement in the AUCt and AUQnf value (AUC means "area under the curve") as compared to the unglycosylated mIFNal control. The AUC value is a measure of drug exposure in a subject. Thus, an interferon with a higher AUC value requires less frequent dosing to a subject to achieve approximately the same results as compared to an interferon with a lower AUC value. Accordingly, the data provided in Table 5 indicate that a subject receiving mN14-CIFN-108 can be dosed less frequently compared to a subject receiving the same amount of the unglycosylated mIFNal control. For example, a subject could be dosed approximately twice a month with the mN14-CIFN-108 compared to the daily dosing needed with the unglycosylated mIFN al control to achieve similar clinical results. Table 5. Improved pharmacokinetics profiles of mN14-CIFN-108 compared to the unglycosylated mIFN al control
Figure imgf000091_0001
[0278] In a pharmacodynamic study, beta2-microglobulin protein level and induction of OASl mRNA in liver are two commonly used biomarkers for the efficacy of interferon treatment. The longer the beta2-microglobulin protein level (or the induction of OASl mRNA in liver) stays above the baseline level, the interferon is active for a longer period of time. As shown in Figure 18B, the beta2-microglobulin protein level in the subject administered with the unglycosylated mIFNal droppped to a level that was less than 0.2 fold increase over baseline level within approximately 50 hours post initial dosing. Comparatively, the beta2-microglobulin protein level in the subject administered with mN14- CIFN-108 were well above 1 fold increase over baseline level with the same period of time and were well over 0.5 fold increase until 100 hours after initial dosing. Also shown in Figure 18B, the induction of OASl mRNA in liver dropped significantly to less than 0.1 fold increase over baseline in subjects administered with the unglycosylated mIFN al. In comparison, the induction stayed well above 0.5 fold increase for 100 hours after initial dosing in the subjects administered with mN14-CIFN-108. This elongated-PD response in animals by mN14-CIFN-108 as compared to the unglycosylated mIFNal indicated that mN14-CIFN-108 remained efficacious in the subject for much longer period of time as compared to the unglycosylated mIFNal. These results further supported that a subject receiving mN14-CIFN-108 can be dosed less frequently compared to a subject receiving the same amount of the unglycosylated mIFN al control.
[0279] It will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure.

Claims

WHAT IS CLAIMED IS:
1. A hyperglycosylated polypeptide variant of a parent polypeptide, wherein the hyperglycosylated polypeptide variant is the parent polypeptide that has been modified to include a peptide extension inserted at a terminal region, wherein the peptide extension is a peptide of 1-200 consecutive amino acids and comprises at least two glycosylation sites, wherein the terminal region is selected from the group consisting of an amino-terminal region that consists of the first 15 amino acids at the amino-terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide and a carboxy-terminal region that consists of the last 15 amino acids at the carboxy-terminus of the parent polypeptide.
2. The hyperglycosylated polypeptide variant of claim 1, wherein the peptide extension comprises at least three glycosylation sites.
3. The hyperglycosylated polypeptide variant of claim 1, wherein the peptide extension comprises at least four glycosylation sites.
4. The hyperglycosylated polypeptide variant of claim 1, wherein the peptide extension comprises at least six glycosylation sites.
5. The hyperglycosylated polypeptide variant of claim 1, wherein the peptide extension comprises at least eight glycosylation sites.
6. The hyperglycosylated polypeptide variant of claim 1, wherein the peptide extension comprises at least ten glycosylation sites.
7. The hyperglycosylated polypeptide variant of any one of claims 1 to 6, wherein the peptide extension is inserted at the amino-terminal region that consists of the first 15 amino acids at the amino-terminus of the parent polypeptide that excludes any signal peptide in the parent polypeptide.
8. The hyperglycosylated polypeptide variant of any one of claims 1 to 6, wherein the peptide extension is inserted at the carboxy-terminal region that consists of the last 15 amino acids at the carboxy-terminus of the parent polypeptide.
9. The hyperglycosylated polypeptide variant of any one of claims 1 to 8, wherein the peptide extension comprises an amino acid motif NB 1 B2 [B 3
]zi, wherein
B1 is an amino acid residue;
B2 is Serine (S) or Threonine (T); B is a sequence of Zl amino acids, wherein Zl is an integer from 1 to 8, wherein each amino acid in the sequence B is independently an amino acid residue.
10. The hyperglycosylated polypeptide variant of any one of claims 1 to 8, wherein the peptide extension comprises an amino acid motif [B4]z2NB5B6[B7]z3, wherein
B4 is a sequence of Z2 amino acids, wherein Z2 is an integer from 1 to 8, wherein each amino acid in the sequence B4 is independently an amino acid residue;
B5 is an amino acid residue;
B6 is Serine (S) or Threonine (T);
η
B is a sequence of Z3 amino acids, wherein Z3 is an integer from 1 to 8, η
wherein each amino acid in the sequence B is independently an amino acid residue.
11. The hyperglycosylated polypeptide variant of any one of claims 9 to 10, wherein the amino acid motif is present one time in the peptide extension.
12. The hyperglycosylated polypeptide variant of any one of claims 9 to 10, wherein the amino acid motif is present at least two times in the peptide extension.
13. The hyperglycosylated polypeptide variant of any one of claims 9 to 10, wherein the amino acid motif is present at least three times in the peptide extension.
14. The hyperglycosylated polypeptide variant of any one of claims 9 to 10, wherein the amino acid motif is present at least four times in the peptide extension.
15. The hyperglycosylated polypeptide variant of any one of claims 9 to 10, wherein the amino acid motif is present at least six times in the peptide extension.
16. The hyperglycosylated polypeptide variant of any one of claims 9 to 10, wherein the amino acid motif is present at least eight times in the peptide extension.
17. The hyperglycosylated polypeptide variant of any one of claims 9 to 10, wherein the amino acid motif is present at least ten times in the peptide extension.
18. The hyperglycosylated polypeptide variant of any one claims 10 to 17, wherein
B4 is Valine (V), Isoleucine (I), Glycine (G), or Alanine (A);
B5 is Valine (V), Isoleucine (I), Glycine (G), or Alanine (A); and B is a sequence selected from the group consisting of Glycine (G), GG, GGG, and GR.
19. The hyperglycosylated polypeptide variant of claim 18, wherein
B4 is Valine (V) or Isoleucine (I); and
η
B is a sequence selected from the group consisting of Glycine (G), GG, GGG, and GR.
20. The hyperglycosylated polypeptide variant of claim 19, wherein the amino acid motif is VNITG.
21. The hyperglycosylated polypeptide variant of claim 19, wherein the amino acid motif is VNITGG.
22. The hyperglycosylated polypeptide variant of claim 19, wherein the amino acid motif is VNITGGG.
23. The hyperglycosylated polypeptide variant of claim 19, wherein the amino acid motif is VNISGR.
24. The hyperglycosylated polypeptide variant of any one of claims 1 to 23, wherein the parent polypeptide is selected from the group consisting of a growth hormone (GH), an insulin-like growth factor (IGF), a granulocyte colony-stimulating factor (G-CSF), an erythropoietin (EPO), an insulin, an antibody Fab fragment, and an antibody scFV fragment.
25. The hyperglycosylated polypeptide variant of claim 24, wherein the parent polypeptide is a growth hormone (GH).
26. The hyperglycosylated polypeptide variant of claim 25, wherein the growth hormone is a human growth hormone (hGH).
27. The hyperglycosylated polypeptide variant of claim 26, wherein the human growth hormone is human growth hormone 1 (hGH-1).
28. The hyperglycosylated polypeptide variant of claim 27, wherein the peptide extension is located between amino acids A26 and F27 of the hGH-1.
29. The hyperglycosylated polypeptide variant of claim 24, wherein the parent polypeptide is an insulin-like growth factor (IGF).
30. The hyperglycosylated polypeptide variant of claim 29, wherein the insulinlike growth factor (IGF) is selected from the group consisting of IGF- 1 and IGF-2.
31. The hyperglycosylated polypeptide variant of claim 29, wherein the insulinlike growth factor (IGF) is human insulin-like growth factor 1A (hIGF-lA).
32. The hyperglycosylated polypeptide variant of claim 31, wherein the peptide extension is located between amino acids K21 and V22 of the hIGF-1 A.
33. The hyperglycosylated polypeptide variant of claim 24, wherein the parent polypeptide is a granulocyte colony-stimulating factor (G-CSF).
34. The hyperglycosylated polypeptide variant of claim 33, wherein the granulocyte colony-stimulating factor (G-CSF) is human G-CSF (hG-CSF).
35. The hyperglycosylated polypeptide variant of claim 34, wherein the peptide extension is located between amino acids E29 and A30 of the hG-CSF.
36. The hyperglycosylated polypeptide variant of claim 24, wherein the parent polypeptide is an erythropoietin (EPO).
37. The hyperglycosylated polypeptide variant of claim 36, wherein the erythropoietin (EPO) is human EPO (hEPO).
38. The hyperglycosylated polypeptide variant of claim 37, wherein the peptide extension is located between amino acids G27 and A28 of the hEPO.
39. The hyperglycosylated polypeptide variant of claim 24, wherein the parent polypeptide is an insulin.
40. The hyperglycosylated polypeptide variant of claim 39, wherein the insulin is human insulin.
41. The hyperglycosylated polypeptide variant of claim 40, wherein the peptide extension is located between amino acids A24 and F25 of the human insulin.
42. The hyperglycosylated polypeptide variant of claim 24, wherein the parent polypeptide is an antibody Fab fragment.
43. The hyperglycosylated polypeptide variant of claim 42, wherein the parent polypeptide is the antibody Fab fragment of an antibody specific for TNF-a.
44. The hyperglycosylated polypeptide variant of claim 42, wherein the parent polypeptide is the antibody Fab fragment of an antibody specific for HER-2 receptor.
45. The hyperglycosylated polypeptide variant of claim 42, wherein the parent polypeptide is the antibody Fab fragment of an antibody specific for VEGF.
46. The hyperglycosylated polypeptide variant of claim 24, wherein the parent polypeptide is an antibody scFV fragment.
47. The hyperglycosylated polypeptide variant of claim 46, wherein the parent polypeptide is the antibody scFV fragment of an antibody specific for TNF-a.
48. The hyperglycosylated polypeptide variant of claim 46, wherein the parent polypeptide is the antibody scFV fragment of an antibody specific for HER-2 receptor.
49. The hyperglycosylated polypeptide variant of claim 46, wherein the parent polypeptide is the antibody scFV fragment of an antibody specific for VEGF.
50. A pharmaceutical composition comprising the hyperglycosylated polypeptide variant of any one of claims 1-49; and a pharmaceutically acceptable excipient.
51. The pharmaceutical composition of claim 50, wherein the pharmaceutically acceptable excipient is suitable for oral delivery.
52. The pharmaceutical composition of claim 50, wherein the pharmaceutically acceptable excipient is suitable for parenteral delivery.
53. An expression cassette for expressing a hyperglycosylated polypeptide, comprising:
a promoter operably linked with a nucleotide sequence encoding a signal peptide;
a first extension sequence under control of the promoter encoding a first peptide extension; and
a restriction site either immediately upstream or downstream of the first extension sequence to allow insertion of a gene encoding a biologically-active polypeptide,
wherein the first peptide extension is a peptide of 1-200 consecutive amino acids and comprises at least two glycosylation sites, and wherein, upon insertion of a gene encoding a biologically-active polypeptide at the restriction site, the expression cassette directs expression of a fusion protein comprising the biologically-active polypeptide linked to the first peptide extension.
54. The expression cassette of claim 53 further comprising a gene encoding a biologically-active polypeptide inserted at the restriction site.
55. The expression cassette of claim 54, wherein the first extension sequence is located between the gene encoding the biologically-active polypeptide and the nucleotide sequence encoding the signal peptide.
56. The expression cassette of claim 54, wherein the gene encoding the biologically-active polypeptide is located between the nucleotide sequence encoding the signal peptide and the first extension sequence.
57. The expression cassette of any one of claims 53 to 56, wherein the first peptide extension comprises at least three glycosylation sites.
58. The expression cassette of any one of claims 53 to 56, wherein the first peptide extension comprises at least four glycosylation sites.
59. The expression cassette of any one of claims 53 to 56, wherein the first peptide extension comprises at least six glycosylation sites.
60. The expression cassette of any one of claims 53 to 56, wherein the first peptide extension comprises at least eight glycosylation sites.
61. The expression cassette of any one of claims 53 to 56, wherein the first peptide extension comprises at least ten glycosylation sites.
62. The expression cassette of any one of claims 53 to 61, wherein the first peptide extension comprises an amino acid motif NB 1 B2 [B 3
]zi, wherein
B1 is an amino acid residue;
B2 is Serine (S) or Threonine (T);
B is a sequence of Zl amino acids, wherein Zl is an integer from 1 to 8, wherein each amino acid in the sequence B is independently an amino acid residue.
63. The expression cassette of any one of claims 53 to 61, wherein the first peptide extension comprises an amino acid motif [B4]z2NB5B6[B7]z3, wherein
B4 is a sequence of Z2 amino acids, wherein Z2 is an integer from 1 to 8, wherein each amino acid in the sequence B4 is independently an amino acid residue;
B5 is an amino acid residue;
B6 is Serine (S) or Threonine (T);
B is a sequence of Z3 amino acids, wherein Z3 is an integer from 1 to 8, η
wherein each amino acid in the sequence B is independently an amino acid residue.
64. The expression cassette of any one of claims 62 to 63, wherein the amino acid motif is present one time in the first peptide extension.
65. The expression cassette of any one of claims 62 to 63, wherein the amino acid motif is present at least two times in the first peptide extension.
66. The expression cassette of any one of claims 62 to 63, wherein the amino acid motif is present at least three times in the first peptide extension.
67. The expression cassette of any one of claims 62 to 63, wherein the amino acid motif is present at least four times in the first peptide extension.
68. The expression cassette of any one of claims 62 to 63, wherein the amino acid motif is present at least six times in the first peptide extension.
69. The expression cassette of any one of claims 62 to 63, wherein the amino acid motif is present at least eight times in the first peptide extension.
70. The expression cassette of any one of claims 62 to 63, wherein the amino acid motif is present at least ten times in the first peptide extension.
71. The expression cassette of any one claims 63 to 70, wherein
B4 is Valine (V), Isoleucine (I), Glycine (G), or Alanine (A);
B5 is Valine (V), Isoleucine (I), Glycine (G), or Alanine (A); and B is a sequence selected from the group consisting of Glycine (G), GG, GGG, and GR.
72. The expression cassette of claim 71 , wherein
B4 is Valine (V) or Isoleucine (I); and
B is a sequence selected from the group consisting of Glycine (G), GG, GGG, and GR.
73. The expression cassette of claim 72, wherein the amino acid motif is VNITG.
74. The expression cassette of claim 72, wherein the amino acid motif is VNITGG.
75. The expression cassette of claim 72, wherein the amino acid motif is VNITGGG.
76. The expression cassette of claim 72, wherein the amino acid motif is VNISGR.
77. The expression cassette of any one of claims 53 to 76, further comprising a second extension sequence encoding a second peptide extension.
78. The expression cassette of claim 77, wherein the gene encoding the biologically-active polypeptide is located between the first extension sequence and the second extension sequence.
79. The expression cassette of any one of claims 77 to 78, wherein the second peptide extension comprises at least three glycosylation sites.
80. The expression cassette of any one of claims 77 to 78, wherein the second peptide extension comprises at least four glycosylation sites.
81. The expression cassette of any one of claims 77 to 78, wherein the second peptide extension comprises at least six glycosylation sites.
82. The expression cassette of any one of claims 77 to 78, wherein the second peptide extension comprises at least eight glycosylation sites.
83. The expression cassette of any one of claims 77 to 78, wherein the second peptide extension comprises at least ten glycosylation sites.
84. The expression cassette of any one of claims 53 to 83, wherein the biologically-active polypeptide is selected from the group consisting of a growth hormone (GH), an insulin-like growth factor (IGF), a granulocyte colony-stimulating factor (G-CSF), an erythropoietin (EPO), an insulin, an antibody Fab fragment, and an antibody scFV fragment.
85. The expression cassette of claim 84, wherein the biologically-active polypeptide is a growth hormone (GH).
86. The expression cassette of claim 85, wherein the growth hormone (GH) is a human growth hormone (hGH).
87. The expression cassette of claim 86, wherein the human growth hormone is human growth hormone 1 (hGH-1).
88. The expression cassette of claim 84, wherein the biologically-active polypeptide is an insulin-like growth factor (IGF).
89. The expression cassette of claim 88, wherein the insulin-like growth factor (IGF) is selected from the group consisting of IGF- 1 and IGF-2.
90. The expression cassette of claim 88, wherein the insulin-like growth factor (IGF) is human insulin-like growth factor 1A (hIGF-lA).
91. The expression cassette of claim 84, wherein the biologically-active polypeptide is a granulocyte colony-stimulating factor (G-CSF).
92. The expression cassette of claim 91, wherein the granulocyte colony- stimulating factor (G-CSF) is human G-CSF (hG-CSF).
93. The expression cassette of claim 84, wherein the biologically-active polypeptide is an erythropoietin (EPO).
94. The expression cassette of claim 93, wherein the erythropoietin (EPO) is human EPO (hEPO).
95. The expression cassette of claim 84, wherein the biologically-active polypeptide is an insulin.
96. The expression cassette of claim 95, wherein the insulin is human insulin.
97. The expression cassette of claim 84, wherein the biologically-active polypeptide is an antibody Fab fragment.
98. The expression cassette of claim 97, wherein the biologically-active polypeptide is the antibody Fab fragment of an antibody specific for TNF-a.
99. The expression cassette of claim 97, wherein the biologically-active polypeptide is the antibody Fab fragment of an antibody specific for HER-2 receptor.
100. The expression cassette of claim 97, wherein the biologically-active polypeptide is the antibody Fab fragment of an antibody specific for VEGF.
101. The expression cassette of claim 84, wherein the biologically-active polypeptide is an antibody scFV fragment.
102. The expression cassette of claim 101, wherein the biologically-active polypeptide is the antibody scFV fragment of an antibody specific for TNF-a.
103. The expression cassette of claim 101, wherein the biologically-active polypeptide is the antibody scFV fragment of an antibody specific for HER-2 receptor.
104. The expression cassette of claim 101, wherein the biologically-active polypeptide is the antibody scFV fragment of an antibody specific for VEGF.
105. An expression vector comprising the expression cassette of any one of claims 53 to 104.
106. A recombinant host cell comprising the expression vector of claim 105.
107. The recombinant host cell of claim 106 is a mammalian cell.
108. The mammalian cell of claim 107 is a human cell.
109. The human cell of claim 108 is a HEK293 cell.
110. A method of producing a hyperglycosylated polypeptide, comprising introducing the expression vector of claim 105 into a mammalian host cell, culturing the mammalian host cell under suitable condition so as to express the hyperglycosylated polypeptide, and recovering the hyperglycosylated polypeptide.
111. A method for producing a hyperglycosylated polypeptide in a mammal, comprising:
introducing the expression vector of Claim 105 into a mammal, wherein the expression vector comprises sequence encoding a biologically-active peptide; and expressing a hyperglycosylated form of said biologically-active peptide in the mammal.
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