WO2004005341A2 - Full-length interferon gamma polypeptide variants - Google Patents
Full-length interferon gamma polypeptide variants Download PDFInfo
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- WO2004005341A2 WO2004005341A2 PCT/DK2003/000426 DK0300426W WO2004005341A2 WO 2004005341 A2 WO2004005341 A2 WO 2004005341A2 DK 0300426 W DK0300426 W DK 0300426W WO 2004005341 A2 WO2004005341 A2 WO 2004005341A2
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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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/57—IFN-gamma
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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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Definitions
- the present invention relates to novel full-length interferon gamma polypeptide variants having interferon gamma (IFNG) activity, methods for their preparation, pharmaceutical compositions comprising the variants and their use in the treatment of diseases, in particular for the treatment of interstitial pulmonary diseases, such as idiopathic pulmonary fibrosis.
- IFNG interferon gamma
- Interferon gamma is a cytokine produced by T-lymphocytes and natural killer cells and exists as a homodimer of two noncovalently bound polypeptide subunits.
- the mature form of each monomer comprises 143 amino acid residues (shown in SEQ ID NO:l) and the precursor form thereof, including the signal sequence, comprises 166 amino acid residues (shown in SEQ ID NO:2).
- Each subunit has two potential N-glycosylation sites (Aggarwal et al., Human
- IFNG subunit polypeptides including one comprising a Cys-Tyr-Cys N-terminal amino acid sequence (positions (-3)-(-l) relative to SEQ ID NO:l), one comprising an N-terminal methionine (position -1 relative to SEQ ID NO:l), and various C-terminally truncated forms comprising 127-134 amino acid residues. It is known that 1-15 amino acid residues may be deleted from the C-terminus without abolishing IFNG activity of the molecule. Furthermore, heterogeneity of the huIFNG C-terminus was described by Pan et al. (Eur. J. Biochem. 166:145-149, 1987). Glycosylation variation in huIFNG has been reported by Curling et al. (Biochem. J.
- huIFNG Polymer-modification of huIFNG was reported by Kita et al. (Drug Des. Deliv. 6 : 157- 167, 1990), and in EP 236987 and US 5109120.
- WO 99/03887 discloses PEGylated variants of polypeptides belonging to the growth hormone superfamily, wherein a non-essential amino acid residue located in a specified region of the polypeptide has been replaced by a cysteine residue.
- IFNG is mentioned as one example of a member of the growth hormone super family, but modification thereof is not discussed in any detail.
- WO 01/36001 discloses novel IFNG conjugates comprising a non-polypeptide moiety attached to an IFNG polypeptide which have been modified by introduction and/or deletion of attachment sites for such non-polypeptide moieties, e.g. PEG and glycosylation sites. These molecules have improved properties, such as improved half-life and/or increased bioavailablity.
- IFNG has been suggested for treatment of interstitial lung diseases (also known as interstitial Pulmonary Fibrosis (IPF) (Ziesche et al. (N. Engl. J. Med.
- IFNG hepatitis B (Hepatogastroenterology 45:2282-2294, 1998), hepatitis C (Int. Hepatol. Communic. 6:264- 273, 1997), septic shock (Nature Medicine 3:678-681, 1997), and rheumatoid arthritis may be treated with IFNG.
- IFNG is presently being clinically evaluated for treatment of ovarian cancer, liver fibrosis, asthma and lymphoma.
- huIFNG is usually applicable via parenteral, preferably via subcutaneous, injection. Maximum serum concentrations have been found after seven hours. It has been reported that the half -life in plasma is 30 minutes after intravenous administration. For this reason efficient treatment with huIFNG involves frequent injections.
- the main adverse effects consist of fever, chills, sweating, headache, myalgia and drowsiness. These effects are associated with injecting huIFNG and are observed within the first hours after injection. Rare side effects are local pain and erythema, elevation of liver enzymes, reversible granulo- and thrombopenia and cardiotoxicity.
- the present inventors have solved the above-mentioned problem by performing selected modifications in the C-terminal part of the IFNG polypeptide. By performing such modifications C-terminal truncation is avoided and, consequently, the truncation can be controlled and homogenous populations of full-length IFNG polypeptides can be obtained.
- the present invention relates to a full-length interferon gamma (IFNG) polypeptide variant exhibiting IFNG activity, wherein said variant comprises
- the present invention relates to a full-length interferon gamma (IFNG) polypeptide variant exhibiting IFNG activity, wherein said variant comprises an amino acid substitution in position R137 and an amino acid substitution in position R140.
- IFNG interferon gamma
- the invention relates to means and methods for preparing an IFNG polypeptide variant of the invention, including nucleotide sequences and expression vectors encoding the variant as well as host cells comprising the vector or nucleotide sequence of the invention.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising the variant of the invention and a pharmaceutically acceptable diluent, carrier or adjuvant; to a variant of the invention for use as a medicament; to use of a variant of the invention for the manufacture of a medicament for the treatment of interstitial pulmonary diseases as well as to a method for treating or preventing interstitial pulmonary diseases, said method comprising administering to a mammal, in particular a human being, in need thereof an effective amount of a variant of the invention.
- the present invention relates to a method for producing a full- length IFNG polypeptide, said method comprising i) cultivating a host cell according to the invention under conditions suitable for production of the IFNG polypeptide, and ii) recovering the IFNG polypeptide.
- Fig. 1 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T]huIFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de- glycosylation with PNGase F. See Example 8 for further details.
- Fig. 2 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+R137P]huIFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 3 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+R139P]huIFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 4 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+S142P]huIFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 5 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+Q143P]huIFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 5 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+Q143P]huIFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 7 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+R137P+ R139P+Q143P]huIFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 8 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+R137P+ R139P+S142P]huLFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 9 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+R137P+ S142P]huIFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 10 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+S132P+R137P+
- R140P]huIFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 11 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+S132P+ R140P]huTFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 12 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+R140P]hulFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- Fig. 13 shows a MALDI-TOF mass spectra of [E38N+S40T+S99T+R137P+ R140P]huIFNG purified from culture media by diafiltration followed by cation exchange, immunoprecipitation and de-glycosylation with PNGase F. See Example 8 for further details.
- conjugated polypeptide is intended to indicate a heterogeneous (in the sense of composite or chimeric) molecule formed by the covalent attachment of one or more polypeptide variant(s) to one or more non-polypeptide moieties.
- covalent attachment means that the polypeptide variant and the non- polypeptide moiety are either directly covalently joined to one another, or else are indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties.
- a conjugated polypeptide variant is soluble at relevant concentrations and conditions, i.e. soluble in physiological fluids such as blood.
- conjugated polypeptide variants of the invention include glycosylated and or PEGylated polypeptides.
- non-conjugated polypeptide may be used about the polypeptide part of the conjugated polypeptide variant.
- non-polypeptide moiety is intended to indicate a molecule that is capable of conjugating to an attachment group of the IFNG variant.
- examples of such molecules include polymer molecules, lipophilic compounds, sugar moieties or organic derivatizing agents. Prefererred examples include polymer molecules, such as PEG, and sugar moieties. It will be understood that the non-polypeptide moiety is linked to the variant through an attachment group of the variant.
- non-polypeptide moieties such as polymer molecule(s)
- every reference to "a non- polypeptide moiety" attached to the IFNG variant or otherwise used in the present invention shall be a reference to one or more non-polypeptide moieties attached to the IFNG variant.
- polymer molecule is defined as a molecule formed by covalent linkage of two or more monomers, wherein none of the monomers is an amino acid residue.
- polymer may be used interchangeably with the term “polymer molecule”.
- sugar moiety is intended to indicate a carbohydrate molecule attached by in vivo or in vitro glycosylation, such as N- or O-glycosylation.
- N-glycosylation site has the sequence N-X-S/T/C", wherein X is any amino acid residue except proline, N is asparagine and S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine.
- N-glycosylation site and “in vivo N-glycosylation site” are used interchangeably herein.
- An "O-glycosylation site” is the OH-group of a serine or threonine residue.
- attachment group is intended to indicate an amino acid residue group capable of coupling to the relevant non-polypeptide moiety such as a polymer molecule or a sugar moiety. Useful attachment groups and their matching non-polypeptide moieties are apparent from the table below.
- attachment group is used in an unconventional way to indicate the amino acid residues constituting an N-glycosylation site (with the sequence N-X-S/T/C, wherein X is any amino acid residue except proline, N is asparagine and S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine).
- N is asparagine
- S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine.
- amino acid residue comprising an attachment group for the non-polypeptide moiety as used in connection with alterations of the amino acid sequence of the IFNG polypeptide is to be understood as one, two or all of the amino acid residues constituting an N-glycosylation site is/are to be altered in such a manner that either a functional N-glycosylation site is introduced into the amino acid sequence, removed from said sequence or a functional N-glycosylation site is retained in the amino acid sequence (e.g. by substituting a serine residue, which already constitutes part of an N-glycosylation site, with a threonine residue and vice versa).
- amino acid names and atom names are used as defined by the Protein DataBank (PDB) (www.pdb.org; which are based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atom names etc.), Eur. J. Biochem., 138, 9-37 (1984) together with their corrections in Eur. J. Biochem., 152, 1 (1985).
- PDB Protein DataBank
- CA is sometimes referred to as C , CB as C ⁇ .
- amino acid residue is intended to indicate an amino acid residue contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or ⁇ ), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. Numbering of amino acid residues in this document is from the N-terminus of wild-type human IFNG (huIFNG) without
- G18 indicates that position 18 is occupied by a glycine residue.
- G18N indicates that the Gly residue of position 18 has been replaced with an Asn residue.
- Multiple substitutions are indicated with a "+", e.g. G18N+S20T means an amino acid sequence which comprises a substitution of the Gly residue in position 18 with an Asn residue and a substitution of the Ser residue in position 20 with a Thr residue.
- Alternative substitutions are indicated with a "/”.
- G18S/T covers the following individual substitutions: G18S and G18T. Deletions are indicated by an asterix.
- G18* indicates that the Gly residue in position 18 has been deleted.
- Insertions are indicated the following way: Insertion of an additional Pro residue after the Gin residue located at position 143 is indicated as Q143QP. Combined substitutions and insertions are indicated in the following way: substitution of the Gin residue at position 143 with a Cys residue and insertion of a Pro residue after the position 143 amino acid residue is indicated as Q143CP.
- the term "nucleotide sequence" is intended to indicate a consecutive stretch of two or more nucleotide molecules.
- the nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
- PCR polymerase chain reaction
- Cell generally refers to a method for amplification of a desired nucleotide sequence in vitro, as described, for example, in US 4,683,195.
- the PCR method involves repeated cycles of primer extension synthesis, using oligonucleotide primers capable of hybridising preferentially to a template nucleic acid.
- Cell “host cell”, “cell line” and “cell culture” are used interchangeably herein and all such terms should be understood to include progeny resulting from growth or culturing of a cell.
- Transformation and “transfection” are used interchangeably to refer to the process of introducing DNA into a cell.
- operably linked refers to the covalent joining of two or more nucleotide sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences ' can be performed.
- the nucleotide sequence encoding a presequence or secretory leader is operably linked to a nucleotide sequence for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide: a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
- operably linked means that the nucleotide sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used, in conjunction with standard recombinant DNA methods.
- introduce is primarily intended to mean substitution of an existing amino acid residue, but may also mean insertion of an additional amino acid residue.
- the term "remove” is primarily intended to mean substitution of the amino acid residue to be removed for another amino acid residue, but may also mean deletion (without substitution) of the amino acid residue to be removed.
- amino acid residue comprising an attachment group for the non-polypeptide moiety is intended to indicate that the amino acid residue is one to which the non-polypeptide moiety binds (in the case of an introduced amino acid residue) or would have bound (in the case of a removed amino acid residue).
- one difference or “differs from” as used in connection with specific modifications is intended to allow for additional differences being present apart from the specified amino acid modification.
- the IFNG variant may, if desired, comprise other modifications that are not related to this property.
- Such other modifications may, for example, include introduction and/or removal of amino acid residues comprising an attachment group for a non-polypeptide moiety, addition of one or more extra residues at the N-terminus, e.g. addition of a Met residue at the N- terminus or addition of the amino acid sequence Cys-Tyr-Cys at the N-terminus, as well as "conservative amino acid substitutions", i.e. substitutions performed within groups of amino acids with similar characteristics, e.g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids. Examples of conservative substitutions in the present invention may, in particular, be selected from the groups listed in the table below.
- AUC SC or "Area Under the Curve when administered subcutaneously” is used in its normal meaning, i.e. as the area under the IFNG activity in serum-time curve, where the IFNG variant has been administered subcutaneously, in particular when administered subcutaneously in rats or non-human primates, such as monkeys.
- the AUC SC may conveniently be calculated by a computer program, such as GraphPad Prism 3.01.
- the term "functional in vivo half-life" is used in its normal meaning, i.e. the time at which 50% of the biological activity of the polypeptide is still present in the body/target organ, or the time at which the activity of the polypeptide is 50% of the initial value.
- the functional in vivo half-life may by determined in rats, cf. the Materials and Method section herein, but is preferably determined in non-human primates, such as monkeys. It is important to note that the term "functional in vivo half-life", when used herein, for a given IFNG variant must be determined for a sample that has been administered intravenously (iv).
- serum half-life may be determined, i.e. the time at which 50% of the polypeptide circulates in the plasma or bloodstream prior to being cleared. Determination of serum half-life is often more simple than determining the functional in vivo half-life and the magnitude of serum half-life is usually a good indication of the magnitude of functional in vivo half-life.
- terms to serum half-life include "plasma half-life", “circulating half-life”, “serum clearance”, “plasma clearance” and "clearance half -life”.
- the serum half -life may be determined in rats, cf. the Materials and Method section herein, but is preferably determined in non-human primates, such as monkeys. It is important to note that the term "serum half -life", when used herein, for a given IFNG variant must be determined for a sample that has been administered intravenously (iv).
- half -life or “in vivo half-life” may refer to both functional in vivo half -life and serum half -life.
- serum is used in its normal meaning, i.e. the term covers blood plasma without fibrinogen and other clotting factors.
- the polypeptide is normally cleared by the action of one or more of the reticuloendothelial systems (RES), kidney, spleen or liver, or by specific or unspecific proteolysis.
- RES reticuloendothelial systems
- renal clearance is used in its normal meaning to indicate any clearance taking place by the kidneys, e.g. by glomerular filtration, tubular excretion or tubular elimination.
- renal clearance depends on physical characteristics of the polypeptide, including molecular weight, size (relative to the cutoff for glomerular filtration), symmetry, shape/rigidity, charge and attached carbohydrate chains. A molecular weight of about 67 kDa is normally considered to be a cut-off -value for renal clearance.
- Renal clearance may be measured by any suitable assay, e.g. an established in vivo assay.
- renal clearance may be determined by administering a labelled (e.g. radiolabelled or fluorescence labelled) conjugated polypeptide to a patient and measuring the label activity in urine collected from the patient.
- Reduced renal clearance is determined relative to the reference molecule, such as glycosylated huIFNG, glycosylated [S99T]huIFNG or Actimmune®.
- the functionality to be retained is normally selected from antiviral, antiproliferative or immunomodulatory activity.
- the term "increased" as used about the functional in vivo half -life or serum half -life is used to indicate that the relevant half -life of the IFNG variant is statistically significantly increased relative to that of a reference molecule, such as glycosylated huIFNG, glycosylated [S99T]huIFNG or Actimmune® determined under comparable conditions.
- a reference molecule such as glycosylated huIFNG, glycosylated [S99T]huIFNG or Actimmune® determined under comparable conditions.
- interesting IFNG variants are such variants, which has an increased functional in vivo half-life or an increased serum half -life as compared to any of the reference molecules mentioned above, when administered intravenously.
- reduced immunogenicity is intended to indicate that the IFNG variant gives rise to a measurably lower immune response than a reference molecule, e.g. glycosylated huIFNG or Actimmune®, as determined under comparable conditions.
- the immune response may be a cell or antibody mediated response (see, e.g., Roitt: Essential Immunology (8th Edition, Blackwell) for further definition of immunogenicity).
- reduced antibody reactivity is an indication of reduced immunogenicity.
- Reduced immunogenicity may be determined by use of any suitable method known in the art, e.g. in vivo or in vitro.
- the term "increased degree of in vivo N-glycosylation” or “increased degree of N-glycosylation” is intended to indicate increased levels of attached carbohydrate molecules, normally obtained as a consequence of increased (or better) utilization of glycosylation site(s). It is well-known (Hooker et al., 1998, J. Interferon and Cytokine Res. 18, 287-295 and Sarenva et al., 1995, Biochem J., 308, 9-14) that when huIFNG is expressed in CHO cells only about 50% of the IFNG molecules utilizes both glycosylation sites, about 40% utilizes one glycosylation site (IN), and about 10% is not glycosylated (ON).
- the increased degree of in vivo N-glycosylation may be determined by any suitable method known in the art, e.g. by SDS-PAGE.
- One convenient assay for determining increased glycosylation is the method described in the section entitled "Determination of Increased Glycosylation" in the Materials and Methods section herein.
- the term "exhibiting IFNG activity" is intended to indicate that the variant has one or more of the functions of native glycosylated huIFNG or Actimmune®, including the capability to bind to an IFNG receptor and cause transduction of the signal transduced upon huIFNG- binding of its receptor as determined in vitro or in vivo (i.e. in vitro or in vivo bioactivity).
- the IFNG receptor has been described by Aguet et al. (Cell 55:273-280, 1988) and Calderon et al. (Proc. Natl. Acad. Sci. USA 85:4837-4841, 1988).
- a suitable assay for testing IFNG activity is the assay entitled "Primary Assay" disclosed herein.
- polypeptide variants "exhibiting IFNG activity” have a specific activity of at least 5% as compared to glycosylated huIFNG, glycosylated [S99T]huIFNG or Actimmune®. It will be understood that depending on which specific modifications are performed, for example whether the variant is PEGylated or not, this may lead to activities over a wide range. Thus, examples of specific activities may range from as low as 5% to as high as 150% as compared to glycosylated huIFNG, glycosylated [S99T]huIFNG or Actimmune®. For example, the specific activity may be at least 10% (e.g.
- 10-125% such as at least 15% (e.g. 15-125%), e.g. at least 20% (such as 20-125%), at least 25% (e.g. 25-125%), at least 30% (e.g. 30-125%), at least 35% (e.g. 35-125%), at least 40% (e.g. 40-125%), at least 45% (e.g. 45-125%), at least 50% (e.g. 50- 125%), at least 55% (e.g. 55-125%), at least 60% (e.g. 60-125%), at least 65% (e.g. 65-125%), at least 70% (e.g. 70-125%), at least 75% (e.g. 75-125%), at least 80% (e.g. 80-125%) or at least 90% (e.g. 90-110%) as compared to the specific activity of glycosylated huIFNG, glycosylated [S99T]huIFNG or Actimmune®.
- the variant may exhibit 1-75% (e.g. 5-75%), such as 1-50% (e.g. 5-50%), e.g. 1-40% (e.g. 5-40%), 1-30% (e.g. 5-30%), 1-20% (e.g. 5-20%) or 1-10% (e.g. 5-10%) of the LFNG activity of glycosylated huIFNG, glycosylated [S99T]huIFNG or Actimunne® when tested in the "Primary Assay" described herein.
- IFNG polypeptide is a polypeptide exhibiting IFNG activity, and is used herein about the polypeptide in monomer or dimeric form, as appropriate. For instance, when specific substitutions are indicated these are normally indicated relative to the IFNG polypeptide monomer. When reference is made to the IFNG polypeptide of the invention this is normally in dimeric form (and thus, e.g., comprises two IFNG polypeptide monomers modified as described).
- the dimeric form of the IFNG polypeptides may be provided by the normal association of two monomers or be in the form of a single chain dimeric IFNG polypeptide. It will be understood that the IFNG polypeptides of the invention may, in addition to the specified modifications, also contain other modifications, e.g.
- LFNG polypeptide also encompasses variant forms of the modified LFNG polypeptides disclosed herein.
- variants which, in addition to substitutions in the C-terminal part of the LFNG polypeptide, comprise further modifications include modifications such as S99T and E38N+S40T. More examples of suitable modifications are given below.
- the variant forms of the LFNG polypeptide differs in 1-15 or 2-15 amino acid residues (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues), e.g. in 1-10 or 2-10 amino acid residues, in 1-8 or 2-8 amino acid residues, in 1-5 or 2-5 amino acid residues, or in 1-3 or 2-3 amino acid residues compared to huLFNG shown in SEQ D NO:l.
- the term "functional site” is intended to indicate one or more amino acid residues which is/are essential for, or otherwise involved in, the function or performance of LFNG. Such amino acid residues are “located at” the functional site.
- the functional site may be determined by methods known in the art and is preferably identified by analysis of a structure of the polypeptide complexed to a relevant receptor, such as the LFNG receptor.
- huLFNG is intended to mean the mature form of wild-type human LFNG having the amino sequence shown in SEQ LD NO:l, including huLFNG produced by recombinant means. If not further specified, the term “huLFNG” may refer to wild-type human LFNG in its glycosylated form (i.e. glycosylated at positions 25 and 97) or in its un-glycosylated form.
- glycosylated indicates that the LFNG polypeptide is produced in a cell capable of glycosylating the polypeptide and, therefore, the LFNG polypeptide is glycosylated at its native N-glycosylation sites (position 25 and 97 of SEQ LD NO:l).
- un-glycosylated indicates that the LFNG polypeptide is not glycosylated at its native N-glycosylation sites (position 25 and 97 of SEQ LD NO:l).
- Such "un- glycosylated” LFNG polypeptides may be obtained by producing the polypeptide in a prokaryotic host cell, such as E. coli, not capable of glycosylation.
- the term “full-length” is intended to mean that the variant is not truncated as compared to the parent polypeptide, which is modified according to the invention.
- the parent protein to be modified as described herein is huLFNG.
- full-length means that the variant contains 143 amino acid residues.
- the parent protein to be modified is Met-huLFNG, the term “full-length” mean that the variant contains 144 amino acid residues.
- C-terminal part covers the last 12 amino acid residues (calculated from the C-terminus) in huLFNG, i.e. amino acid residues S132-Q143.
- Actimmune® refers to the 140 amino acid form of LFNG (disclosed in SEQ LD NO:3) achieved by fermentation of a genetically engineered E.coli bacterium. Actimmune® is un-glycosylated. Further information of Actimmune® is available on www.actimmune.com. Ln the present context the term “non-positively charged amino acid residue” covers the following amino acid residues: A, V, L, I, M, F, W, P, G, S, T, C, Y, N, Q, D and E.
- the present invention relates to a full-length LFNG polypeptide variant exhibiting LFNG activity, wherein said variant comprises (a) at least one substitution in a position selected from the group consisting of S132 and S142; and (b) at least one amino acid substitution in a position selected from the group consisting of R137, R139 and R140.
- the present invention also relates to a substantially homogenous population of a full-length LFNG variant, wherein said full-length LFNG variant exhibits IFNG activity and wherein said full-length LFNG variant comprises (a) at least one substitution in a position selected from the group consisting of S 132 and S142; and (b) at least one amino acid substitution in a position selected from the group consisting of R137, R139 and R140.
- the present invention also relates to a composition
- a composition comprising a substantially homogenous population of a full-length LFNG variant, wherein said full-length LFNG variant exhibits LFNG activity and wherein said full-length LFNG variant comprises (a) at least one substitution in a position selected from the group consisting of S132 and S142; and (b) at least one amino acid substitution in a position selected from the group consisting of R137, R139 and R140.
- the term "substantially homogenous population” is defined as a population giving rise to a mass spectroscopic profile characterized by a single, dominant peak having an area under the curve (AUC) that is at least 3-fold higher than the AUC of any other peak appearing in the profile.
- the AUC of the dominant peak is at least 4-fold higher, such as at least 5-fold higher than the AUC of any other peak appearing in the profile.
- the population of LFNG variants may contain at least 75% of the full- length LFNG variant of the invention (as compared to the total amount of LFNG variants in the population), preferably at least 80%, such as at least 85%, e.g. at least 90%, more preferably at least 95%, such as at least 96%, e.g. at least 97%, even more preferably at least 98%, such as at least 99%.
- R140 may be substituted with any amino acid residue, including lysine
- R137 and R139 may be substituted with any amino acid residue, except lysine.
- the amino acid residue to be introduced in one or more of the positions R137, R139 and/or R 140 is a non-positively charged amino acid residue.
- Amino acid residue to be introduced in one or more of the positions R137, R139 and/or R140 may be selected from the group consisting of small amino acid residues, such as Ala, Gly, Ser, Cys and Thr; acidic amino acid residues, such as Asp and Glu; hydrophobic amino acid residues, such as He, Leu, Met, Pro and Val; aromatic amino acid residues, such as Phe, Trp and Tyr; and polar amino acid residues, such as Asn and Gin. Ln a highly preferred embodiment of the invention the amino acid residue to be introduced in one or more of the positions R137, R139 and/or R140 is a proline residue.
- Amino acid residue to be introduced in position S132 or S142 may be selected from the group consisting of small amino acid residues, such as Ala, Gly, Ser, Cys and Thr; acidic amino acid residues, such as Asp and Glu: hydrophobic amino acid residues, such as Ue, Leu, Met, Pro and Val; aromatic amino acid residues, such as Phe, Trp and Tyr; and polar amino acid residues, such as Asn and Gin.
- the amino acid residue to be introduced in position S132 or S 142 is Pro.
- the variant comprises a substitution selected from the group consisting of S 132P, S 142P and S 132P+S 142P, in particular S 132P or S 142P.
- the C-terminal part of the LFNG variant should, in addition to a substitution in position S132 and/or S142, contain at least one further substitution in a position selected from the group consisiting of R137, R139, R140 and combinations thereof.
- the C-terminal part of the variant shall contain a substitution in a position selected from the group consisting of R137, R139, R140, R137+R139, R137+R140, R139+R140 and R137+R139+R140.
- substitutions in posititon R137 include R137A, R137N, R137L,
- substitutions in posititon R139 include R139A, R139N, R139L, R139I, R139M, R139F, R139W, R139P, R139G, R139S, R139T, R139C, R139Y, R139 ⁇ , R139Q, R139D and R139E, preferably R139P
- substitutions in posititon R140 include R140A, R140V, R140L, R140I, R140M, R140F, R140W, R140P, R140G, R140S, R140T, R140C, R140Y, R140N, R140Q, R140D, R140E and R140K, preferably R140P.
- the C-terminal part of the variant contains a substitution in a position selected from the group consisting of R137P, R139P, R140P, R137P+R139P, R137P+R140P, R139P+R140P and R137P+R139P+R140P.
- the present invention relates to a full-length LFNG polypeptide variant exhibiting LFNG activity, wherein said variant comprises an amino acid substitution in position R137 and an amino acid substitution in position R140. Furthermore, the present invention also relates to a substantially homogenous population of a full-length LFNG variant, wherein said full-length LFNG variant exhibits LFNG activity and wherein said full-length LFNG variant comprises an amino acid substitution in position 137 and an amino acid substitution in position R140.
- the present invention also relates to a composition
- a composition comprising a substantially homogenous population of a full-length LFNG variant, wherein said full-length LFNG variant exhibits LFNG activity and wherein said full-length LFNG variant comprises an amino acid substitution in position R137 and an amino acid substitution in position R140.
- substantially homogenous population is defined as a population giving rise to a mass spectroscopic profile characterized by a single, dominant peak having an area under the curve (AUC) that is at least 3 -fold higher than the AUC of any other peak appearing in the profile.
- AUC area under the curve
- the AUC of the dominant peak is at least 4-fold higher, such as at least 5 -fold higher than the AUC of any other peak appearing in the profile.
- the population of LFNG variants may contain at least 75% of the full- length LFNG variant of the invention (as compared to the total amount of LFNG variants in the population), preferably at least 80%, such as at least 85%, e.g. at least 90%, more preferably at least 95%, such as at least 96%, e.g. at least 97%, even more preferably at least 98%, such as at least 99%.
- Amino acid residue to be introduced in the positions R137 and R140 may independently be selected from the group consisting of small amino acid residues, such as Ala, Gly, Ser, Cys and Thr; acidic amino acid residues, such as Asp and Glu; hydrophobic amino acid residues, such as He, Leu, Met, Pro and Val; aromatic amino acid residues, such as Phe, Trp and Tyr; and polar amino acid residues, such as Asn and Gin.
- small amino acid residues such as Ala, Gly, Ser, Cys and Thr
- acidic amino acid residues such as Asp and Glu
- hydrophobic amino acid residues such as He, Leu, Met, Pro and Val
- aromatic amino acid residues such as Phe, Trp and Tyr
- polar amino acid residues such as Asn and Gin.
- Ln a preferred embodiment of the invention at least one of the amino acid residues to be introduced in the positions R137 and R140 is Pro.
- the variant comprises the substitutions R137X + R140P, wherein X is any amino acid residue, except arginine and lysine.
- the variant comprises the substitutions R137P + R140X, wherein X is any amino acid residue except arginine.
- the amino acid residue to be introduced in both of positions R137 and R140 is Pro, i.e. in a highly preferred embodiment of the invention, the variant comprises the substitutions R137P + R140P.
- the variant comprises at least one further modification in the C-terminal part of the variant from amino acid residue S132 to amino acid Q143.
- introduction of a non-naturally occurring residue in a human polypeptide may give rise to an epitope capable of inducing a response from the human immune system.
- This problem may be effectively solved by "shielding" the introduced residues, e.g. by introducing an in vivo N-glycosylation site or other non-polypeptide moieties, such as PEG, in the vicinity of the introduced residues.
- an amino acid residue comprising an attachment group for a non-polypeptide moiety (which is capable of screening the potential epitope) may be introduced in the vicinity of the introduced residues.
- One particular preferred amino acid residue comprising an attachment group for a non-polypeptide moiety, such as PEG, is cysteine.
- the introduced cyeteine residue may, when covalently attached to a non-polypeptide moiety, such as PEG, confer additional advantageous properties to the LFNG polypeptide, such as increased functional in vivo half-life and/or increased AUC SC .
- N-glycosylation sites in the C-terminal part of the LFNG polypeptide are also contemplated according to the present invention, this approach is not particularly preferred since full utilization of N-glycosylation sites in the C-terminal part of LFNG may prove difficult.
- the variant further comprises at least one cysteine residue in the C-terminal part of the variant from amino acid residue S 132 to amino acid Q143.
- substitutions which introduce a cysteine residue in the C- terminal part of the LFNG variant include substitutions selected from the group consisting of S132C, Q133C, M134C, L135C, F136C, R137C, G138C, R139C, R140C, A141C, S142 and Q143. It will be understood that cysteine residues should not be introduced in positions which are essential for obtaining the full-length LFNG gamma variant.
- cysteine residues may be introduced in the C-terminal part of the LFNG variant it is preferred that only a single cysteine residue is introduced in this region of the molecule.
- the introduced cysteine residue is preferably covalently attached (conjugated) to a non-polypeptide moiety, such as a polymer molecule, preferably PEG or more preferably mPEG having a molecular weight from 1-20 kDa, such as 1 kDa, 2 kDa, 5 kDa, 10 kDa, 12 kDa or 20 kDa.
- a polymer molecule preferably PEG or more preferably mPEG having a molecular weight from 1-20 kDa, such as 1 kDa, 2 kDa, 5 kDa, 10 kDa, 12 kDa or 20 kDa.
- the conjugation between the cysteine-containing LFNG polypeptide and the polymer molecule may be achieved in any suitable manner, e.g. as described in the section entitled "Conjugation to a polymer molecule", e.g. in using a one step method or in the stepwise manner referred
- the preferred method for PEGylating the LFNG polypeptide is to covalently attach PEG to cysteine residues using cysteine-reactive PEGs.
- cysteine-reactive PEGs with different groups (e.g. maleimide, vinylsulfone and orthopyridyl-disulfide) and different size PEGs (2-20 kDa) are commercially available, e.g. from Shearwater Polymers Lnc, Huntsville, AL, USA.
- this part of the sequence may be identical to residue no. 1 to residue no. 131 of huLFNG and may be glycosylated (e.g. by producing the variant in a glycosylation host cell) or un-glycosy- lated (e.g. by producing the variant in a prokaryotic host cell, such as E. coli).
- the variant comprises an amino acid sequence from residue no. 1 to residue no. 131, which further comprises 1-10, such as 1-7, e.g. 1-5 or 1-3 modifications, preferably substitutions, compared to amino acid residue no. 1 to residue no. 131 of huLFNG.
- 1-10 such as 1-7
- substitutions preferably substitutions, compared to amino acid residue no. 1 to residue no. 131 of huLFNG.
- substitution S99T leading to a more efficient utilization of the position 97 N-glycosylation site.
- Other interesting substitutions include E38N+S40T (leading to an increased AUC SC ), in particular E38N+S40+S99T.
- Such variants may be glycosylated or un-glycosylated. Preferably, such variants are glycosylated.
- amino acid sequence from residue no. 1 to residue no. 131 preferably comprises 1-10, such as 1-7, e.g. 1-5 or 1-3, modifications, preferably substitutions, compared to amino acid residue no. 1 to residue no. 131 of huLFNG.
- modifications preferably substitutions, compared to amino acid residue no. 1 to residue no. 131 of huLFNG.
- modifications which serve to optimise the glycosylation of a given glycosylation site.
- glycosylation of the naturally occurring N- glycosylation site located in position 97 of huLFNG may be increased, i.e. an increased fraction of fully, or substantially fully, glycosylated LFNG polypeptides may be obtained, by substituting the serine residue located in position 99 of huLFNG with a threonine residue.
- S99T substitution it has been found that about 90% of the polypeptides present in the harvested medium utilize both N-glycosylation sites, whereas only about 60% of the huLFNG polypeptides present in the harvested medium were fully glycosylated.
- the LFNG variant of the invention comprises the substitution S99T.
- in vivo glycosylation sites which may have been introduced into the sequence (see the section entitled "IFNG variants wherein the non- polypeptide moiety is a sugar moiety") may be optimised.
- the in vivo glycosylation site is an N-glycosylation site, but also an O-glycosylation site is contemplated as relevant for the present invention.
- This optimisation may be achieved by performing a modification, preferably a substitution, in a position, which is located close to a glycosylation site, in particular close to an in vivo N-glycosylation site.
- 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 is attached, preferably in position -1, +1, or +3, in particular in position +1 or +3.
- amino acid residue located close to an in vivo N-glycosylation site (having the sequence N-X-S/T/C) may be located in position -4, -3, -2, -1, +1, +2, +3 or +4 relative to the N-residue.
- the amino acid residue in said position must be either Ser, Thr or Cys.
- the modification of the amino acid residue in position +2 relative to the in vivo N-glycosylation site is a substitution where the amino acid residue in question is replaced with a Thr residue. If, on the other hand, said amino acid residue is already a Thr residue it is normally not preferred or necessary to perform any substitutions in that position.
- X should not be Pro and preferably not Trp, Asp, Glu and Leu.
- the amino acid residue to be introduced is preferably selected form the group consisting of Phe, Asn, Gin, Tyr, Val, Ala, Met, Lie, Lys, Gly, Arg, Thr, His, Cys and Ser, more preferably Ala, Met, lie, Lys, Gly, Arg, Thr, His, Cys and Ser, in particular Ala or Ser.
- the amino acid residue to be introduced is preferably selected from the group consisting of His, Asp, Ala, Met, Asn, Thr, Arg, Ser and Cys, more preferably Thr, Arg, Ser and Cys.
- Such modifications are particular relevant if the X residue is a Ser residue.
- the N-glycosylation site at position 97 may be further optimised by performing a modification, such as a substitution, in a position selected from the group consisting of E93, K94, L95, T96, Y98, N100 and T101 (i.e. at position -4, -3, -2, -1, +1, +3 or +4 relative to ⁇ 97).
- a modification such as a substitution
- substitutions performed in position 98 include Y98F, Y98N, Y98Q, Y98N, Y98A, Y98M, Y98I, Y98K, Y98G, Y98R, Y98T, Y98H, Y98C and Y98S, preferably Y98A, Y98M, Y98I, Y98K, Y98G, Y98R, Y98T, Y98H, Y98C and Y98S, in particular Y98S.
- substitutions performed in position 100 include N100H, N100D, N100A, N100M, V100 ⁇ , V100T, V100R, V100S, or V100C, in particular N100T, N100R, N100S or NIOOC.
- this site may be further optimised by performing a modification, such as a substitution, in a position selected from the group consisting of D21, N22, A23, D24, G26, L28 andF29 (i.e. at position -4, -3, -2, -1, +1, +3 or +4 relative to ⁇ 25).
- a modification such as a substitution
- substitutions performed in position 26 include G26F, G26N, G26Y, G26Q, G26V, G26A, G26M, G26I, G26 , G26R, G26T, G26H, G26C and G26S, preferably G26A, G26M, G26I, G26K, G26R, G26T, G26H, G26C and G26S, more preferably G26A and G26S, in particular G26A.
- substitutions performed in position 28 include G28H, G28D, G28A, G28M, G28N, G28T, G28R, G28S, or G28S, in particular G28A, G28T, G28R, G28S or G28C.
- any of the modifications mentioned in connection with optimisation of glycosylation at position 97 may be combined with any of the mentioned in connection with optimisation of glycosylation at position 25.
- IFNG variants with increased All C sc and/ or increased half -life Another class of interesting modifications that may be introduced into the amino acid sequence from residue no. 1 to residue no. 131 include modifications, in particular substitutions, which serve to increase the AUC SC and/or the serum half-life/functional in vivo half -life when administered intravenously.
- the LFNG variant comprises, in the amino acid sequence from residue no. 1 to residue no. 131, at least one introduced and/or at least one removed amino acid residue comprising an attachment group for a non-polypeptide moiety.
- the amino acid sequence from residue no. 1 to residue no. 131 comprises at least one introduced amino acid residue comprising an attachment group for a non-polypeptide moiety.
- Such variants typically exhibit an increased functional in vivo half-life and/or an increased AUC SC .
- interesting LFNG variants are such variants where the ratio between the serum half-life (or functional in vivo half -life) of said variant and the serum half -life (or functional in vivo half-life) of glycosylated huLFNG or glycosylated [S99T]huLFNG is at least 1.25, more preferably at least 1.50, such as at least 1.75, e.g. at least 2, even more preferably at least 3, such as at least 4, e.g. at least 5, when administered intravenously, in particular when administered intraveniously in non-human primates, such as monkeys.
- LFNG variants are such variants where the ratio between the serum half-life (or functional in vivo half -life) of said variant and the serum half-life (or functional in vivo half -life) of Actimmune® is at least 2 more preferably at least 3, such as at least 4, e.g. at least 5, even more preferably at least 6, such as at least 7, e.g. at least 8, most preferably at least 9, such as at least 10, when administered intravenously, in particular when administered intraveniously into rats or non-human primates, such as monkeys.
- the term "increased" as used about the AUC SC is used to indicate that the Area Under the Curve for an LFNG variant of the invention, when administered subcutaneously, is statistically significantly increased relative to that of a reference molecule, such as glycosylated huLFNG, glycosylated [S99T]huLFNG or Actimmune®, determined under comparable conditions.
- a reference molecule such as glycosylated huLFNG, glycosylated [S99T]huLFNG or Actimmune®, determined under comparable conditions.
- preferred LFNG variants are such variants, which have an increased AUC SC , as compared to any of the reference molecules mentioned above.
- the same amount of LFNG activity should be administered for the LFNG variant of the invention and the reference molecule.
- Particular preferred LFNG variants are such variants where the ratio between the AUC SC of said variant and the AUC SC of glycosylated huLFNG or glycosylated [S99T]huLFNG is at least 1.25, such as at least 1.5, e.g. at least 2, more preferably at least 3, such as at least 4, e.g. at least 5 or at least 6, even more preferably at least 7, such as at least 8, e.g. at least 9 or at least 10, most preferably at least 12, such as at least 14, e.g. at least 16, at least 18 or at least 20, in particular when administered (subcutaneously) in rats.
- LFNG variants are such variants where the ratio between the AUC SC of said variant and the AUC SC of Actimmune® is at least 100, more preferably at least 150, such as at least 200, e.g. at least 250, even more preferably at least 300, such as at least 400 e.g. at least 500, most preferably at least 750, such as at least 1000, e.g. at least 1500 or at least 2000, in particular when administered (subcutaneously) in rats.
- the total number of amino acid residues to be modified in accordance with this embodiment of the invention typically does not exceed 10.
- the amino acid sequence from residue no. 1 to residue no. 131 comprises 1-10, such as 1-7, e.g. 1-5 or 1-3 modifications compared to residue no. 1 to residue no. 131 of huLFNG.
- the LFNG variant comprises an amino acid sequence (from residue no. 1 to residue no. 131) which differs from the amino acid sequence of huLFNG (from residue no. 1 to residue no. 131) in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.
- the modification(s) is/are a substitution(s).
- the LFNG polypeptide is boosted or otherwise altered in the content of the specific amino acid residues to which the relevant non-polypeptide moiety binds, whereby a more efficient, specific and/or extensive conjugation is achieved.
- removal of one or more attachment groups it is possible to avoid conjugation to the non-polypeptide moiety in parts of the polypeptide in which such conjugation is disadvantageous, e.g. to an amino acid residue located at or near a functional site of the polypeptide (since conjugation at such a site may result in inactivation of the resulting conjugated polypeptide due to impaired receptor recognition). Further, it may be advantageous to remove an attachment group located closely to another attachment group in order to avoid heterogeneous conjugation to such groups.
- more than one amino acid residue of the LFNG polypeptide is altered, e.g. the alteration embraces removal as well as introduction of amino acid residues comprising attachment sites for the non- polypeptide moiety of choice.
- This embodiment is considered of particular interest in that it is possible to specifically design the LFNG polypeptide so as to obtain an optimal conjugation to the non-polypeptide moiety.
- polypeptide may comprise other modifications, e.g. substitutions, that are not related to introduction and/or removal of amino acid residues comprising an attachment group for the non-polypeptide moiety.
- modifications include conservative amino acid substitutions and/or introduction of Cys-Tyr-Cys or Met at the N-terminus.
- the exact number of attachment groups available for conjugation and present in the LFNG polypeptide in dimeric form is dependent on the effect desired to be achieved by the conjugation.
- the effect to be obtained is, e.g., dependent on the nature and degree of conjugation (e.g. the identity of the non-polypeptide moiety, the number of non-polypeptide moieties desirable or possible to conjugate to the polypeptide, where they should be conjugated or where conjugation should be avoided, etc.).
- amino acid residue comprising an attachment group for a non-polypeptide moiety is selected on the basis of the nature of the non-polypeptide moiety part of choice and, in most instances, on the basis of the conjugation method to be used.
- the non-polypeptide moiety is a polymer molecule such as a polyethylene glycol- or polyalkylene oxide-derived molecule
- amino acid residues capable of functioning as an attachment group may be selected from the group consisting of cysteine, lysine, aspartic acid, glutamic acid and arginine.
- cysteine is preferred.
- the attachment group is, e.g., an in vivo glycosylation site, preferably an N-glycosylation site.
- the position is preferably located at the surface of the LFNG polypeptide, and more preferably occupied by an amino acid residue that has more than 25% of its side chain exposed to the surface, in particular more than 50% of its side chain exposed to the surface, as determined on the basis of a 3D structure or model of LFNG in its dimeric form, the structure or model optionally further comprising one or two LFNG receptor molecules.
- Such positions are listed in Example 1 herein.
- loop regions of LFNG may be of interest to modify one or more amino acid residues located in the loop regions of LFNG since most amino acid residues within these loop regions are exposed to the surface and located sufficiently far away from functional sites so that non-polypeptide moieties, such as polymer molecules, in particular PEG molecules, and or N-glycosylation sites, may be introduced without impairing the function of the molecule.
- non-polypeptide moieties such as polymer molecules, in particular PEG molecules, and or N-glycosylation sites
- amino acid residues constituting said loop regions are residues N16-K37 (the "A-B loop"), F60-S65 (the “B-C loop”), N83-S84 (the "C-D loop”) and Y98-L103 (the "D-E loop”).
- attachment groups located at the receptor-binding site of LFNG may be removed, preferably by substitution of the amino acid residue comprising such group.
- the amino acid residues constituting the LFNG receptor-binding site are Ql, D2, Y4, V5, E9, K12, G18, H19, S20, D21, N22, A23, D24, ⁇ 25, G26, T27, L30, K34, K37, K108, Hill, El 12, 1114, Q115, A118 and El 19 (see also Example 2 herein).
- a single chain LFNG polypeptide it may be sufficient to remove attachment groups in the receptor-binding site of only one of the monomers and thereby obtain a single chain LFNG polypeptide conjugate with one active and one inactive receptor-binding site.
- the distance between amino acid residues located at the surface of the FNG polypeptide is calculated on the basis of a 3D structure of the LFNG dimeric polypeptide. More specifically, the distance between the CB's of the amino acid residues comprising such attachment groups, or the distance between the functional group (NZ for lysine, CG for aspartic acid, CD for glutamic acid, SG for cysteine) of one and the CB of another amino acid residue comprising an attachment group are determined.
- CA is used instead of CB.
- the LFNG polypeptide part of the invention any of said distances is preferably more than 8 A, in particular more than 10A in order to avoid or reduce heterogeneous conjugation.
- the amino acid sequence of the LFNG polypeptide variant may differ from the huLFNG amino acid sequence in that one or more amino acid residues constituting part of an epitope has been removed, preferably by substitution to an amino acid residue comprising an attachment group for the non-polypeptide moiety, so as to destroy or inactivate the epitope.
- Epitopes of huLFNG may be identified by use of methods known in the art, also known as epitope mapping, see, e.g.
- IgGl antibodies from specific antisera towards huLFNG are purified by immunoprecipitation and the reactive phages are identified by immunoblotting.
- the sequence of the oligopeptide can be determined followed by localization of the sequence on the 3D-structure of the LFNG.
- the thereby identified region on the structure constitutes an epitope that then can be selected as a target region for introduction of an attachment group for the non- polypeptide moiety.
- the LFNG polypeptide of the invention has a molecular weight of at least 67 kDa, in particular at least 70 kDa as measured by SDS-PAGE according to Laemmli, U.K., Nature Vol 227 (1970), p680-85.
- LFNG has a MW in the range of about 34-50 kDa, and therefore additional about 20-40kDa is required to obtain the desired effect. This may, e.g., be provided by 2-4 lOkDa PEG molecules or by a combination of additional in vivo glycosylation sites and additional PEG molecules, or as otherwise described herein.
- a conjugated LFNG polypeptide according to the invention comprises 1-10 (additional) non-polypeptide moieties, such as 1-8, 2-8, 1-5 or 2-5 (additional) non-polypeptide moieties.
- the polypeptide will comprise 1-3 (additional) non-polypeptide moieties, such as 1, 2 or 3 (additional) non-polypeptide moieties.
- LFNG exists as a dimeric polypeptide.
- the polypeptide is normally in homodimeric form (e.g. prepared by association of two LFNG polypeptide molecules prepared as described herein).
- the LFNG polypeptide may be provided in single chain form, wherein two LFNG polypeptide monomers are linked via a peptide bond or a peptide linker.
- Providing the LFNG polypeptide in single chain form has the advantage that the two constituent LFNG polypeptides may be different which can be advantageous, e.g., to enable asymmetric mutagenesis of the polypeptides.
- PEGylation sites can be removed from the receptor-binding site from one of the monomers, but retained in the other. Thereby, after PEGylation one monomer has an intact receptor-binding site, whereas the other may be fully PEGylated (and thus provide significantly increased molecular weight).
- IFNG variants wherein the non-polypeptide moiety is a sugar moiety
- the LFNG variant comprises, in the amino acid sequence from residue no. 1 to residue no. 131, at least one introduced and/or at least one removed glycosylation site, i.e. the non-polypeptide moiety is a sugar moiety.
- the glycosylation site is an in vivo glycosylation site, e.g. an O-linked or N-linked sugar moiety, preferably an N-linked sugar moiety.
- said LFNG variant comprises, in the amino acid sequence from residue no. 1 to residue no. 131, at least one introduced glycosylation site, in particular an introduced in vivo N-glycosylation site.
- the introduced glycosylation site is introduced by a substitution.
- an in vivo N-glycosylation site may be introduced into a position (from residue no. 1 to residue no. 131) of the LFNG variant comprising an amino acid residue exposed to the surface.
- said surface-exposed amino acid residue has at least 25% of the side chain exposed to the surface, in particular at least 50% of its side chain exposed to the surface. Details regarding determination of such positions can be found in Example 1 herein.
- the N-glycosylation site is introduced in such a way that the N-residue of said site is located in said position.
- an O-glycosylation site is introduced so that the S or T residue making up such site is located in said position.
- the in vivo glycosylation site in particular the N residue of the N-glycosylation site or the S or T residue of the O-glycosylation site, is located within the 118 N-terminal amino acid residues of the LFNG polypeptide, more preferably within the 97 N-terminal amino acid residues. Still more preferably, the in vivo glycosylation site is introduced into a position wherein only one mutation is required to create the site (i.e. where any other amino acid residues required for creating a functional glycosylation site is already present in the molecule).
- substitutions that lead to introduction of an additional N-glycosylation site at positions exposed at the surface of the LFNG polypeptide and occupied by amino acid residues having at least 25% of the side chain exposed to the surface include: Q1N+P3S/T, P3N+V5S/T, K6N+A8S/T, E9N+L11S/T, K12S/T, K13N+F15S/T, Y14N+N16S/T, G18S/T, G18N, G18N+S20T, H19N+D21S/T, D21N+A23S/T, G26N+L28S/T, G31N+L33S/T, K34N+W36S/T, K37S/T, K37N+E39S/T, E38N, E38N+S40T, E39N+D41S/T, S40N+R42S/T, K55N+F57S
- S/T indicates a substitution to a serine or threonine residue, preferably a threonine residue.
- substitutions that lead to introduction of an additional N-glycosylation site at positions exposed at the surface of the LFNG polypeptide having at least 50% of the side chain exposed to the surface (in a structure with receptor molecule) include:
- Substitutions where only one amino acid substitution is required to introduce an N- glycosylation site include K12S/T, G18S/T, G18N, K37S/T, E38N, M45N, I49N, K61S/T, D63N, Q67N, V70N, K80S/T, F82N, N85S/T, K87S/T, K94N, Q106S/T, E119N, A124N, K130N and R140N, in particular G18N, G18S/T, K37S/T, E38N, K61S/T, D63N, Q67N, K80S/T, N85S/T, K94N, Q106S/T, A124N and K130N (positions with more than 25% of its site chain exposed to the surface in a structure without receptor molecule), or more preferably G18N, E38N, D63N, Q67N, K94N, A124N and K130N (positions with more than 50% of its side chain exposed
- the mutations Q1N+P3S/T, E9N+L11S/T, G18N, G18N+S20T, H19N+D21S/T, D21N+A23S/T, G26N+L28S/T, K34N+W36S/T, K37N+E39S/T, El 19N and El 19N+S121T should normally not be performed, unless a reduced receptor affinity is desired.
- the positive cluster K128, R129, K130 and R131 is required for activity and should normally not be modified.
- Particular preferred LFNG variants of invention include at least one further substitution selected from the group consisting of G18S, G18T, E38N, E38N+S40T, K61S, K61T, S65N+Q67S, S65N+Q67T, N85S, N85T, K94N, Q106S and Q106T, more preferably selected from the group consisting of G18T, E38N+S40T, K61T, S65N+Q67T, N85T, K94N and Q106T, even more preferably selected from the group consisting of G18T, E38N+S40T, K61T, S65N+Q67T and N85T, in particular E38N+S40T.
- specific examples of interesting full-length variants of the invention include variants selected from the group consisting of
- [N85T+S99T+R137P+R140P]huLFNG more preferably selected from the group consisting of [G18T+S99T+R137P+R140P]huLFNG,
- the LFNG polypeptide variant of the invention preferably contains a single additional in vivo glycosylation site in the amino acid sequence from residue no. 1 to residue no. 131.
- the polypeptide variant comprises more than one additional in vivo N-glycosylation site, in particular 2-7 or 2-5 additional in vivo N- glycosylation sites, such as 2, 3, 4, 5, 6 or 7 in vivo N-glycosylation sites.
- additional in vivo N- glycosylation sites are preferably introduced by one or more substitutions described in any of the above lists.
- the LFNG polypeptide comprises at least two introduced glycosylation sites, in particular at least two introduced N- glycosylation sites in the amino acid sequence from residue no. 1 to residue no. 131.
- the at least two modifications, in particular substitutions, leading to the introduction of the at least two introduced N-glycosylation sites may preferably be selected from the group consisting of G18S, G18T, E38N, E38N+S40T, K61S, K61T, S65N+Q67S, S65N+Q67T, N85S, N85T, K94N, Q106S and Q106T, more preferably selected from the group consisting of
- G18T, E38N+S40T, K61T, S65N+Q67T, N85T, K94N and Q106T even more preferably selected from the group consisting of G18T, E38N+S40T, K61T, S65N+Q67T and N85T.
- substitutions giving rise to an LFNG variant comprising at least two additional N-glycosylation sites in the amino acid sequence from residue no. 1 to residue no. 131 include: G18T+E38N+S40T, G18T+K61T, G18T+ S65N+Q67T, G18T+N85T,
- any of the above-mentioned modified LFNG variants further comprises the substitution S99T.
- the naturally occurring N-glycosylation site located at position 25 may be removed. This may be done by removing the N25 residue and/or by removing the T27 residue, preferably by substitution. Preferably, the N-glycosylation site located at position 25 may be removed by the substitution N25G+T27P.
- the LFNG variant comprises, in the amino acid sequence from residue no. 1 to residue no. 131, at least one introduced cysteine residue.
- the cysteine residue is introduced by substitution.
- a cysteine residue may be introduced into a position of the LFNG polypeptide (from residue no. 1 to residue no. 131), which comprises an amino acid residue exposed to the surface.
- said surface-exposed amino acid residue has at least 25% of the side chain exposed to the surface, in particular at least 50% of its side chain exposed to the surface. Details regarding determination of such positions can be found in Example 1 herein.
- substitutions that lead to introduction of a cysteine residue at positions exposed at the surface of the LFNG polypeptide and occupied by amino acid residue having at least 25% of the side chain exposed to the surface include: QIC, D2C, P3C, K6C, E9C, N10C, K13C, Y14C, N16C, G18C, H19C, D21C, N25C, G26C, G31C, K34C, N35C, K37C, E38C, E39C, S40C, K55C, K58C, N59C, K61C, D62C, D63C, Q64C, S65C, Q67C, K68C, E71C, T72C, K74C, E75C, N78C, V79C, K80C, N83C, S84C, N85C, K86C, K87C, D90C, E93C, K94C, T101C, D102C, L103
- Substitutions that lead to introduction of a cysteine residue at positions exposed at the surface of the LFNG polypeptide and occupied by amino acid residue having at least 50% of the side chain exposed to the surface include: P3C, K6C, N10C, K13C, N16C, D21C, N25C, G26C, G31C, K34C, K37C, E38C, E39C, K55C, K58C, N59C, D62C, Q64C, S65C, K68C, E71C, E75C, N83C, S84C, K86C, K87C, K94C, T101C,
- cysteine residue and subsequently attaching these cysteine residue to a non-polypeptide moiety in the region constituting the receptor binding site (except in special cases, cf. the section entitled "Variants with a reduced receptor affinity”). Accordingly, the mutations QIC, E9C, G18C, H19C, D21C, G26C, K34C, K37C and
- E119C should normally not be performed, unless a reduced receptor affinity is desired.
- the positive cluster K128, R129, K130 and R131 is required for activity and should normally not be modified.
- said cysteine residue is introduced by a substitution selected from the group consisting of N10C, N16C, E38C, N59C, S65C, N83C, K94C, N104C and A124C, such as N16C, N59C and N16C+N59C.
- said cysteine residue is introduced by the substitution N16C or N59C.
- [N59C+S99T+R137P+R140P]huLFNG most preferably selected from the group consisting of [N16C+S99T+R137P-rR140P]huLFNG and [N59C+S99T+R137P+R140P]huLFNG.
- the LFNG variant of the invention preferably contains a single cysteine residue in the amino acid sequence from residue no. 1 to residue no. 131.
- the polypeptide comprises more than one cysteine, in particular 2-7 or 2-5 cysteine residues, such as 2, 3, 4, 5, 6 or 7 cysteine residues.
- cysteine residues are preferably introduced by one or more substitutions described in any of the above lists.
- the LFNG variant comprises at least two introduced cysteine residues in the amino acid sequence from residue no. 1 to residue no. 131.
- the at least two modifications, in particular substitutions, leading to the introduction of the at least two cysteine residues may preferably be selected from the group consisting of N10C, N16C, E38C, N59C, S65C, N83C, K94C, N104C and A124C. Specific examples of such substitutions giving rise to an LFNG polypeptide comprising at least two cysteine residues (in the amino acid sequence from residue no.l to residue no.
- 131) include: N10C+N16C, N10C+E38C, N10C+N59C, N10C+S65C, N10C+N83C, N10C+K94C, N10C+N104C, N10C+A124C, N16C+E38C, N16C+N59C, N16C+S65C, N16C+N83C, N16C+K94C, N16C+N104C, N16C+A124C, E38C+N59C, E38C+S65C, E38C+N83C, E38C+K94C, E38C+N104C, E38C+A124C, N59C+S65C, N59C+N83C, N59C+K94C, N59C+N104C, N59C+A124C, S65C+N83C, S65C+K94C, S65C+N104C, S65C+A124C, N83C, N83C+K94
- any of the above-mentioned modified LFNG polypeptides further comprises the substitution S99T.
- the naturally occurring N-glycosylation site located at position 25 may be removed. This may be done by removing the N25 residue and/or by removing the T27 residue, preferably by substitution.
- the N-glycosylation site located at position 25 may be removed by the substitution N25G+T27P.
- the introduced cysteine residue(s) may preferably be conjugated to a non-polypeptide moiety, such as PEG or more preferably mPEG.
- the conjugation between the cysteine-containing polypeptide and the polymer molecule may be achieved in any suitable manner, e.g. as described in the section entitled "Conjugation to a polymer molecule", e.g. in using a one step method or in the stepwise manner referred to in said section.
- the preferred method for PEGylating the LFNG polypeptide is to covalently attach PEG to cysteine residues using cysteine-reactive PEGs.
- a number of highly specific, cysteine-reactive PEGs with different groups e.g.
- maleimide, vinylsulfone and orthopyridyl-disulfide) and different size PEGs are commercially available, e.g. from Shearwater Polymers Inc., Hunts ville, AL, USA).
- IFNG variants wherein a first non-polypeptide moiety is a sugar moiety and a second non- polypeptide moiety is a molecule, which has cysteine as an attachment group
- the LFNG variant comprises, in the amino acid sequence from residue no. 1 to residue no. 131, at least one introduced N-glycosylation site and at least one introduced cysteine residue.
- the cysteine residue and the N- glycosylation site is introduced by substitution.
- Such polypeptides may be prepared by selecting the residues described in the two preceding sections describing suitable positions for introducing N-glycosylation sites and cysteine residues, respectively.
- said LFNG variants comprises substitutions selected from the group consisting of G18T+N10C, G18T+E38C, G18T+N59C, G18T+S65C, G18T+N83C, G18T+K94C, G18T+N104C, G18T+A124C, E38N+S40T+N10C, E38N+S40T+N16C, E38N+S40T+N59C, E38N+S40T+S65C, E38N+S40T+N83C, E38N+S40T+K94C, E38N+S40T+N104C, E38N+S40T+A124C, K61T+N10C, K61T+N16C, K61T+E38C, K61T+S65C, K61T+N83C, K61T+K94C, K61T+N104C, K61T+A124C, N85T+N
- any of the above-mentioned modified LFNG polypeptides further comprises the substitution S99T.
- the LFNG variant comprises, in the amino acid sequence from residue no. 1 to residue no. 131, at least one removed N- glycosylation site and at least one introduced cysteine residue.
- the cysteine residue is introduced by substitution and the N-glycosylation site is removed by substitution.
- the removed N-glycosylation site is the N-glycosylation site located at position 25. This may be done by removing the N25 residue and or by removing the T27 residue, preferably by the substitution N25G+T27P.
- One way to increase the serum half -life or the functional in vivo half-life of an LFNG polypeptide would be to decrease the receptor-mediated intemalisation and thereby decrease the receptor-mediated clearance.
- the receptor-mediated intemalisation is dependent upon the affinity of the LFNG dimer for the LFNG receptor complex and, accordingly, an LFNG polypeptide with a decreased affinity to the LFNG receptor complex is expected to be internalised, and hence cleared, to a lesser extent.
- the affinity of the LFNG dimer to its receptor complex may be decreased by performing one or more modifications, in particular substitutions, in the recpetor binding region of the LFNG polypeptide.
- the amino acid residues which constitute the receptor binding region is defined in Example 2 herein.
- One class of substitutions that may be performed is conservative amino acid substitutions.
- the modification performed gives rise to the introduction of an N-glycosylation site.
- the LFNG polypeptide comprises, in the amino acid sequence from residue no. 1 to residue no. 131, at least one modification in the receptor binding site (as defined herein). More particularly, the LFNG polypeptide comprises at least one substitution, preferably a substitution, which creates an in vivo N-glycosylation site, in said receptor binding region.
- substitutions may be selected from the group consisting of Q1N+P3S/T, D2N+Y4S/T, Y4N+K6S/T, V5N+E7S/T, E9N+L11S/T, K12N+Y14S/T, G18N, G18N+S20T, H19N+D21S/T, S20N+N22S/T, D21 ⁇ +A23S/T, V22N+D24S/T, D24N+G26S/T, G26N+L28S/T, L30N+I32S/T, K34N+W36S/T, K37N+E39S/T, K108N+I110S/T, H111N+L113S/T, E112N+I114S/T, I114N+N116S/T, Q115 ⁇ +M117S/T, A118N+L120S/T, E119N and E119N+S121T, preferably from the group consisting
- Such variants are contemplated to exhibit a reduced receptor affinity as compared to glycosylated huLFNG, glycosylated [S99T]huLFNG or Actimmune®.
- the receptor affinity may be measured by any suitable assay and will be known to the person skilled in the art.
- One example of a suitable assay for determining the receptor binding affinity is the BIAcore® assay described in Michiels et al. Int. J. Biochem. Cell Biol. 30:505-516 (1998).
- LFNG polypeptides considered useful for the purposes described herein are such LFNG polypeptides, wherein the binding affinity (Kj) is 1-95% of the K -value of glycosylated huLFNG, glycosylated [S99T]huLFNG or Actimmune®.
- the K d -value of the LFNG polypeptide may be 1-75% or 1-50%, such as 1-25%, e.g. 1-20% or even as low as 1-15%, 1-10% or 1-5% of the K d -value of glycosylated huLFNG, glycosylated [S99T]huLFNG or Actimmune®.
- such LFNG polypeptides having reduced receptor affinity will exhibit a reduced LFNG activity, e.g. when tested in the "Primary Assay" described herein.
- the LFNG polypeptide may exhibit 1-95% (e.g. 5-95%) of the LFNG activity of glycosylated huLFNG, glycosylated [S99T]huLFNG or Actimmune®, e.g. 1-75% (e.g. 5-75%), such as 1- 50% (e.g. 5-50%), e.g. 1-20% (e.g. 5-20%) or 1-10% (e.g. 5-10%) of the LFNG activity of glycosylated huLFNG, glycosylated [S99T]huLFNG or Actimmune®.
- LFNG polypeptides are contemplated to possess an increased half -life due to the reduced receptor-mediated clearence. Therefore, the LFNG polypeptides according to this aspect of the invention are contemplated to fulfil the requirements with respect to increased half-life described previously herein in connection with the definition of increased half-life.
- cysteine variant wherein said cysteine is covalently attached to a non-polypeptide moiety, such as PEG, also exhibit a reduced receptor-binding affinity and hence a lowered LFNG activity when tested in the "Primary Assay" described herein. It is envisaged that this property may be achieved independently of whether the cysteine (and hence the non-polypeptide moiety) is introduced in the receptor binding site since the non-polypeptide moiety is normally of such size that it may interact partially impair binding to the LFNG polypeptide to its receptor independently of whether said moiety is introduced in the receptor binding site or not.
- a non-polypeptide moiety such as PEG
- any of the above-mentioned modifications giving rise to a reduced receptor binding affinity may be combined with any of the other modifications disclosed herein, in particular the modifications mentioned in the sections entitled “IFNG variants with optimised N-glycosylation sites", "IFNG variants wherein the non-polypeptide moiety is a sugar moiety", “IFNG variants wherein the non-polypeptide moiety is a molecule, which has cysteine as an attachment group” and "IFNG variants wherein the first non-polypeptide moiety is a sugar moiety and the second non-polypeptide moiety is a molecule, which has cysteine as an attachment group", such as the modifications selected from the group consisiting of E38N+S40T, S99T and combinations thereof.
- Determination of C-terminal truncation of purified samples of LFNG polypeptides can be carried out in a number of ways.
- One way of elucidating C-terminal truncations of IFNG polypeptides relies on accurate mass determinations by mass spectrometry.
- the glycosylation of LFNG is heterogeneous thus making it extremely difficult to determine an accurate mass directly on the glycoprotein. Therefore, different levels of enzymatic deglycosylation are typically used in combination with mass spectrometry.
- the entire glycan part of the LFNG polypeptide is cleaved of using the endo-glycosidase PNGase F followed by accurate mass determination using either ESI mass spectrometry or MALDI-TOF mass spectrometry. Comparing the experimental masses to the known amino acid sequence of LFNG makes it possible to determine the sites of C-terminal truncation.
- sialic acid of the glycan part of the LFNG polypeptide is cleaved off instead of the entire glycan. In some cases this is sufficient to reduce the heterogeneity of the sample to a level where the sites of C-terminal truncations can be deduced following accurate mass determination using either ESI mass spectrometry or MALDI- TOF mass spectrometry.
- LFNG polypeptide mapping in combination with mass spectrometry and chemical amino acid sequencing.
- the LFNG polypeptide is degraded with a protease of known specificity (e.g. Asp-N protease) followed by peptide separation using RP-HPLC. Fractions can then by mass analysed either on-line using ESI mass spectrometry or off-line using MALDI-TOF mass spectrometry. Comparing the masses obtained for peptides with the known amino acid sequence of LFNG makes it possible to determine the likely sites of C-terminal truncation. Verification can then be obtained through amino acid sequencing.
- the non-polypeptide moiety is preferably selected from the group consisting of a sugar moiety (e.g. by way of in vivo N-glycosylation), a polymer molecule, a lipophilic compound and an organic derivatizing agent. All of these agents may confer desirable properties to the LFNG polypeptide, in particular increased AUC SC , increased serum half-life, increased functional in vivo half-life when administered intravenously, reduced immunogenicity and/or increased bioavailability.
- the polypeptide is normally conjugated to only one type of non-polypeptide moiety, but may also be conjugated to two or more different types of non-polypeptide moieties, e.g.
- the polypeptide and the lipophilic compound may be conjugated to each other, either directly or by use of a linker.
- the lipophilic compound may be a natural compound such as a saturated or unsaturated fatty acid, a fatty acid diketone, a terpene, a prostaglandin, a vitamine, a carotenoide or steroide, or a synthetic compound such as a carbon acid, an alcohol, an amine and sulphonic acid with one or more alkyl-, aryl-, alkenyl- or other multiple unsaturated compounds.
- the conjugation between the polypeptide and the lipophilic compound, optionally through a linker may be done according to methods known in the art, e.g. as described by Bodanszky in Peptide Synthesis, John Wiley, New York, 1976 and in WO 96/12505.
- the polymer molecule to be coupled to the polypeptide may be any suitable polymer molecule, such as a natural or synthetic homo-polymer or heteropolymer, typically with a molecular weight in the range of 300-100,000 Da or 1000-50,000 Da, such as in the range of 2000- 40,000 Da or 2000-30,000 Da, e.g. in the range of 2000-20,000 Da, 2000-10,000 Da or 1000-5000 Da. More specifically, the polymer molecule, such as PEG, in particular mPEG, will typically have a molecular weight of about 2, 5, 10, 12, 15, 20, 30, 40 or 50 kDa. Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine (i.e.
- a hetero-polymer is a polymer, which comprises one or more different coupling groups, such as, e.g., a hydroxyl group and an amine group.
- suitable polymer molecules include polymer molecules selected from the group consisting of polyalkylene oxide (PAO), including polyalkylene glycol (PAG), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, poly-vinyl alcohol (PVA), poly-carboxylate, poly-(vmylpyrolidone), polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextran including carboxymethyl-dextran, or any other biopolymer suitable for reducing immunogenicity and/or increasing functional in vivo half-life and/or serum half-life and/or increasing the AUC SC .
- PAO polyalkylene oxide
- PAG polyalkylene glycol
- PEG polyethylene glycol
- PPG polypropylene glycol
- PVA poly-vinyl alcohol
- PVA poly-carboxylate
- poly-(vmylpyrolidone) polyethylene-co-maleic acid anhydr
- polymer molecule is human albumin or another abundant plasma protein.
- polyalkylene glycol-derived polymers are biocompatible, non-toxic, non-antigenic, non-immunogenic, have various water solubility properties, and are easily secreted from living organisms.
- PEG is the preferred polymer molecule to be used, since it has only few reactive groups capable of cross-linking compared, e.g., to polysaccharides such as dextran, and the like.
- monofunctional PEG e.g., monomethoxypolyethylene glycol (mPEG)
- mPEG monomethoxypolyethylene glycol
- the hydroxyl end groups of the polymer molecule must be provided in activated form, i.e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl proprionate (SPA), succinimidy carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)).
- reactive functional groups include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl proprionate (SPA), succinimidy carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), al
- activated polymer molecules are commercially available, e.g. from Shearwater Polymers, Inc., Huntsville, AL, USA.
- the polymer molecules can be activated by conventional methods known in the art, e.g. as disclosed in WO 90/13540.
- Specific examples of activated linear or branched polymer molecules for use in the present invention are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogue (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference).
- Specific examples of activated PEG polymers include the following linear PEGs: NHS-PEG (e.g.
- SPA-PEG SSPA-PEG, SB A- PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM-PEG), and NOR-PEG
- BTC-PEG EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, NS-PEG, IODO-PEG, and MAL-PEG
- branched PEGs such as PEG2- ⁇ HS and those disclosed in US 5,932,462 and US 5,643,575, both of which references are incorporated herein by reference.
- the conjugation of the polypeptide variant and the activated polymer molecules is conducted by use of any conventional method, e.g. as described in the following references (which also describe suitable methods for activation of polymer molecules): Harris and Zalipsky, eds., Poly(ethylene glycol) Chemistry and Biological Applications, AZC, Washington; R.F. Taylor, (1991), “Protein immobilisation. Fundamental and applications", Marcel Dekker, N.Y.; S.S. Wong, (1992), “Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; G.T. Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques", Academic Press, N.Y.).
- the activation method and/or conjugation chemistry to be used depends on the attachment group(s) of the variant polypeptide (examples of which are given further above), as well as the functional groups of the polymer (e.g. being amine, hydroxyl, carboxyl, aldehyde, sulfydryl, succinimidyl, maleimide, vinysulfone or haloacetate).
- the PEGylation may be directed towards conjugation to all available attachment groups on the variant polypeptide (i.e. such attachment groups that are exposed at the surface of the polypeptide) or may be directed towards one or more specific attachment groups, e.g.
- the N- terminal amino group as described in US 5,985,265 or to cysteine residues may be achieved in one step or in a stepwise manner (e.g. as described in WO 99/55377).
- PEGylation is achieved by co ⁇ jugatin the PEG group(s) to introduced cysteine residues.
- activated PEG polymers particularly preferred for coupling to cysteine residues include the following linear PEGs: vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG); maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) and orthopyridyl-disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG).
- vinylsulfone-PEG VS-PEG
- MAL-PEG maleimide-mPEG
- OPSS-PEG orthopyridyl-disulfide-mPEG
- PEG or mPEG polymers will have a size of about 5 kDa, about 10 kD, about 12 kDa or about 20 kDa.
- the LFNG variant is usually treated with a reducing agent, such as dithiothreitol (DDT) prior to PEGylation.
- DDT dithiothreitol
- the reducing agent is subsequently removed by any conventional method, such as by desalting. Conjugation of PEG to a cysteine residue typically takes place in a suitable buffer at pH 6-9 at temperatures varying from 4°C to 25°C for periods up to 16 hours.
- the PEGylation is designed so as to produce the optimal molecule with respect to the number of PEG molecules attached, the size and form (e.g. whether they are linear or branched) of such molecules, and where in the polypeptide such molecules are attached.
- the molecular weight of the polymer to be used may be chosen on the basis of the desired effect to be achieved. For instance, if the primary purpose of the conjugation is to achieve a conjugate having a high molecular weight (e.g. to reduce renal clearance) it may be desirable to conjugate as few high Mw polymer molecules as possible to obtain the desired molecular weight.
- a sufficiently high number of low molecular weight polymer e.g. with a molecular weight of about 5,000 Da
- 2-8 such as 3-6 such polymers may be used.
- the polymer molecule which may be linear or branched, has a high molecular weight, e.g. about 20 kDa.
- the polymer conjugation is performed under conditions aiming at reacting all available polymer attachment groups with polymer molecules.
- the molar ratio of activated polymer molecules to polypeptide is 1000-1, in particular 200-1, preferably 100-1, such as 10-1 or 5-1 in order to obtain optimal reaction.
- equimolar ratios may be used.
- linker it is also contemplated according to the invention to couple the polymer molecules to the polypeptide through a linker.
- Suitable linkers are well known to the skilled person.
- a preferred example is cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; US 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.
- the coupling of a sugar moiety may take place in vivo or in vitro.
- the nucleotide sequence encoding the polypeptide variant must be inserted in a glycosylating, eukaryotic expression host.
- the expression host cell may be selected from fungal (filamentous fungal or yeast), insect or animal cells or from transgenic plant cells.
- the glycosylation may be achieved in the human body when using a nucleotide sequence encoding the polypeptide of the invention in gene therapy.
- the host cell is a mammalian cell, such as a CHO cell, a BHK cell or a HEK cell, e.g. a HEK293 cell, or an insect cell, such as an SF9 cell, or a yeast cell, e.g. Saccharomyces cerevisiae, Pichia pastoris or any other suitable glycosylating host, e.g. as described further below.
- sugar moieties attached to the LFNG polypeptide by in vivo glycosylation are further modified by use of glycosyltransferases, e.g. using the glycoAdvanceTM technology marketed by Neose, Horsham, PA, USA.
- glycosyltransferases e.g. using the glycoAdvanceTM technology marketed by Neose, Horsham, PA, USA.
- Covalent in vitro coupling of glycosides to amino acid residues of LFNG may be used to modify or increase the number or profile of carbohydrate substituents.
- the sugar(s) may be attached to a) arginine and histidine, b) free carboxyl groups, c) free sulfhydryl groups such as those of cysteine, d) free hydroxyl groups such as those of serine, threonine, tyrosine or hydroxyproline, e) aromatic residues such as those of phenylalanine or tryptophan or f) the amide group of glutamine.
- These amino acid residues constitute examples of attachment groups for a sugar moiety, which may be introduced and/or removed in the LFNG polypeptide.
- TGases transglutaminases
- Covalent modification of the LFNG polypeptide may be performed by reacting (an) attachment group(s) of the polypeptide with an organic derivatizing agent.
- organic derivatizing agent Suitable derivatizing agents and methods are well known in the art. For example, cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroaceta ide, to give carboxymefhyl or carboxyamidomethyl derivatives.
- Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ -(4- imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitro ⁇ henol, or chloro-7-nitrobenzo-2-oxa-l,3-diazole.
- Histidyl residues are derivatized by reaction with diefhylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
- Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
- Suitable reagents for derivatizing ⁇ -amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.
- Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, and ninhydrin.
- Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine guanidino group.
- aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
- the conjugation between the polypeptide and the non-polypeptide moiety is conducted under conditions where the functional site of the LFNG polypeptide is blocked by a helper molecule capable of binding to the functional site of the polypeptide.
- the helper molecule is one, which specifically recognizes a functional site of the polypeptide, such as a receptor.
- the helper molecule may be an antibody, in particular a monoclonal antibody recognizing the polypeptide exhibiting LFNG activity.
- the helper molecule may be a neutralizing monoclonal antibody.
- the polypeptide is allowed to interact with the helper molecule before effecting conjugation. This ensures that the functional site of the polypeptide is shielded or protected and consequently unavailable for derivatization by the non-polypeptide moiety such, as a polymer. Following its elution from the helper molecule, the conjugate between the non-polypeptide moiety and the polypeptide can be recovered with at least a partially preserved functional site.
- the subsequent conjugation of the polypeptide having a blocked functional site to a polymer, a lipophilic compound, a sugar moiety, an organic derivatizing agent or any other compound is conducted in the normal way, e.g. as described in the sections above entitled "Conjugation to " and "Coupling to ... " '.
- the helper molecule is first covalently linked to a solid phase such as column packing materials, for instance Sephadex or agarose beads, or a surface, e.g. reaction vessel. Subsequently, the polypeptide is loaded onto the column material carrying the helper molecule and conjugation carried out according to methods known in the art, e.g. as described in the sections above entitled “Conjugation to " and “Coupling to ... ". This procedure allows the conjugated polypeptide to be separated from the helper molecule by elution. The conjugated polypeptide is eluted by conventional techniques under physico- chemical conditions that do not lead to a substantive degradation of the conjugated polypeptide.
- a solid phase such as column packing materials, for instance Sephadex or agarose beads, or a surface, e.g. reaction vessel.
- the fluid phase containing the conjugated polypeptide is separated from the solid phase to which the helper molecule remains covalently linked.
- the separation can be achieved in other ways:
- the helper molecule may be derivatized with a second molecule (e.g. biotin) that can be recognized by a specific binder (e.g. streptavidin).
- the specific binder may be linked to a solid phase thereby allowing the separation of the conjugated polypeptide from the helper molecule-second molecule complex through passage over a second helper-solid phase column which will retain, upon subsequent elution, the helper molecule-second molecule complex, but not the polypeptide conjugate.
- the conjugated polypeptide may be released from the helper molecule in any appropriate fashion.
- De-protection may be achieved by providing conditions in which the helper molecule dissociates from the functional site of the LFNG to which it is bound.
- a complex between an antibody to which a polymer is conjugated and an anti-idiotypic antibody can be dissociated by adjusting the pH to an acid or alkaline pH.
- the LFNG polypeptide variant preferably in glycosylated form, may be produced by any suitable method known in the art. Such methods include constructing a nucleotide sequence encoding the polypeptide and expressing the sequence in a suitable transformed or transfected host. However, polypeptides of the invention may be produced, albeit less efficiently, by chemical synthesis or a combination of chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.
- the nucleotide sequence of the invention encoding an LFNG polypeptide may be constructed by isolating or synthesizing a nucleotide sequence encoding the parent LFNG, such as huLFNG with the amino acid sequence SEQ LD NO:l, and then changing the nucleotide sequence so as to effect introduction (i.e. insertion or substitution) or deletion (i.e. removal or substitution) of the relevant amino acid residue(s).
- nucleotide sequence is conveniently modified by site-directed mutagenesis in accordance with well-known methods, see, e.g., Mark et al., "Site-specific Mutagenesis of the Human Fibroblast Interferon Gene", Proc. Natl. Acad. Sci. USA, 81, pp. 5662-66 (1984); and US 4,588,585.
- the nucleotide sequence is prepared by chemical synthesis, e.g. by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced.
- oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced.
- several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and assembled by PCR, ligation or ligation chain reaction (LCR).
- LCR ligation or ligation chain reaction
- the nucleotide sequence encoding the polypeptide is inserted into a recombinant vector and operably linked to control sequences necessary for expression of the LFNG in the desired transformed host cell.
- control sequences necessary for expression of the LFNG in the desired transformed host cell.
- not all vectors and expression control sequences function equally well to express the nucleotide sequence encoding an LFNG polypeptide described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it or be able to integrate into the chromosome.
- the vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
- an expression control sequence a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the nucleotide sequence encoding the polypeptide, particularly as regards potential secondary structures.
- Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the nucleotide sequence, their secretion characteristics, their ability to fold the polypeptide correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the nucleotide sequence.
- the recombinant vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
- the vector is one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
- the vector is preferably an expression vector, in which the nucleotide sequence encoding the LFNG polypeptide is operably linked to additional segments required for transcription of the nucleotide sequence.
- the vector is typically derived from plasmid or viral DNA.
- suitable expression vectors for expression in the host cells mentioned herein are commercially available or described in the literature.
- Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus.
- Specific vectors are, e.g., pCDNA3.1(+) ⁇ Hyg (Invitrogen, Carlsbad, CA, USA) and pCI-neo (Stratagene, La Jola, CA, USA).
- Useful expression vectors for bacterial hosts include known bacterial plas ids, such as plasmids from E. coli, including pBR322, ⁇ T3a and ⁇ ET12a (both from Novagen Inc., WI, USA), wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g.
- Useful expression vectors for yeast cells include the 2 ⁇ plasmid and derivatives thereof, the POT1 vector (US 4,931,373), the pJSO37 vector described in (Okkels, Ann. New York Acad. Sci. 782, 202-207, 1996) and pPICZ A, B or C (Invitrogen).
- Useful vectors for insect cells include ⁇ VL941, pBG311 (Gate et al., "Isolation of the Bovine and Human Genes for Mullerian inhibiting Substance And Expression of the Human Gene In Animal Cells", Cell, 45, pp. 685-98 (1986), pBluebac 4.5 and pMelbac (both available from Invitrogen) as well as PVL1392 (available from Pharmingen).
- vectors for use in this invention include those that allow the nucleotide sequence encoding the LFNG polypeptide to be amplified in copy number.
- amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, "Construction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient Expression", Mol. Cell. Biol, 2, pp.
- the recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
- a DNA sequence enabling the vector to replicate in the host cell in question.
- An example of such a sequence is the SV40 origin of replication.
- suitable sequences enabling the vector to replicate are the yeast plasmid 2 ⁇ replication genes REP 1-3 and origin of replication.
- the vector may also comprise a selectable marker, e.g.
- DHFR dihydrofolate reductase
- Schizosaccharomyces po be TPI gene one which confers resistance to a dmg, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
- selectable markers include amdS, pyrG, arcB, niaD, sC.
- control sequences is defined herein to include all components, which are necessary or advantageous for the expression of the LFNG polypeptide.
- Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide.
- control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activating sequence, signal peptide sequence, and transcription terminator.
- the control sequences include a promoter.
- a wide variety of expression control sequences may be used in the present invention.
- Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors as well as any sequence known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
- control sequences for directing transcription in mammalian cells include the early and late promoters of SN40 and adenovirus, e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovirus immediate- early gene promoter (CMN), the human elongation factor l (EF-l ⁇ ) promoter, the Drosophila minimal heat shock protein 70 promoter, the Rous Sarcoma Nirus (RSN) promoter, the human ubiquitin C (UbC) promoter, the human growth hormone terminator, SN40 or adenovirus Elb region polyadenylation signals and the Kozak consensus sequence (Kozak, M. JMol Biol 1987 Aug 20;196(4):947-50).
- adenovirus 2 major late promoter e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovirus immediate- early
- a synthetic intron may be inserted in the 5' untranslated region of the nucleotide sequence encoding the LF ⁇ G polypeptide.
- An example of a synthetic intron is the synthetic intron from the plasmid pCI- ⁇ eo (available from Promega Corporation, WI, USA).
- control sequences for directing transcription in insect cells include the polyhedrin promoter, the P10 promoter, the Autographa calif ornica polyhedrosis virus basic protein promoter, the baculovirus immediate early gene 1 promoter and the baculovirus 39K delayed-early gene promoter, and the SN40 polyadenylation sequence.
- control sequences for use in yeast host cells include the promoters of the yeast ⁇ -mating system, the yeast triose phosphate isomerase (TPI) promoter, promoters from yeast glycolytic genes or alcohol dehydogenase genes, the ADH2-4c promoter and the inducible GAL promoter.
- TPI yeast triose phosphate isomerase
- suitable control sequences for use in filamentous fungal host cells include the ADH3 promoter and terminator, a promoter derived from the genes encoding Aspergillus oryzae TAKA amylase triose phosphate isomerase or alkaline protease, an A. niger ⁇ -amylase, A. niger or A. nidulans glucoamylase, A. nidulans acetamidase, Rhizomucor miehei aspartic proteinase or lipase, the TPI1 terminator and the ADH3 terminator.
- suitable control sequences for use in bacterial host cells include promoters of the lac system, the trp system, the TAC or TRC system and the major promoter regions of phage lambda.
- the nucleotide sequence of the invention may or may not also include a nucleotide sequence that encode a signal peptide.
- the signal peptide is present when the polypeptide is to be secreted from the cells in which it is expressed. Such signal peptide, if present, should be one recognized by the cell chosen for expression of the polypeptide.
- the signal peptide may be homologous (e.g. be that normally associated with huLFNG) or heterologous (i.e. originating from another source than huLFNG) to the polypeptide or may be homologous or heterologous to the host cell, i.e.
- the signal peptide may be prokaryotic, e.g. derived from a bacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian, or insect or yeast cell.
- the presence or absence of a signal peptide will, e.g., depend on the expression host cell used for the production of the polypeptide, the protein to be expressed (whether it is an intracellular or intracellular protein) and whether it is desirable to obtain secretion.
- the signal peptide may conveniently be derived from a gene encoding an Aspergillus sp.
- amylase or glucoamylase a gene encoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosa lipase.
- the signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral ⁇ -amylase, A. niger acid-stable amylase, or A. niger glucoamylase.
- the signal peptide may conveniently be derived from an insect gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipokinetic hormone precursor, (cf.
- a preferred signal peptide for use in mammalian cells is that of huLFNG or the murine Ig kappa light chain signal peptide (Coloma, M (1992) J. Imm. Methods 152:89-104).
- suitable signal peptides have been found to be the ⁇ -factor signal peptide from S. cereviciae. (cf. US 4,870,008), the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al, Nature 289, 1981, pp. 643-646), a modified carboxypeptidase signal peptide (cf. L.A. Nails et al., Cell 48, 1987, pp.
- yeast BAR1 signal peptide cf. WO 87/02670
- yeast aspartic protease 3 YAP3
- Any suitable host may be used to produce the LF ⁇ G polypeptide, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants.
- bacterial host cells include grampositive bacteria such as strains of Bacillus, e.g. B. brevis or B. subtilis, Pseudomonas or Streptomyces, or gramnegative bacteria, such as strains of E. coli.
- the introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff- Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thome, 1987, Journal of Bacteriology 169: 5771-5278).
- protoplast transformation see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115
- competent cells see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubn
- filamentous fungal host cells examples include strains of Aspergillus, e.g. A. oryzae, A. niger, or A. nidulans, Fusarium or Trichoderma.
- Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se.
- Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and US 5,679,543.
- Suitable methods for transforming Fusarium species are described by Malardier et al, 1989, Gene 78: 147-156 and WO 96/00787.
- Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. ⁇ .
- yeast host cells examples include strains of Saccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Kluyveromyces, Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H. Polymorpha or Yarrowia. Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides therefrom are disclosed by Clontech Laboratories, Inc, Palo Alto, CA, USA (in the product protocol for the
- YeastmakerTM Yeast Tranformation System Kit YeastmakerTM Yeast Tranformation System Kit
- suitable insect host cells include a Lepidoptora cell line, such as Spodopterafrugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (High Five) (US 5,077,214). Transformation of insect cells and production of heterologous polypeptides therein may be performed as described by Invitrogen.
- Suitable mammalian host cells include Chinese hamster ovary (CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL-1573)), as well as plant cells in tissue culture.
- COS Green Monkey cell lines
- BHK Baby Hamster Kidney
- HEK 293 ATCC CRL-1573
- Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
- the mammalian cell such as a CHO cell
- sialyltransferase e.g. 1,6-sialyltransferase, e.g. as described in US 5,047,335, in order to provide improved glycosylation of the LFNG polypeptide.
- Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, electroporation, DEAE-dextran mediated transfection, liposome-mediated transfection, viral vectors and the transfection method described by Life Technologies Ltd, Paisley, UK using Lipofectamin 2000. These methods are well known in the art and e.g. described by Ausbel et al. (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. The cultivation of mammalian cells are conducted according to established methods, e.g. as disclosed in (Animal Cell Biotechnology, Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc, Totowa, New Jersey, USA and Harrison MA and Rae LF, General Techniques of Cell Culture, Cambridge University Press 1997).
- a eukaryotic host cell e.g. of the type mentioned above, is preferably used.
- the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art.
- the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
- the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art.
- suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection).
- the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates. The resulting polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.
- the polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). Specific methods for purifying polypeptides exhibiting LFNG activity are disclosed in EP 110044 and unexamined Japanese patent application No. 186995/84.
- the biological activity of the LFNG polypeptide can be assayed by any suitable method known in the art.
- assays include antibody neutralization of antiviral activity, induction of protein kinase, oligoadenylate 2,5-A synthetase or phosphodiesterase activities, as described in EP 0 041 313 Bl.
- assays also include immunomodulatory assays (see, e.g., US 4,753,795), growth inhibition assays, and measurement of binding to cells that express interferon receptors.
- a specific assay (entitled "Primary Assay") is described in the Materials and Methods section herein.
- the present invention relates to improved methods of treating, in particular, inflammatory diseases, e.g. interstitial lung diseases, such as idiopathic pulmonary fibrosis, but also granulomatous diseases; cancer, in particular ovarian cancer; infections such as pulmonary atypical mycobacterial infections; bone disorders (e.g.
- the molecule of the invention is preferably administered in a composition including a pharmaceutically acceptable carrier or excipient.
- “Pharmaceutically acceptable” means a carrier or excipient that does not cause any untoward effects in patients to whom it is administered.
- the molecules of the invention can be used "as is" and/or in a salt form thereof.
- Suitable salts include, but are not limited to, salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, as well as e.g. zinc salts. These salts or complexes may by present as a crystalline and/or amorphous structure.
- the polypeptide of the invention is administered at a dose approximately paralleling that employed in therapy with known commercial preparations of LFNG, such as Actimmune®, or as specified in EP 0795 332. The exact dose to be administered depends on the circumstances. Normally, the dose should be capable of preventing or lessening the severity or spread of the condition or indication being treated.
- an effective amount of the LFNG polypeptide or composition of the invention depends, inter alia, upon the disease, the dose, the administration schedule, whether the polypeptide or composition is administered alone or in conjunction with other therapeutic agents, the serum half- life/functional in vivo half -life of the compositions, and the general health of the patient.
- the present invention also relates to an LFNG polypeptide according to the present invention or a pharmaceutical composition according to the present invention for use as a medicament.
- the invention also relates to the use of i) an LFNG variant according to the present invention, or ) a pharmaceutical composition of the invention, for the manufacture of a medicament, a pharmaceutical composition or a it-of -parts for the treatment of diseases selected from the group consisting of inflammatory diseases, such as interstitial lung diseases, in particular idiopathic pulmonary fibrosis; cancer, in particular ovarian cancer; infections, such as pulmonary atypical mycobacterial infections; bone disorders (e.g.
- a bone metabolism disorder so as malignant osteopetrosis
- granulomatous diseases such as rheumatoid arthritis; multiresistent tuberculosis; cryptococcal meningitis; cystic fibrosis and liver fibrosis, in particular liver fibrosis secondary to hepatitis C; asthma and lymphoma.
- the disease is an interstitial lung disease, in particular idiopathic pulmonary fibrosis.
- a glucocorticoid such as prednisolone may also be included.
- the preferred dosing is 1- 4, more preferably 2-3, ⁇ g/kg patient weight of the polypeptide component per dose.
- the preferred dosing is 100-350, more preferably 100-150 ⁇ g glucocorticoid/kg patient weight per dose.
- the invention also relates to a kit of parts suitable for the treatment of interstitial lung diseases comprising a first pharmaceutical composition comprising the active components i) or ii) mentioned above and a second pharmaceutical composition comprising at least one glucocorticoid, each optionally together with a pharmaceutically acceptable carrier and/or excipient.
- a kit of parts suitable for the treatment of interstitial lung diseases comprising a first pharmaceutical composition comprising the active components i) or ii) mentioned above and a second pharmaceutical composition comprising at least one glucocorticoid, each optionally together with a pharmaceutically acceptable carrier and/or excipient.
- the variant of the invention can be formulated into pharmaceutical compositions by well-known methods. Suitable formulations are described by Remington's Pharmaceutical
- the pharmaceutical composition may be formulated in a variety of forms, including liquid, gel, lyophilized, powder, compressed solid, or any other suitable form. The preferred form will depend upon the particular indication being treated and will be apparent to one of skill in the art. However, the LFNG polypeptide of the invention is preferably formulated as a liquid pharmaceutical composition.
- the pharmaceutical composition may be administered orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other acceptable manner, e.g. using
- the formulations can be administered continuously by infusion, although bolus injection is acceptable, using techniques well known in the art, such as pumps or implantation. In some instances the formulations may be directly applied as a solution or spray.
- the preferred mode of administration will depend upon the particular indication being treated and will be apparent to one of skill in the art, but usually subcutaneous administration is preferred as this mode of administration can typically be conducted by the patient himself.
- the pharmaceutical composition of the invention may be administered in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the polypeptide of the invention, either concurrently or in accordance with any other acceptable treatment schedule.
- the polypeptide or pharmaceutical composition of the invention may be used as an adjunct to other therapies. In particular, combinations with glucocorticoids as described in EP 0 795 332 are considered.
- parenterals An example of a pharmaceutical composition is a solution designed for parenteral administration. Although in many cases pharmaceutical solution formulations are provided in liquid form, appropriate for immediate use, such parenteral formulations may also be provided in frozen or in lyophilized form. In the former case, the composition must be thawed prior to use. The latter form is often used to enhance the stability of the active compound contained in the composition under a wider variety of storage conditions, as it is recognized by those skilled in the art that lyophilized preparations are generally more stable than their liquid counterparts. Such lyophilized preparations are reconstituted prior to use by the addition of one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.
- suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.
- parenterals In case of parenterals, they are prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the polypeptide having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art (all of which are termed "excipients"), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives.
- excipients typically employed in the art
- Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are typically present at a concentration ranging from about 2 mM to about 50 mM Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid- sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid- sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fuma
- Preservatives are added to retard microbial growth, and are typically added in amounts of about 0.2%- 1% (w/v).
- Suitable preservatives for use with the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g. benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.
- Isotonicifiers are added to ensure isotonicity of liquid compositions and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
- Polyhydric alcohols can be present in an amount between 0.1% and 25% by weight, typically 1% to 5%, taking into account the relative amounts of the other ingredients.
- Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall.
- Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, omithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as
- proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins
- hydrophilic polymers such as polyvinylpyrrolidone
- monosaccharides such as xylose, mannose, fructose and glucose
- disaccharides such as lactose, maltose and sucrose
- trisaccharides such as raffinose, and polysaccharides such as dextran.
- Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on the active protein weight.
- Non-ionic surfactants or detergents may be present to help solubilize the therapeutic agent as well as to protect the therapeutic polypeptide against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the polypeptide.
- Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers (Tween®-20, Tween®-80, etc.).
- Additional miscellaneous excipients include bulking agents or fillers (e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) and cosol vents.
- bulking agents or fillers e.g. starch
- chelating agents e.g. EDTA
- antioxidants e.g., ascorbic acid, methionine, vitamin E
- cosol vents e.g., ascorbic acid, methionine, vitamin E
- the active ingredient may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example hydroxymethylcellulose, gelatin or poly-(methylmethacylate) microcapsules, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
- colloidal drug delivery systems for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
- macroemulsions for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
- Parenteral formulations to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.
- said pharmaceutical composition comprises the i) LFNG variant of the invention, ii) a buffering agent, in particular a salt of an organic acid, capable of maintaining the pH between 4.5-7.5, iii) a stabilizer, in particular an organic sugar or sugar alcohol, iv) a non-ionic surfactant, and v) sterile water.
- the buffering agent is capable of maintaining the pH between 5.0-7.5, more preferably between 5.0-7.0, in particular between 5.0-6.5.
- the buffering agent is selected from the group consisting of acetate, succinate and citrate, the stabilizer is mannitol or sorbitol, the non-ionic surfactant is Tween®-20 or Tween®-80.
- the phamaceutical composition does not include any preservatives.
- the pharmaceutical composition comprises an sulfoalkyl ether cyclodextrin derivative, such as any of the derivatives described in US 5,874,418, US 5,376,645 and US 5,134,127, the contents of which are incorporated herein by reference.
- the sulfoalkyl ether cyclodextrin is a compound of the Formula (I):
- n 4, 5 or 6
- Ri, R 2 , R 3 , R , R 5 , R 6 , R , R 8 , and R 9 are each, independently, -O- or a -O-(C 2 -C 6 alkylene)- SO 3 - group, wherein at least one of Ri and R 2 is independently a -O-(C 2 -C 6 alkylene)-SO 3 - group, and
- Si, S 2 , S 3 , S 4 , S 5 , S 6 , S 7 , S 8 , and S 9 are each, independently, a pharmaceutically acceptable cation.
- n is 5. In a still further embodiment n is 6. In a further embodiment at least one of Ri and R 2 is -O-(CH 2 ) m -SO -, and m is 2, 3, 4, 5 or 6. In a further embodiment Ri and R 2 is independently selected from
- At least one of R , R 6 , and R 8 is independently, -O-(C 2 -C 6 alkylene)-SO 3 -; and R 5 , R , and R 9 are all -O-.
- Si, S 2 , S 3 , S 4 , S 5 , S 6 , S 7 , S 8 , and S 9 are each, independently, a pharmaceutically acceptable cation selected from FT 1" , alkali metals (e.g. Li + , Na + , K + ), alkaline earth metals (e.g., Ca +2 , Mg +2 ), ammonium ions and amine cations such as the cations of ( -
- Si, S 2 , S 3 , S , S 5 , S 6 , S 7 , S 8 , and S 9 are independently selected from alkaline metal cation, alkaline earth metal cation, quaternary ammonium cation, tertiary ammonium cation, and secondary ammonium cation.
- At least one of R , R 6 , and R 8 is independently, -O-(C 2 -C 6 alkylene)-SO 3 -; and R 5 , R 7 , and R 9 are all -O-.
- alkylene and alkyl as used herein (e.g., in the -O-(C 2 -C 6 -alkylene)SO 3 - group or in the alkylamines), include linear, cyclic, and branched, saturated and unsaturated (i.e., containing a double bond) divalent alkylene groups and monovalent alkyl groups, respectively.
- alkanol in this text likewise includes both linear, cyclic and branched, saturated and unsaturated alkyl components of the alkanol groups, in which the hydroxyl groups may be situated at any position on the alkyl moiety.
- cycloalkanol includes unsubstituted or substituted (e.g., by methyl or ethyl) cyclic alcohols.
- the presently preferred sulfoalkyl ether cyclodextrin derivative is a salt of beta cyclodextrin sulfobutyl ether (in particular the sodium salt thereof also termed SBE7- ⁇ -CD which is available as Captisol®) (Cydex, Overland Park, Kansas 66213, US).
- sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the variant, the matrices having a suitable form such as a film or microcapsules.
- sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the ProLease® technology or Lupron Depot® (i ⁇ jectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
- polyesters for example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)
- polylactides copolymers
- polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for long periods such as up to or over 100 days
- certain hydrogels release proteins for shorter time periods.
- encapsulated polypeptides remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved.
- stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
- the pharmaceutical composition may be in solid or liquid form, e.g. in the form of a capsule, tablet, suspension, emulsion or solution.
- the pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of the active ingredient.
- a suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but can be determined by persons skilled in the art using routine methods.
- Solid dosage forms for oral administration may include capsules, tablets, suppositories, powders and granules.
- the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch.
- Such dosage forms may also comprise, as is normal practice, additional substances, e.g. lubricating agents such as magnesium stearate.
- additional substances e.g. lubricating agents such as magnesium stearate.
- the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
- the variants may be admixed with adjuvants such as lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
- adjuvants such as lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and
- the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
- compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc., e.g. as disclosed elsewhere herein.
- Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water.
- Such compositions may also comprise adjuvants such as wetting agents, sweeteners, flavoring agents and perfuming agents.
- Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.
- liquid or semi-liquid preparations suitable for penetration through the skin e.g., liniments, lotions, ointments, creams, or pastes
- drops suitable for administration to the eye, ear, or nose e.g., liniments, lotions, ointments, creams, or pastes
- Formulations suitable for use with a nebulizer will typically comprise the polypeptide dissolved in water at a concentration of, e.g., about 0.01 to 25 mg of variant per mL of solution, preferably about 0.1 to 10 mg/mL.
- the formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure), and/or human serum albumin ranging in concentration from 0.1 to 10 mg/ml.
- buffers that may be used are sodium acetate, citrate and glycine.
- the buffer will have a composition and molarity suitable to adjust the solution to a pH in the range of 3 to 9.
- buffer molarities of from 1 mM to 50 mM are suitable for this purpose.
- sugars which can be utilized are lactose, maltose, mannitol, sorbitol, trehalose, and xylose, usually in amounts ranging from 1% to 10% by weight of the formulation.
- the nebulizer formulation may also contain a surfactant to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.
- a surfactant to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.
- Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty acid esters. Amounts will generally range between 0.001% and 4% by weight of the formulation.
- An especially preferred surfactant for purposes of this invention is polyoxyethylene sorbitan monooleate.
- Formulations for use with a metered dose inhaler device will generally comprise a finely divided powder.
- This powder may be produced by lyophilizing and then milling a liquid variant formulation and may also contain a stabilizer such as human serum albumin (HSA). Typically, more than 0.5% (w/w) HSA is added.
- HSA human serum albumin
- one or more sugars or sugar alcohols may be added to the preparation if necessary. Examples include lactose maltose, mannitol, sorbitol, sorbitose, trehalose, xylitol, and xylose.
- the amount added to the formulation can range from about 0.01 to 200% (w/w), preferably from approximately 1 to 50%, of the variant present. Such formulations are then lyophilized and milled to the desired particle size.
- the properly sized particles are then suspended in a propellant with the aid of a surfactant.
- the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof.
- Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant. This mixture is then loaded into the delivery device.
- An example of a commercially available metered dose inhaler suitable for use in the present invention is the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.
- Formulations for powder inhalers will comprise a finely divided dry powder containing variant and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50% to 90% by weight of the formulation.
- the particles of the powder shall have aerodynamic properties in the lung corresponding to particles with a density of about 1 g/cm 2 having a median diameter less than 10 micrometers, preferably between 0.5 and 5 micrometers, most preferably of between 1.5 and 3.5 micrometers.
- An example of a powder inhaler suitable for use in accordance with the teachings herein is the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.
- the powders for these devices may be generated and/or delivered by methods disclosed in US 5,997,848, US 5,993,783, US 5,985,248, US 5,976574, US 5,922,354, US 5,785,049 and US 5,654,007.
- Mechanical devices designed for pulmonary delivery of therapeutic products include but are not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those of skill in the art.
- nebulizers include but are not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those of skill in the art.
- Specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St.
- the invention provides compositions and methods for treating bacterial and viral infections, cancers or tumors, interstitial pulmonary diseases such as idiopathic pulmonary fibrosis, granulomatous diseases, bone disorders (e.g. a bone metabolism disorder so as malignant osteopetrosis) and autoimmune diseases such rheumatoid arthritis.
- interstitial pulmonary diseases such as idiopathic pulmonary fibrosis, granulomatous diseases, bone disorders (e.g. a bone metabolism disorder so as malignant osteopetrosis) and autoimmune diseases such rheumatoid arthritis.
- the invention in a further aspect relates to a method of treating a mammal having circulating antibodies against huLFNG, which method comprises administering a compound which has the bioactivity of LFNG and which does not react with said antibodies.
- the compound is preferably a variant as described herein and the mammal is preferably a human being.
- the mammals to be treated may suffer from any of the diseases listed above for which LFNG is a useful treatment.
- the invention relates to a method of making a pharmaceutical product for use in treatment of mammals having circulating antibodies against huLFNG, wherein a compound which has the bioactivity of LFNG and which does not react with such is formulated into an injectable or otherwise suitable formulation.
- the term "circulating antibodies" is intended to indicate autoantibodies formed in a mammal in response to having been treated with any of the commercially available LFNG preparations.
- nucleotide sequence encoding an LFNG polypeptide of the invention in gene therapy applications.
- Gene therapy applications contemplated include treatment of those diseases in which the polypeptide is expected to provide an effective therapy.
- Local delivery of LFNG using gene therapy may provide the therapeutic agent to the target area while avoiding potential toxicity problems associated with non-specific administration.
- Liposome-mediated DNA transfer e.g., as disclosed by Caplen et al., "Liposome- mediated CFTR Gene Transfer to the Nasal Epithelium Of Patients With Cystic Fibrosis" 5 Nature Med., 3, pp. 39-46 (1995); Crystal, "The Gene As A Drug", Nature Med., 1, pp.- 15-17 (1995); Gao and Huang, "A Novel Cationic Liposome Reagent For Efficient Transfection of Mammalian Cells", Biochem.Biophys Res. Comm, 179, pp. 280-85 (1991);
- Retrovirus-mediated DNA transfer e.g., as disclosed by Kay et al., "In vivo Gene Therapy of Hemophilia B: Sustained Partial Correction In Factor LX-Deficient Dogs", Science, 10 262, pp. 117-19 (1993); Anderson, “Human Gene Therapy", Science, 256, pp.808-13(1992);
- DNA viruses include adenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses (preferably herpes simplex virus based vectors), and parvoviruses (preferably "defective" or non-autonomous parvovirus based vectors, more preferably adeno-associated virus based vectors, most preferably AAV-2 based 15 vectors).
- adenoviruses preferably Ad-2 or Ad-5 based vectors
- herpes viruses preferably herpes simplex virus based vectors
- parvoviruses preferably "defective" or non-autonomous parvovirus based vectors, more preferably adeno-associated virus based vectors, most preferably AAV-2 based 15 vectors.
- CHO-K1 cells available from American Type Culture Collection (ATCC #CCL-61)).
- HeLa cells available from American Type Culture Collection (ATCC #CCL-2)). 25 ISRE-Luc was obtained fromStratagene, La Jolla USA. pCDNA 3.1/hygro was obtained from Invitrogen, Carlsbad USA.
- Restricion enzymes and polymerases were obtained from New England Biolabs Inc., Beverly,
- DMEM medium Dulbecco's Modified Eagle Media (DMEM), 10% fetal bovine serum and 30 Hygromycin B were obtained from Life Technologies A/S, Copenhagen, Denmark.
- LucLite substrate was obtained from Packard Bioscience, Groningen, The Netherlands.
- TopCount luminometer was obtained from Packard Bioscience, Groningen, The Netherlands.
- Biotinylated polyclonal anti-human LFNG antibody, BAF285 was obtained available from R&D Systems Inc., Minneapolis, USA.
- TMB blotting reagent was obtained from KEM-EN-TEC, Copenhagen, Denmark.
- ISRE Interferon Stimulated Response Element
- HeLa cells are co-transfected with ISRE-Luc and pCDNA 3.1/hygro and foci (cell clones) are created by selection in DMEM media containing Hygromycin B. Cell clones are screened for luciferase activity in the presence or absence of LFNG. Those clones showing the highest ratio of stimulated to unstimulated luciferase activity are used in further assays.
- To screen polypeptides 15,000 cells/well are seeded in 96 well culture plates and incubated overnight in DMEM media. The next day the polypeptides as well as a known standard are added to the cells in various concentrations.
- LucLite substrate (Packard Bioscience, Groningen, The Netherlands) is subsequently added to each well. Plates are sealed and luminescence measured on a TopCount luminometer (Packard) in SPC (single photon counting) mode. Each individual plate contains wells incubated with LFNG as a stimulated control and other wells containing normal media as an unstimulated control. The ratio between stimulated and unstimulated luciferase activity serves as an internal standard for both LFNG activity and experiment-to-experiment variation.
- a SDS-PAGE gel is run under standard conditions and transferred to a nitrocellulose membrane.
- Western blotting is done according to standard procedures using a biotinylated polyclonal anti-human LFNG antibody (BAF285 from R & D Systems) as primary antibody and Horse Radish Peroxidase-conjugated streptavidin (P0397from DAKO) as secondary antibody followed by staining with TMB blotting reagent (KEM-EN-TEC, Copenhagen, Denmark).
- TMB blotting reagent KEM-EN-TEC, Copenhagen, Denmark.
- the AUC sc is determined by one 200 ⁇ l bolus subcutaneous administration of equal amount (on an activity basis) of the LFNG polypeptide of the invention in rats.
- rats Female Sprag-Dawley rats, weiging between 220-260 grams, are used.
- the LFNG polypeptide is formulated in sodium succinate (720 mg/1), mannitol 40 g/1), polysorbat 20 (100 mg/1) at pH 6.0.
- one blood sample is drawn in the tail-vein to ensure that no background LFNG activity can be detected.
- blood samples are withdrawn from the tail vein after 10 min, 20 min, 40 min, 60 min, 120 min, 240 min, 480 min, 720 min, 1440 min, 1620 min, 1920 min and 2880 min (sometimes also 3600 min).
- Serum is prepared by letting the blood sample coagulate for 20 min at room temperature followed by centrifugation at 5000g, 20 min at room temperature. The serum is then isolated and stored at - 80 ° C until determination of LFNG activity using the "Primary Assay" described above. The amount of units in serum (U/ml) against time (min) is then plotted and the AUC SC is calculated using GraphPad Prism 3.01.
- the functional in vivo half -life is determined by one 200 ⁇ l bolus intravenous administration of equal amount (on an activity basis) of the LFNG polypeptide of the invention in rats.
- rats Female Sprag-Dawley rats, weiging between 220-260 grams, are used.
- the LFNG polypeptide is formulated in sodium succinate (720 mg/1), mannitol 40 g/1), polysorbat 20 (100 mg/1) at pH 6.0.
- one blood sample is drawn in the tail-vein to ensure that no background LFNG activity can be detected.
- blood samples are withdrawn from the other tail vein after 5 min, 10 min, 20 min, 40 min, 60 min, 120 min, 240 min, 480 min, 720 min, 1440 min, 1620 min, 1920 min and 2880 min.
- Serum is prepared by letting the blood sample coagulate for 20 min at room temperature followed by centrifugation at 5000g, 20 min at room temperature. The serum is then isolated and stored at -80 ° C until determination of LFNG activity using the "Primary Assay" described above.
- ASA Accessible Surface Area
- ASA accessible surface area
- the fractional ASA of the side chain atoms is computed by division of the sum of the ASA of the atoms in the side chain with a value representing the ASA of the side chain atoms of that residue type in an extended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton (1991) J.Mol.Biol.: 220,507-530.
- the CA atom is regarded as a part of the side chain of Glycine residues but not for the remaining residues.
- the following table are used as standard 100% ASA for the side chain:
- Residues not detected in the structure are defined as having 100% exposure as they are thought to reside in flexible regions.
- the distance between atoms was determined using molecular graphics software e.g. InsightH v. 98.0, MSI LNC.
- the receptor-binding site is defined as comprising of all residues having their accessible surface area changed upon receptor binding. This is determined by at least two ASA calculations; one on the isolated ligand(s) in the ligand(s)/receptor(s) complex and one on the complete ligand(s)/receptor(s) complex.
- the X-ray structure used was of an LFNG homo-dimer in complex with two molecules of a soluble form of the LFNG receptor having a third molecule of the LFNG receptor in the structure not making interactions with the IFNG homodimer reported by Thiel et.al. Structure 8:927-936 (2000).
- the structure consists of the LFNG homodimer wherein the two molecules are labeled A and B.
- the M0 is removed from the structure in all the calculations of this example.
- the structure of the two LFNG monomers has very weak electron density after residue 120 and residues were only modeled until residue T126. Therefore, residues S121-T126 were removed from the structure prior to the calculations in this example.
- the two receptor fragments labeled C and D make direct interactions with the LFNG homodimer and a third receptor molecule labeled E makes no contact with the LFNG homodimer and are not included in these calculations.
- Residues not determined in the structure are treated as fully surface exposed, i.e. residues S121, P122, A123, A124, K125, T126, G127, K128, R129, K130, R131, S132, Q133, M134, L135, F136, R137, G138, R139, R140, A141, S142, Q143.
- residues S121, P122, A123, A124, K125, T126, G127, K128, R129, K130, R131, S132, Q133, M134, L135, F136, R137, G138, R139, R140, A141, S142, Q143 are also constitute separate targets for introduction of attachment groups in accordance with the present invention (or may be viewed as belonging to the group of surface exposed amino acid residues, e.g. having more than 25% or more than 50% exposed side chains).
- GenBank accession number X13274 encompassing a full length cDNA encoding mature huLFNG with its native signal peptide, was modified in order to facilitate high expression in CHO cells. Codons of the huLFNG nucleotide sequence were modified by making a bias in the codon usage towards the codons frequently used in homo sapiens. Subsequently, certain nucleotides in the sequence were substituted with others in order to introduce recognition sites for DNA restriction endonucleases. Primers were designed such that the gene could be synthesised. The primers were assembled to the synthetic gene by one step PCR using Platinum Pfx- polymerase kit (Life Technologies) and standard three-step PCR cycling parameters.
- the assembled gene was amplified by PCR using the same conditions and has the sequence shown in SEQ LD NO:4.
- the synthesised gene was cloned into pcDNA3.1/hygro (InVitrogen) between the BamHI at the 5' end and the Xbal at the 3' end, resulting in pIGY-22.
- oligonucleotides were designed in such a way that PCR generated changes could be introduced in the expression plasmid (pIGY-22) by classical two-step PCR followed by subcloning the PCR fragment using BamHI and Xbal.
- ADJ013 5'-GATGGCTGGCAACTAGAAG-3' (antisense downstream vector primer)
- ADJ014 5'-TGTACGGTGGGAGGTCTAT-3' (sense upstream vector primer)
- ADJ091 5'- CATGATCTTCCGATCGGTCTCGTTCTTCCAATT-3'
- ADJ092 5'- AATTGGAAGAACGAGACCGATCGGAAGATCATG-3'
- the S99T variant was generated by classical two-step PCR as described above, using ADJ013 and ADJ014 as vector primers, ADJ093 and ADJ094 as mutation primers, and pIGY- 22 as template.
- the 447 bp PCR fragment was subcloned into pcDNA3.1/Hygro (InVitrogen) using BamHI and Xbal, leading to plasmid pIGY-48.
- the E38N+S40T+S99T variant was generated by classical two-step PCR as described above, using ADJ013 and ADJ014 as vector primers, ADJ091 and ADJ092 as mutation primers, and pIGY-48 as template.
- the 447 bp PCR fragment was subcloned into pcDNA3.1/Hygro (InVitrogen) using BamHI and Xbal, leading to plasmid pIGY-54.
- C-terminally modified LFNG variants were generated by one-step PCR using pIGY-54 as template (i.e. including the E38N+S40T+S99T mutations).
- An 'upstream' oligonucleotide containing the start codon (preceded by a BamAI site for cloning and the sequence
- GCCGCCACC in order to optimise mRNA translation
- a 'downstream' oligonucleotide containing the desired mutation(s) and a Xbal site were used as primers.
- PF033 pcDNA3.1(+)/Hygro(InVitrogen)-derivative plasmid containing an intron from pCI-Neo (Stratagene) was used as expression vector.
- This vector termed PF033, was constructed by PCR amplification of the intron from pCI-Neo using
- LFNG-encoding plasmids were transfected into the cells using Lipofectamine 2000 (Life Technologies) according to the manufacturer's specifications. 24 hrs after transfection, culture media were collected and assayed for LFNG activity. Furthermore, in order to quantify the relative number of glycosylation sites utilized, Western blotting is performed using harvested culture medium.
- Stable clones expressing the LFNG polypeptide are generated by transfection of CHO Kl cells with LFNG-encoding plasmids followed by incubation of the cells in media containing 0.36 mg/ l hygromycin. Stably transfected cells are isolated and sub-cloned by limited dilution. Clones producing high levels of LFNG are identified by ELISA.
- Stable cell lines expressing the LFNG polypeptide are grown in Dulbecco's MEM/Nut- mix F-12 (Ham) L-glutamine, 15 mM Hepes, pyridoxine-HCl (Life Technologies Cat # 31330- 038), 1:10 FBS (BioWhittaker Cat # 02-701F), 1:100 penicillin and streptomycin (BioWhittaker Cat # 17-602E) in 1700 cm2 roller bottles (Corning, # 431200) until confluence. The media is then changed to 300 ml UltraCHO with L-glutamine (BioWhittaker Cat # 12- 724Q) with the addition of 1:500 EX-CYTE VLE (Serological Proteins Inc.
- the filtrate was microfiltrated (0.22 ⁇ m) before ultrafiltration to approximately 1/20 volume using a Millipore TFF system.
- the concentrate was diafiltrated using 10 mM Tris, pH 7.6.
- Ammonium sulphate was added to a concentration of 2.1 M and after stirring the precipitate was removed by centrifugation at 8000 rpm for 25 minutes in a Sorvall centrifuge using a GS3 rotor.
- the LFNG variant was then applied onto a Q-sepharose FF (Amersham Biosciences) column previously equilibrated in 10 mM Tris, pH 8.8. After application the column was washed with 3 column volumes of 10 mM Tris, pH 8.8 before eluting the bound LFNG variant in a gradient from 0-30% 10 mM Tris-HCl, 1 M NaCl, pH 8.8, over 25 column volumes. Fractions containing the LFNG variant were pooled and buffer exchanged into 5 mM sodium succinate, 4% mannitol, pH 6.0, using a VivaSpin20 column (VivaScience) and Tween 20 was subsequently added to a concentration of 0.01%. The LFNG variant was sterile filtered and stored at -80°C.
- the above-mentioned variants were constructed to study the C-terminal truncation in more detail.
- the variants were purified from serum-free media as described in Example 5, except that stabile clones from pooled clones were used instead of selected high-expressing single clones.
- 2500 to 5000 ml media was used to purify the individual variants.
- the sterile-filtered media (0.22 ⁇ m) were concentrated to approx. 1/15 volume and subsequently diafiltered (to a conductivity ⁇ 2 mS/cm) using 5 mM sodium phosphate, pH 6.2, on a PALL FLLTRON system.
- the concentrated/diafiltered media was filtered (0.22 ⁇ m) to clear the sample from any precipitated material prior to further purification.
- the pH in the filtrate was adjusted to 6.2 before application onto a 2 ml CM-sepharose Fast Flow (Pharmacia) previously equilibrated in 10 mM sodium phosphate, pH 6.2.
- the column was washed with 10- 15 column volumes 10 mM sodium phosphate, pH 6.2, before step-eluting bound variants with 2-3 column volumes 100 mM sodium phosphate, 500 mM NaCl, pH 7.0.
- the step-eluted variants were filtered (0.45 ⁇ m) before being immunoprecipitated with an IFNG antibody affinity column.
- the antibody affinity column was prepared according to the manufacture's instructions by coupling 10 mg monoclonal mouse anti-human LFNG antibody (catalog no. MD-2, U-CyTech, Holland) onto approx. 1.3 ml activated CNBr-sepharose
- CM-sepharose column The filtered sample from the CM-sepharose column was applied onto the antibody affinity column previously equilibrated with phosphate-buffered saline. The column was then washed with 5 column volumes phosphate buffered saline and the variant was subsequently eluted into a vial already containing 0.15 ml 500 mM sodium phosphate, pH 7.2, using 2 column volumes 100 mM glycine, pH 3.5.
- N-linked carbohydrate moieties attached to Asn-residues were removed by treatment of 30 ⁇ l of the affinity-purified variant with 1 mU PNGase F (Roche) at 37°C for 16 h.
- a 1 ⁇ l sample aliquot was mixed with 1 ⁇ l matrix solution (saturated ⁇ -cyano-4-hydroxy cinnamic acid in 50% acetonitril, 0.1% TFA).
- Half of the mixture was spotted onto a Thin-Layer of ⁇ - cyano-4-hydroxy cinnamic acid on the target plate and air-dried.
- the Thin-Layer coating was preformed on the target plate by crystallisation of ⁇ -cyano-4-hydroxy cinnamic acid from the Thin-Layer matrix solution (saturated ⁇ -cyano-4-hydroxy cinnamic acid in 100% acetone)).
- the sample spot was washed twice with 1 ⁇ l HPLC grade water and air-dried.
- the sample spot was added 0.2 ⁇ l matrix solution and air-dried. Following introduction of the target plate into the mass spectrometer, spectra were recorded at threshold laser power.
- Fig. 7 shows that further inclusion of the substitution Q143P in [E38N+S40T+S99T+ R137P+R139P]huLFNG does not alter the C-terminal processing notably.
- R139P+Q143P]huLFNG is still the protein having Alal41 as C-terminal amino acid residue.
- Fig. 8 shows that further inclusion of the substitution S142P in [E38N+ S40T+S99T+R137P+R139P]huLFNG alters the C-terminal processing notably as the only protein present in significant amount is the full-length protein having Q143 as the C-terminal amino acid residue.
- Fig. 9 clearly shows that inclusion of the substitution S142P in [E38N+S40T+S99T+ R137P]huLFNG leads to a significantly smaller degree of C-terminal processing and that the full-length protein having Q143 as the C-terminal amino acid residue is the major species present (comapare to Fig. 2).
- Fig. 10 evidently shows that the variant [E38N+S40T+S99T+S132P+R137P+R140P] huLFNG is hardly C-terminally processed. Only trace amounts of other species than the full- length protein can be identified. In a similar way, Fig. 11 shows that the full-length form of the variant [E38N+S40T+
- S99T+S132P+S140P]huLFNG is the major species.
- Fig. 13 shows that further inclusion of the substitution R140P in [E38N+ S40T+S99T+R137P]huLFNG alters the C-terminal processing notably as the only protein present in significant amount is the full-length protein having Q143 as the C-terminal amino acid residue.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2003239774A AU2003239774A1 (en) | 2002-07-03 | 2003-06-23 | Full-length interferon gamma polypeptide variants |
EP03732252A EP1539814A2 (en) | 2002-07-03 | 2003-06-23 | Full-length interferon gamma polypeptide variants |
US10/521,008 US7524931B2 (en) | 2002-07-03 | 2003-06-23 | Full-length interferon gamma polypeptide variants |
CA002491178A CA2491178A1 (en) | 2002-07-03 | 2003-06-23 | Full-length interferon gamma polypeptide variants |
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US39412002P | 2002-07-03 | 2002-07-03 | |
US60/394,120 | 2002-07-03 | ||
US41521402P | 2002-09-30 | 2002-09-30 | |
US60/415,214 | 2002-09-30 | ||
US41739902P | 2002-10-09 | 2002-10-09 | |
US60/417,399 | 2002-10-09 |
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PCT/DK2003/000426 WO2004005341A2 (en) | 2002-07-03 | 2003-06-23 | Full-length interferon gamma polypeptide variants |
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US (1) | US7524931B2 (en) |
EP (1) | EP1539814A2 (en) |
AU (1) | AU2003239774A1 (en) |
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CA2644127A1 (en) * | 2006-03-08 | 2007-09-13 | Biomethodes | Human interferon-gamma (infgamma) variants |
US9580719B2 (en) | 2007-04-27 | 2017-02-28 | Pfenex, Inc. | Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins |
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WO2023182947A1 (en) * | 2022-03-22 | 2023-09-28 | Chiang Mai University | A modified protein of interferon gamma and its use thereof |
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US7625555B2 (en) | 2007-06-18 | 2009-12-01 | Novagen Holding Corporation | Recombinant human interferon-like proteins |
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US10538565B2 (en) | 2007-06-18 | 2020-01-21 | Novagen Holding Corporation | Method of treating diseases with recombinant human interferon-like proteins |
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US10442845B2 (en) | 2009-12-31 | 2019-10-15 | Biorion Technologies B.V. | Interferon analogs |
Also Published As
Publication number | Publication date |
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CA2491178A1 (en) | 2004-01-15 |
AU2003239774A1 (en) | 2004-01-23 |
US7524931B2 (en) | 2009-04-28 |
EP1539814A2 (en) | 2005-06-15 |
WO2004005341A3 (en) | 2004-02-26 |
US20060099175A1 (en) | 2006-05-11 |
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