WO2008137471A2 - Modified interferon beta polypeptides and their uses - Google Patents

Modified interferon beta polypeptides and their uses Download PDF

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Publication number
WO2008137471A2
WO2008137471A2 PCT/US2008/062083 US2008062083W WO2008137471A2 WO 2008137471 A2 WO2008137471 A2 WO 2008137471A2 US 2008062083 W US2008062083 W US 2008062083W WO 2008137471 A2 WO2008137471 A2 WO 2008137471A2
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ifn beta
amino acid
beta polypeptide
polypeptide
group
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PCT/US2008/062083
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English (en)
French (fr)
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WO2008137471A3 (en
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Vadim Kraynov
Nick Knudsen
Anna-Maria A. Hays Putnam
Denise Krawitz
Jason Pinkstaff
Heather Myler
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Ambrx, Inc.
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Priority to AU2008247815A priority Critical patent/AU2008247815B2/en
Priority to JP2010506615A priority patent/JP2010525821A/ja
Priority to BRPI0810622-3A priority patent/BRPI0810622A2/pt
Priority to EP08747233.8A priority patent/EP2076533B1/en
Priority to NZ580686A priority patent/NZ580686A/xx
Priority to MX2009011870A priority patent/MX2009011870A/es
Application filed by Ambrx, Inc. filed Critical Ambrx, Inc.
Priority to CA002685596A priority patent/CA2685596A1/en
Priority to CN200880014461.7A priority patent/CN101675071B/zh
Publication of WO2008137471A2 publication Critical patent/WO2008137471A2/en
Publication of WO2008137471A3 publication Critical patent/WO2008137471A3/en
Priority to IL201791A priority patent/IL201791A/en
Priority to HK09111900.7A priority patent/HK1134822A1/xx

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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]
    • C07K14/565—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to interferon beta polypeptides optionally modified with at least one non- naturally-encoded amino acid.
  • Interferons are a well-known family of cytokines secreted by a large variety of eukaryotic cells. Interferons have a variety of biological activities, including antiviral, immunomodulatory, immunoregulatory, neoplastic, and antiproliferative properties, and have been utilized as therapeutic agents for treatment of diseases such as cancer, and various viral diseases. Interferons have demonstrated utility in the treatment of a variety of diseases, and are in widespread use for the treatment of multiple sclerosis and viral hepatitis; the most common therapeutic applications are currently treatment of hepatitis C and multiple sclerosis. Interferons are members of the growth hormone (GH) supergene family (Bazan, F.
  • GH growth hormone
  • some members of the family include the following: growth hormone, prolactin, placental lactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-IO, IL-I l, IL- 12 (p35 subunit), IL- 13, IL-15, oncostatin M, ciliary neurotrophic factor, leukemia inhibitory factor, alpha interferon, beta interferon, gamma interferon, omega interferon, tau interferon, epsilon interferon, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and cardiotrophin-1 (CT-I) ("the GH supergene family”
  • GH supergene family have similar secondary and tertiary structures, despite the fact that they generally have limited amino acid or DNA sequence identity.
  • the shared structural features allow new members of the gene family to be readily identified.
  • Four helical bundle and interferon polypeptides are described in WO 2005/074650 entitled “Modified Human Four Helical Bundle Polypeptides and Their Uses," WO 2005/074524 entitled “Modified Human Interferon Polypeptides and Their Uses," WO 2006/133089 and WO 2006/133088 entitled “Improved Human Interferon Polypeptides and Their Uses,” which are all incorporated by reference herein in their entirety.
  • Interferons include a number of related proteins, such as interferon-alpha (IFN- ⁇ ), interferon-beta (IFN- ⁇ ), interferon-gamma IFN- ⁇ ) interferon-kappa (IFN-K, also known as interferon-epsilon or IFN- ⁇ ), interferon-tau (IFN- ⁇ ), and interferon-omega (IFN- ⁇ ).
  • IFN- ⁇ interferon-alpha
  • IFN- ⁇ interferon-beta
  • IFN-K interferon-gamma IFN- ⁇
  • IFN-K interferon-kappa
  • IFN- ⁇ interferon-epsilon or IFN- ⁇
  • IFN- ⁇ interferon-tau
  • IFN- ⁇ interferon-omega
  • interferon proteins are produced in a variety of cell types: IFN- ⁇ (leukocytes), IFN- ⁇ (fibroblasts), IFN- ⁇ (lymphocytes), IFN- ⁇ or K (keratinocytes), IFN- ⁇ (leukocytes) and IFN- ⁇ (trophoblasts).
  • IFN- ⁇ , IFN- ⁇ , IFN- ⁇ or K, IFN- ⁇ , and IFN- ⁇ are classified as type I interferons, while IFN- ⁇ is classified as a type II interferon.
  • Interferon alpha is encoded by a multi-gene family, while the other interferons appear to each be coded by a single gene in the human genome. Furthermore, there is some allelic variation in interferon sequences among different members of the human population.
  • Interferons are relatively small, single-chain glycoproteins released by cells invaded by viruses or exposed to certain other substances. Interferons are presently grouped into three major classes, designated: 1) leukocyte interferon (interferon-alpha, ⁇ -interferon, IFN- ⁇ ), 2) fibroblast interferon (interferon-beta, ⁇ -interferon, IFN- ⁇ ), and 3) immune interferon (interferon-gamma, ⁇ -interferon, IFN- ⁇ ). In response to viral infection, lymphocytes synthesize primarily ⁇ -interferon (with omega interferon, IFN- ⁇ ), while infection of fibroblasts usually induces production of ⁇ -interferon.
  • leukocyte interferon interferon-alpha, ⁇ -interferon, IFN- ⁇
  • fibroblast interferon interferon-beta
  • IFN- ⁇ immune interferon
  • lymphocytes synthesize primarily ⁇ -interferon (with omega interferon, IFN
  • IFN ⁇ and IFN ⁇ share about 20-30 percent amino acid sequence homology.
  • the gene for human IFN- ⁇ lacks introns, and encodes a protein possessing 29% amino acid sequence identity with human IFN- ⁇ , suggesting that IFN- ⁇ and IFN- ⁇ genes have evolved from a common ancestor (Taniguchi et al., Nature 285 547-549 (1980)).
  • IFN- ⁇ is synthesized by lymphocytes in response to mitogens. Pestka et al. in Annu. Rev. Immunol.
  • interferons IFN- ⁇ , - ⁇ , - ⁇ , -K, - ⁇ , - ⁇ , - ⁇ , and - ⁇
  • interferon-like molecules such as limitin, IL-28A, IL-28B, and IL-29
  • the interferons have different species and many allelic variants.
  • interferons with novel activities and mutant sequences have been isolated from cells from patients with various diseases.
  • Interferons were originally derived from naturally occurring sources, such as buffy coat leukocytes and fibroblast cells, optionally using inducing agents to increase interferon production. Interferons have also been produced by recombinant DNA technology. The cloning and expression of mature IFN ⁇ is described by Goeddel et al., Nucleic Acids Res. 8, 4057 (1980).
  • Type-I interferons all appear to bind a common receptor, type I IFN-R, composed of IFNARl and IFN AR2 subunits. The exact binding mode and downstream signal transduction cascades differ somewhat among the type I interferons. However, in general, the JAK/STAT signal transduction pathway is activated following binding of interferon to the interferon receptor. STAT transcription factors then translocate to the nucleus, leading to the expression of a number of proteins with antiviral, antineoplastic, and immunomodulatory activities. [06] The properties of naturally occurring type I interferon proteins are not optimal for therapeutic use. Type I interferons induce injection site reactions and a number of other side effects.
  • Interferons are highly immunogenic, eliciting neutralizing and non-neutralizing antibodies in a significant fraction of patients. Interferons are poorly absorbed from the subcutaneous injection site and have short serum half-lives. Finally, type I interferons do not express solubly in prokaryotic hosts, thus necessitating more costly and difficult refolding or mammalian expression protocols.
  • IFN ⁇ - Ia (Avonex ® , and Rebif ® ), IFN ⁇ - Ib (Betaseron ® ), IFN alfacon-1 (Infergen ® ), IFN ⁇ -2 (Intron A ® ), IFN ⁇ -2a (Roferon-A ® ), Peginterferon alfa-2a (PEGASYS ® ), and Peginterferon alfa-2b (PEG-Intron ® ).
  • PEGASYS ® is comprised of nine identifiable isoforms, which specific isoforms differing in anti-viral activity (Foser et al., Pharmacogenomics J 2003; 3:312).
  • the present invention is directed to identification of interferon proteins with improved properties. A number of groups have generated modified interferons with improved properties; the references below are all expressly incorporated by reference in their entirety.
  • Cysteine-depleted variants have been generated to minimize formation of unwanted inter- or intra-molecular disulfide bonds (U.S. Patent Nos. 4,518,584; 4,588,585; and 4,959,314, which are incorporated by reference in their entirety).
  • Methionine-depleted variants have been generated to minimize susceptibility to oxidation (EPO 260350, which is incorporated by reference herein).
  • Patent No. 6,531,122 which is incorporated by reference.
  • Expression and generation of IFN beta variants and conjugates have been discussed in US Patent Nos. 7,144,574 and 6,531,111, which are incorporated by reference herein in their entirety. Modifications discussed included glycosylation sites that were introduced into IFN beta or removed from the polypeptide, substitutions near glycosylation sites, conjugation to lysine or cysteine residues, and introduction or removal of amino acids.
  • Interferon beta variants with enhanced stability have been discussed, in which the hydrophobic core was optimized using rational design methods (WO 00/68387, which is incorporated by reference). Alternate formulations that promote interferon stability or solubility have also been disclosed (U.S. Patent Nos. 4,675,483; 5,730,969; 5,766,582; WO 02/38170, which are incorporated by reference).
  • Immunogenicity is a major limitation of current interferon (including but not limited to, interferon beta) therapeutics. Although immune responses are typically most severe for non-human proteins, even therapeutics based on human proteins, such as interferon beta, are often observed to be immunogenic. Immunogenicity is a complex series of responses to a substance that is perceived as foreign and may include production of neutralizing and non- neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, and anaphylaxis. A number of patients develop neutralizing antibodies to IFN beta (Int.
  • variants engineered for improved solubility may also possess reduced immunogenicity.
  • Cysteine-depleted variants have been generated to minimize formation of unwanted inter- or intra-molecular disulfide bonds (U.S. Patent Nos. 4,518,584; 4,588,585; 4,959,314, which are incorporated by reference); such variants show a reduced propensity for aggregation.
  • Interferon beta variants with enhanced stability have been made, in which the hydrophobic core was optimized using rational design methods (WO 00/68387, which is incorporated by reference); in some cases solubility may be enhanced by improvements in stability.
  • Immunogenicity may limit the efficacy and safety of interferon therapeutics in multiple ways.
  • Therapeutic efficacy may be reduced directly by the formation of neutralizing antibodies.
  • Efficacy may also be reduced indirectly, as binding to either neutralizing or non- neutralizing antibodies may alter serum half-life.
  • Unwanted immune responses may take the form of injection site reactions, including but not limited to delayed-type hypersensitivity reactions. It is also possible that anti-interferon beta neutralizing antibodies may cross-react with endogenous interferon beta and block its function.
  • interferon proteins with improved properties, including but not limited to increased efficacy, decreased side effects, decreased immunogenicity, increased solubility, and enhanced soluble prokaryotic expression.
  • interferon polypeptides that require less frequent injection and/or result in decreased risk of developing neutralizing antibodies.
  • Improved interferon therapeutics could may be useful for the treatment of a variety of diseases and conditions, including autoimmune diseases, viral infections, and, inflammatory diseases, cell proliferation diseases, bacterial infections, enhancing fertility, and cancer, among others and transplant rejection.
  • interferons may be used to promote the establishment of pregnancy in certain mammals.
  • interferon beta one member of the interferon family
  • interferon-beta-la trademarked and sold as AVONEX ® , mfg. Biogen, Inc., Cambridge, Mass.
  • REB IF ® REB IF ®
  • interferon-beta-la or "IFN-beta-la” or “IFN- ⁇ -la” or “interferon- ⁇ -Ia”, or in various hyphenated and unhyphenated forms, used interchangeably.
  • a currently marketed formulation of AVONEX has 30 ug/dose (200 MIU/mg) and provides CHO-derived (Chinese Hamster Ovary) IFN-beta Ia given intramuscularly four times per week.
  • a currently marketed formulation of REBIF has 44 ⁇ g/dose (270 MIU/mg) given subcutanelous TIW and also provides CHO-derived IFN beta Ia.
  • interferon-beta-lb (trademarked and sold as BETASERON ® Berlex, Richmond, Calif.), hereinafter, "interferon-beta-lb”.
  • BETASERON ® Berlex, Richmond, Calif.
  • a currently marketed formulation of BETASERON has 250 ⁇ g/dose (32 MIU/mg) and provides E. C ⁇ /z-derived IFN-beta Ib given subcutaneously every other day or three times daily.
  • Interferon beta- Ia is produced in mammalian cells using the natural human gene sequence and is glycosylated, whereas interferon beta- Ib is produced in E.
  • C 17S cysteine-to-serine substitution at amino acid position 17
  • Common side effects include, but are not limited to, fever, headaches, fatigue, anxiety, depression, liver disorders, and injection site reactions.
  • Yong et al. Neurology (1998) 51 :682- 689 discuss the use of interferon beta in the treatment of multiple sclerosis and indicate the accumulation rate of disability from MS is reduced.
  • Karpusas et al. in Proc Natl Acad Sci 1997 94:11813-11818. This protein is glycosylated at a single site (Asn80). The protein has a tendency to aggregate when produced in E. coli (Mitsui et al. Pharmacol Ther 1993 58:93-132).
  • Karpusas et al. describe the production of human interferon beta in Chinese hamster ovary cells (CHO) and purification of the secreted protein using blue Sepharose and SP-Sepharose (ion exchange).
  • Alpha and beta interferons have been used in the treatment of the acute viral disease herpes zoster (T. C. Merigan et al, N. Engl. J. Med. 298, 981-987 (1978); E. Heidemann et al., Onkologie 7, 210-212 (1984)), chronic viral infections, e.g. hepatitis C and hepatitis B infections (R. L. Knobler et al., Neurology 34(10): 1273-9 (1984); M. A. Faerkkilae et al., Act. Neurol. Sci. 69, 184-185 (1985)).
  • Human IFN ⁇ is a regulatory polypeptide with a molecular weight of about 22 kDa consisting of 166 amino acid residues. It can be produced by most cells in the body, in particular fibroblasts, in response to viral infection or exposure to other agents. It binds to a multimeric cell surface receptor, and productive receptor binding results in a cascade of intracellular events leading to the expression of IFN ⁇ inducible genes which in turn produces effects which can be classified as anti-viral, antiproliferative and immunomodulatory. [27] The amino acid sequence of human IFN ⁇ is known (Taniguchi, Gene 10:11-15,
  • IFN ⁇ may have include, but are not limited to, inhibitory effects on the proliferation of leukocytes and antigen presentation, modulation of the profile of cytokine production towards an anti-inflammatory phenotype, and reduction of T-cell migration by inhibiting the activity of T-cell matrix metalloproteases to account for the mechanism of IFN ⁇ in MS (Neurol. 51 :682-689, 1998).
  • IFN beta may be used in the treatment of a number of diseases including, but not limited to, osteosarcoma, basal cell carcinoma, cervical dysplasia, glioma, acute myeloid leukemia, multiple myeloma, Hodgkin's disease, breast carcinoma, melanoma, and viral infections, including but not limited to, papilloma virus, viral hepatitis, herpes genitalis, herpes zoster, herpetic keratitis, herpes simplex, viral encephalitis, cytomegalovirus pneumonia, and rhinovirus.
  • Side effects of current IFN ⁇ therapeutics include injection site reactions, fever, chills, myalgias, arthralgias, and other flu-like symptoms (Clin. Therapeutics, 19:883-893, 1997).
  • Interferon-beta molecules of the invention may retain all or most of their biological activities and the following properties may result: altered pharmacokinetics and pharmacodynamics leading to increased half-life and alterations in tissue distribution (e.g., ability to stay in the vasculature for longer periods of time), increased stability in solution, reduced immunogenicity, protection from proteolytic digestion and subsequent abolition of activity.
  • interferon- beta Such molecules would be a substantial advance in the pharmaceutical and medical arts and would make a significant contribution to the management of various diseases in which interferon has sor ⁇ e utility, such as multiple sclerosis, fibrosis, and other inflammatory or autoimmune diseases, cancers, hepatitis and other viral diseases.
  • diseases in which interferon has sor ⁇ e utility such as multiple sclerosis, fibrosis, and other inflammatory or autoimmune diseases, cancers, hepatitis and other viral diseases.
  • Conjugates formed between interferon beta comprising a non-naturally encoded amino acid and another molecule, including but not limited to a polymer could result in modulated thermal stability of the conjugate.
  • modulated thermal stability may be an advantage when formulating interferon- beta in powder form for use in subsequent administration via inhalation.
  • Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated PEG is a method of increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time of many biologically active molecules, including proteins, peptides, and particularly hydrophobic molecules.
  • PEG has been used extensively in pharmaceuticals, on artificial implants, and in other applications where biocompatibility, lack of toxicity, and lack of immunogenicity are of importance.
  • the total molecular weight and hydration state of the PEG polymer or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics typically associated with PEG polymer attachment, such as increased water solubility and circulating half life, while not adversely impacting the bioactivity of the parent molecule.
  • PEG derivatives are frequently linked to biologically active molecules through reactive chemical functionalities, such as lysine, cysteine and histidine residues, the N-terminus and carbohydrate moieties.
  • Proteins and other molecules often have a limited number of reactive sites available for polymer attachment. Often, the sites most suitable for modification via polymer attachment play a significant role in receptor binding, and are necessary for retention of the biological activity of the molecule.
  • indiscriminate attachment of polymer chains to such reactive sites on a biologically active molecule often leads to a significant reduction or even total loss of biological activity of the polymer-modified molecule.
  • Reactive sites that form the loci for attachment of PEG derivatives to proteins are dictated by the protein's structure.
  • Proteins, including enzymes are composed of various sequences of alpha-amino acids, which have the general structure H 2 N-CHR-COOH.
  • the alpha amino moiety (H 2 N-) of one amino acid joins to the carboxyl moiety (--COOH) of an adjacent amino acid to form amide linkages, which can be represented as -(NH-CHR-CO) n — , where the subscript "n" can equal hundreds or thousands.
  • the fragment represented by R can contain reactive sites for protein biological activity and for attachment of PEG derivatives.
  • PEGylation is that the PEG derivatives can undergo undesired side reactions with residues other than those desired.
  • Histidine contains a reactive imino moiety, represented structurally as — N(H)-, but many chemically reactive species that react with epsilon -NH 2 can also react with ⁇ N(H)--.
  • the side chain of the amino acid cysteine bears a free sulfhydryl group, represented structurally as -SH.
  • the PEG derivatives directed at the epsilon - -NH 2 group of lysine also react with cysteine, histidine or other residues.
  • a cysteine residue can be introduced site-selectively into the structure of proteins using site-directed mutagenesis and other techniques known in the art, and the resulting free sulfhydryl moiety can be reacted with PEG derivatives that bear thiol-reactive functional groups.
  • This approach is complicated, however, in that the introduction of a free sulfhydryl group can complicate the expression, folding and stability of the resulting protein.
  • PEG derivatives have been developed that are more stable (e.g., U.S. Patent 6,602,498, which is incorporated by reference herein) or that react selectively with thiol moieties on molecules and surfaces (e.g., U.S. Patent 6,610,281, which is incorporated by reference herein).
  • U.S. Patent 6,602,498, which is incorporated by reference herein or that react selectively with thiol moieties on molecules and surfaces
  • thiol moieties e.g., U.S. Patent 6,610,281, which is incorporated by reference herein.
  • a number of new amino acids with novel chemical, physical or biological properties including photoaffinity labels and photoisomerizable amino acids, photocrosslinking amino acids (see, e.g., Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U. S. A 1 99:11020-11024; and, Chin, J. W., et al., (2002) J. Am. Chem. Soc. 124:9026-9027), keto amino acids, heavy atom containing amino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E. coli and in yeast in response to the amber codon, TAG, using this methodology. See, e.g., J. W.
  • Certain chemical functional groups are known to be inert to the functional groups found in the 20 common, genetically-encoded amino acids but react cleanly and efficiently to form stable linkages.
  • Azide and acetylene groups are known in the art to undergo a Huisgen [3+2] cycloaddition reaction in aqueous conditions in the presence of a catalytic amount of copper. See, e.g., Tornoe, et al., (2002) L Org. Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41 :2596-2599.
  • an azide moiety By introducing an azide moiety into a protein structure, for example, one is able to incorporate a functional group that is chemically inert to amines, sulfhydryls, carboxylic acids, hydroxyl groups found in proteins, but that also reacts smoothly and efficiently with an acetylene moiety to form a cycloaddition product. Importantly, in the absence of the acetylene moiety, the azide remains chemically inert and unreactive in the presence of other protein side chains and under physiological conditions.
  • the present invention addresses, among other things, problems associated with the activity and production of IFN beta polypeptides, and also addresses the production of a IFN beta polypeptide with improved biological or pharmacological properties, such as enhanced antiviral activity and/or improved therapeutic half-life.
  • This invention provides IFN beta polypeptides comprising one or more non- naturally encoded amino acids.
  • the IFN beta polypeptide comprises one or more post- translational modifications. In some embodiments, the IFN beta polypeptide is linked to a linker, polymer, or biologically active molecule. In some embodiments, the IFN beta polypeptide is linked to a bifunctional polymer, bifunctional linker, or at least one additional IFN beta polypeptide. In some embodiments, the IFN beta polypeptide comprises one or more post-translational modifications to the C17S mutant form of IFN beta.
  • the non-naturally encoded amino acid is linked to a water soluble polymer.
  • the water soluble polymer comprises a poly(ethylene glycol) moiety.
  • the non-naturally encoded amino acid is linked to the water soluble polymer with a linker or is bonded to the water soluble polymer.
  • the poly(ethylene glycol) molecule is a bifunctional polymer.
  • the bifunctional polymer is linked to a second polypeptide.
  • the second polypeptide is a IFN beta polypeptide.
  • the present invention also includes each of the above embodiments in addition to the C17S mutation in IFN beta.
  • the IFN beta polypeptide comprises at least two amino acids linked to a water soluble polymer comprising a poly(ethylene glycol) moiety. In some embodiments, at least one amino acid is a non-naturally encoded amino acid. In some embodiments, the IFN beta polypeptide comprising at least two amino acids linked to a water soluble polymer comprising a poly(ethylene glycol) moiety includes the C17S mutation, in other embodiments any mutation at position 17 is included in addition to one or more non-naturally encoded amino acids in an IFN beta polypeptide.
  • one or more non-naturally encoded amino acids are incorporated in one or more of the following positions in IFN beta: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102
  • one or more non-naturally encoded amino acids are incorporated at any position in one or more of the following regions corresponding to secondary structures in interferon beta as follows: Helix A (2-22); Helix B (51-71); Helix C (80-107); Helix D (118-136); Helix E (139-162); AB loop: ABl (23-35); AB2 (36-40); AB3 (41-50) from SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 3, 4.
  • the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 25-35, 80-100, and 121-135 of interferon beta (SEQ ID NO: 1 or the corresponding amino acids from SEQ ID NO: 3, 4). In other embodiments, the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 41-49 from interferon beta from SEQ ID NO: 1 or the corresponding amino acids from SEQ ID NO: 3, 4.
  • one or more non-naturally encoded amino acids are incorporated at one or more of the following positions of IFN beta: 28, 36, 76, 80, 107, 108, 111, 8, 15, 19, 36, 42, 46, 48, 49, 80, 108, 111, 113, 155, and any combination thereof (SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4).
  • one or more non-naturally encoded amino acids are incorporated at one or more of the following positions of IFN beta: 28, 36, 76, 80, 107, 108, 111, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4.
  • one or more non- naturally encoded amino acids are incorporated at one or more of the following positions of IFN beta: 8, 15, 19, 36, 42, 46, 48, 49, 80, 108, 111, 113, 155, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4.
  • one or more non-naturally encoded amino acids are incorporated at one or more of the following positions of IFN beta: 15, 42, 80, 108, 111, 155, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4).
  • one or more non-naturally encoded amino acids are incorporated at one or more of the following positions of IFN beta: 36, 111 of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4).
  • the polypeptide of the invention comprises one or more natural amino acid substitution, addition, or deletion.
  • the polypeptide of the invention comprises a C17S substitution (serine for a cysteine at position 17) of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NO: 3, 4.
  • the polypeptide of the invention comprises a C17S substitution (serine for a cysteine at position 17) and one or more natural amino acid substitution, addition, or deletion.
  • the polypeptide of the invention comprises one or more non-naturally encoded amino acid substitution, addition, or deletion in the signal sequence. In some embodiments, the polypeptide of the invention comprises one or more non-naturally encoded amino acid substitution, addition, or deletion in the signal sequence of SEQ ID NO: 4. In some embodiments, the polypeptide of the invention comprises one ore more naturally encoded amino acid substitution, addition, or deletion in the signal sequence of SEQ ID NO: 4. In some embodiments, one or more non-natural amino acids are incorporated in the leader or signal sequence of SEQ ID NOs: 4 or other IFN beta sequence. The present invention also includes each of the above embodiments in addition to the C17S mutation in IFN beta.
  • the non-naturally occurring amino acid at one or more of these positions is linked to a water soluble polymer, including but not limited to, positions: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99
  • the non-naturally occurring amino acid in one or more of these regions is linked to a water soluble polymer, including but not limited to: Helix A (2-22); Helix B (51-71); Helix C (80-107); Helix D (118-136); Helix E (139-162); AB loop: ABl (23- 35); AB2 (36-40); AB3 (41-50) from SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 3, 4.
  • a water soluble polymer including but not limited to: Helix A (2-22); Helix B (51-71); Helix C (80-107); Helix D (118-136); Helix E (139-162); AB loop: ABl (23- 35); AB2 (36-40); AB3 (41-50) from SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 3, 4.
  • the non-naturally occurring amino acid in one or more of these regions is linked to a water soluble polymer, including but not limited to, residues 25-35, 80-100, and 121-135 of interferon beta (SEQ ID NO: 1 or the corresponding amino acids from SEQ ID NO: 3, 4).
  • the non-naturally occurring amino acid in one or more of these regions is linked to a water soluble polymer, including but not limited to, residues 41-49 from interferon beta from SEQ ID NO: 1 or the corresponding amino acids from SEQ ID NO: 3, 4.
  • the non-naturally occurring amino acid at one or more of these positions is linked to a water soluble polymer, including but not limited to, positions: 28, 36, 76, 80, 107, 108, 111, 8, 15, 19, 36, 42, 46, 48, 49, 80, 108, 111, 113, 155, and any combination thereof (SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4).
  • the non-naturally occurring amino acid at one or more of these positions is linked to a water soluble polymer, including but not limited to, positions: 28, 36, 76, 80, 107, 108, 111, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4.
  • the non-naturally occurring amino acid at one or more of these positions is linked to a water soluble polymer, including but not limited to, positions: 8, 15, 19, 36, 42, 46, 48, 49, 80, 108, 111, 113, 155, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4).
  • the non- naturally occurring amino acid at one or more of these positions is linked to a water soluble polymer, including but not limited to, positions: 15, 42, 80, 108, 111, 155, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4).
  • the non-naturally occurring amino acid at one or more of these positions is linked to a water soluble polymer, including but not limited to, positions: 36, 111 of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4).
  • the non-naturally occurring amino acid in the signal or leader sequence is linked to a water soluble polymer (SEQ ID NO: 4 or other IFN beta sequence).
  • the present invention also includes each of the above embodiments in addition to the C17S mutation in IFN beta.
  • the IFN beta polypeptide comprises a substitution, addition or deletion that modulates affinity of the IFN beta polypeptide for a IFN polypeptide receptor or binding partner, including but not limited to, a protein, polypeptide, small molecule, or nucleic acid.
  • the IFN beta polypeptide comprises a substitution, addition, or deletion that increases the stability of the IFN beta polypeptide when compared with the stability of the corresponding IFN beta without the substitution, addition, or deletion. Stability and/or solubility may be measured using a number of different assays known to those of ordinary skill in the art. Such assays include but are not limited to SE-HPLC and RP-HPLC.
  • the IFN beta polypeptide comprises a substitution, addition, or deletion that modulates the immunogenicity of the IFN beta polypeptide when compared with the immunogenicity of the corresponding IFN beta without the substitution, addition, or deletion. In some embodiments, the IFN beta polypeptide comprises a substitution, addition, or deletion that modulates serum half-life or circulation time of the IFN beta polypeptide when compared with the serum half-life or circulation time of the corresponding IFN beta without the substitution, addition, or deletion.
  • the IFN beta polypeptide comprises a substitution, addition, or deletion that increases the aqueous solubility of the IFN beta polypeptide when compared to aqueous solubility of the corresponding IFN beta without the substitution, addition, or deletion.
  • the IFN beta polypeptide comprises a substitution, addition, or deletion that increases the solubility of the IFN beta polypeptide produced in a host cell when compared to the solubility of the corresponding IFN beta without the substitution, addition, or deletion.
  • the IFN beta polypeptide comprises a substitution, addition, or deletion that increases the expression of the IFN beta polypeptide in a host cell or increases synthesis in vitro when compared to the expression or synthesis of the corresponding IFN beta without the substitution, addition, or deletion.
  • the IFN beta polypeptide comprising this substitution retains agonist activity and retains or improves expression levels in a host cell.
  • the IFN beta polypeptide comprises a substitution, addition, or deletion that increases protease resistance of the IFN beta polypeptide when compared to the protease resistance of the corresponding IFN beta without the substitution, addition, or deletion.
  • the IFN beta pol ypeptide comprises a substitution, addition, or deletion that modulates signal transduction activity of the IFN receptor when compared with the activity of the receptor upon interaction with the corresponding IFN beta polypeptide without the substitution, addition, or deletion.
  • the IFN beta polypeptide comprises a substitution, addition, or deletion that modulates its binding to another molecule such as a receptor when compared to the binding of the corresponding IFN beta polypeptide without the substitution, addition, or deletion.
  • the IFN beta polypeptide comprises a substitution, addition, or deletion that modulates its anti-viral activity compared to the anti-viral activity of the corresponding IFN beta polypeptide without the substitution, addition, or deletion.
  • the IFN beta polypeptide comprises a substitution, addition, or deletion that enhances its anti-viral activity compared to the anti-viral activity of the corresponding IFN beta polypeptide without the substitution, addition, or deletion.
  • the IFN beta polypeptide comprises a substitution, addition, or deletion that increases compatibility of the IFN beta polypeptide with pharmaceutical preservatives (e.g., w-cresol, phenol, benzyl alcohol) when compared to compatibility of the corresponding IFN beta without the substitution, addition, or deletion.
  • pharmaceutical preservatives e.g., w-cresol, phenol, benzyl alcohol
  • This increased compatibility would enable the preparation of a preserved pharmaceutical formulation that maintains the physiochemical properties and biological activity of the protein during storage.
  • one or more engineered bonds Eire created with one or more non-natural amino acids.
  • the intramolecular bond may be created in many ways, including but not limited to, a reaction between two amino acids in the protein under suitable conditions (one or both amino acids may be a non-natural amino acid); a reaction with two amino acids, each of which may be naturally encoded or non-naturally encoded, with a linker, polymer, or other molecule under suitable conditions; etc.
  • one or more amino acid substitutions in the IFN beta polypeptide may be with one or more naturally occurring or non-naturally occurring amino acids. In some embodiments the amino acid substitutions in the IFN beta polypeptide may be with naturally occurring or non-naturally occurring amino acids, provided that at least one substitution is with a non-naturally encoded amino acid. In some embodiments, one or more amino acid substitutions in the IFN beta polypeptide may be with one or more naturally occurring amino acids, and additionally at least one substitution is with a non-naturally encoded amino acid.
  • the non-naturally encoded amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.
  • the non-naturally encoded amino acid comprises a carbonyl group. In some embodiments, the non-naturally encoded amino acid has the structure:
  • n is 0-10;
  • R 1 is an alkyl, aryl, substituted alkyl, or substituted aryl;
  • R 2 is H, an alkyl, aryl, substituted alkyl, and substituted aryl;
  • R 3 is H, an amino acid, a polypeptide, or an amino terminus modification group, and
  • R 4 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.
  • the non-naturally encoded amino acid comprises an aminooxy group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazide group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazine group. In some embodiments, the non-naturally encoded amino acid residue comprises a semicarbazide group.
  • the non-naturally encoded amino acid residue comprises an azide group.
  • the non-naturally encoded amino acid has the structure: wherein n is 0-10; Rj is an alkyl, aryl, substituted alkyl, substituted aryl or not present; X is O, N, S or not present; m is 0-10; R 2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R 3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.
  • the non-naturally encoded amino acid comprises an alkyne group. In some embodiments, the non-naturally encoded amino acid has the structure:
  • the polypeptide is a IFN beta polypeptide agonist, partial agonist, antagonist, partial antagonist, or inverse agonist.
  • the IFN beta polypeptide agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-naturally encoded amino acid linked to a water soluble polymer.
  • the water soluble polymer comprises a poly(ethylene glycol) moiety.
  • the IFN beta polypeptide agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-naturally encoded amino acid and one or more post-translational modification, linker, polymer, or biologically active molecule.
  • the present invention also provides isolated nucleic acids comprising a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 2 or nucleic acids that encode polypeptides of SEQ ID NOs: 3, 4.
  • the present invention also provides isolated nucleic acids comprising a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 2 or polynucleotides that hybridize under stringent conditions to polynucleotides that encode polypeptides shown as SEQ ID NOs: 3, 4 wherein the polynucleotide comprises at least one selector codon.
  • the present invention also provides isolated nucleic acids comprising a polynucleotide that encodes the polypeptides shown as SEQ ID NOs.: 1, 3, 4.
  • the present invention also provides isolated nucleic acids comprising a polynucleotide that encodes the polypeptides shown as SEQ ID NOs.: 1, 3, 4, with one or more non-naturally encoded amino acids. It is readily apparent to those of ordinary skill in the art that a number of different polynucleotides can encode any polypeptide of the present invention.
  • the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, a five-base codon, and a four-base codon.
  • the present invention also provides methods of making a IFN beta polypeptide linked to a water soluble polymer.
  • the method comprises contacting an isolated IFN beta polypeptide comprising a non-naturally encoded amino acid with a water soluble polymer comprising a moiety that reacts with the non-naturally encoded amino acid.
  • the non-naturally encoded amino acid incorporated into the IFN beta polypeptide is reactive toward a water soluble polymer that is otherwise unreactive toward any of the 20 common amino acids.
  • the non-naturally encoded amino acid incorporated into the IFN beta polypeptide is reactive toward a linker, polymer, or biologically active molecule that is otherwise unreactive toward any of the 20 common amino acids.
  • the IFN beta polypeptide linked to the water soluble polymer is made by reacting a IFN beta polypeptide comprising a carbonyl-containing amino acid with a poly(ethylene glycol) molecule comprising an aminooxy, hydrazine, hydrazide or semicarbazide group.
  • the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the poly (ethylene glycol) molecule through an amide linkage.
  • the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the poly(ethylene glycol) molecule through a carbamate linkage.
  • the IFN beta polypeptide linked to the water soluble polymer is made by reacting a poly(ethylene glycol) molecule comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid that comprises an aminooxy, hydrazine, hydrazide or semicarbazide group.
  • the IFN beta polypeptide linked to the water soluble polymer is made by reacting a IFN beta polypeptide comprising an alkyne-containing amino acid with a poly(ethylene glycol) molecule comprising an azide moiety.
  • the azide or alkyne group is linked to the poly(ethylene glycol) molecule through an amide linkage.
  • the IFN beta polypeptide linked to the water soluble polymer is made by reacting a IFN beta polypeptide comprising an azide-containing amino acid with a poly(ethylene glycol) molecule comprising an alkyne moiety.
  • the azide or alkyne group is linked to the poly(ethylene glycol) molecule through an amide linkage.
  • the poly(ethylene glycol) molecule has a molecular weight of between about 0.1 kDa and about 100 kDa. In some embodiments, the poly(ethylene glycol) molecule has a molecular weight of between 0.1 kDa and 50 kDa.
  • the poly(ethylene glycol) molecule is a branched polymer.
  • each branch of the poly(ethylene glycol) branched polymer has a molecular weight of between 1 kDa and 100 kDa, or between 1 kDa and 50 kDa.
  • the water soluble polymer linked to the IFN beta polypeptide comprises a polyalkylene glycol moiety.
  • the non-naturally encoded amino acid residue incorporated into the IFN beta polypeptide comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine, a semicarbazide group, an azide group, or an alkyne group.
  • the non-naturally encoded amino acid residue incorporated into the IFN beta polypeptide comprises a carbonyl moiety and the water soluble polymer comprises an aminooxy, hydrazide, hydrazine, or semicarbazide moiety.
  • the non-naturally encoded amino acid residue incorporated into the IFN beta polypeptide comprises an alkyne moiety and the water soluble polymer comprises an azide moiety.
  • the non-naturally encoded amino acid residue incorporated into the IFN beta polypeptide comprises an azide moiety and the water soluble polymer comprises an alkyne moiety.
  • compositions comprising a IFN beta polypeptide comprising a non-naturally encoded amino acid and a pharmaceutically acceptable carrier.
  • the non-naturally encoded amino acid is linked to a water soluble polymer.
  • the present invention also provides cells comprising a polynucleotide encoding the IFN beta polypeptide comprising a selector codon.
  • the cells comprise an orthogonal RNA synthetase and/or an orthogonal tRNA for substituting a non-naturally encoded amino acid into the IFN beta polypeptide.
  • the present invention also provides methods of making a IFN beta polypeptide comprising a non-naturally encoded amino acid.
  • the methods comprise culturing cells comprising a polynucleotide or polynucleotides encoding a IFN beta polypeptide, an orthogonal RNA synthetase and/or an orthogonal tRNA under conditions to permit expression of the IFN beta polypeptide; and purifying the IFN beta polypeptide from the cells and/or culture medium.
  • the present invention also provides methods of increasing therapeutic half-life, serum half-life or circulation time of IFN beta polypeptides.
  • the present invention also provides methods of modulating immunogenicity of IFN beta polypeptides.
  • the methods comprise substituting a non-naturally encoded amino acid for any one or more amino acids in naturally occurring IFN beta polypeptides and/or linking the IFN beta polypeptide to a linker, a polymer, a water soluble polymer, or a biologically active molecule.
  • the present invention also provides methods of treating a patient in need of such treatment with an effective amount of a IFN beta molecule of the present invention.
  • the methods comprise administering to the patient a therapeutic ally-effective amount of a pharmaceutical composition comprising a IFN beta polypeptide comprising a non- naturally-encoded amino acid and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising a IFN beta polypeptide comprising a non- naturally-encoded amino acid and a pharmaceutically acceptable carrier.
  • the non-naturally encoded amino acid is linked to a water soluble polymer.
  • the IFN beta polypeptide is glycosylated. In some embodiments, the IFN beta polypeptide is not glycosylated.
  • the present invention also provides IFN beta polypeptides comprising a sequence shown in SEQ ID NO: 1, 3, 4, or any other IFN beta polypeptide sequence, except that at least one amino acid is substituted by a non-naturally encoded amino acid.
  • the present invention also provides IFN beta polypeptides comprising a sequence shown as SEQ ID NO: 1, 3, 4.
  • the non-naturally encoded amino acid is linked to a water soluble polymer.
  • the water soluble polymer comprises a poly(ethylene glycol) moiety.
  • the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.
  • the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a IFN beta polypeptide comprising the sequence shown in SEQ ID NO: 1, 3, 4, or any other IFN beta polypeptide sequence, wherein at least one amino acid is substituted by a non-naturally encoded amino acid.
  • the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a IFN beta polypeptide comprising the sequence shown in SEQ ID NO: 1, 3, 4.
  • the non-naturally encoded amino acid comprises a saccharide moiety.
  • the water soluble polymer is linked to the polypeptide via a saccharide moiety.
  • a linker, polymer, or biologically active molecule is linked to the IFN beta polypeptide via a saccharide moiety.
  • the present invention also provides a IFN beta polypeptide comprising a water soluble polymer linked by a covalent bond to the IFN beta polypeptide at a single amino acid.
  • the water soluble polymer comprises a poly(ethylene glycol) moiety.
  • the amino acid covalently linked to the water soluble polymer is a non- naturally encoded amino acid present in the polypeptide.
  • the present invention provides a IFN beta polypeptide comprising at least one linker, polymer, or biologically active molecule, wherein said linker, polymer, or biologically active molecule is attached to the polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into the polypeptide.
  • the polypeptide is monoPEGylated.
  • the present invention also provides a IFN beta polypeptide comprising a linker, polymer, or biologically active molecule that is attached to one or more non-naturally encoded amino acid wherein said non-naturally encoded amino acid is ribosomally incorporated into the polypeptide at pre-selected sites.
  • IFN beta leader or signal sequence joined to an IFN beta coding region as well as a heterologous signal sequence joined to an IFN beta coding region.
  • the heterologous leader or signal sequence selected should be one that is recognized and processed, e.g. by host cell secretion system to secrete and possibly cleaved by a signal peptidase, by the host cell.
  • a method of treating a condition or disorder with the IFN beta of the present invention is meant to imply treating with IFN beta with or without a signal or leader peptide.
  • the present invention also provides methods of inducing an increase in anti-viral activity in cells, said method comprising administering IFN beta to said cells in an amount effective to induce an increase in anti-viral activity.
  • conjugation of the IFN beta polypeptide comprising one or more non-naturally occurring amino acids to another molecule provides substantially purified IFN beta due to the unique chemical reaction utilized for conjugation to the non-natural amino acid.
  • Conjugation of IFN beta comprising one or more non-naturally encoded amino acids to another molecule, such as PEG may be performed with other purification techniques performed prior to or following the conjugation step to provide substantially pure IFN beta.
  • Figure 2 A model of the crystal structure of IFN beta is shown. Selected sites for substitution with a non-naturally encoded amino acid are shown.
  • Figure 3 SDS PAGE analysis of the expression of IFN beta polypeptides of the present invention is shown.
  • Figure 6A - A graph of pharmacokinetic data from Example 27, 3 ⁇ g/kg dose level group, showing serum IFN beta concentration measured in ng/mL over time elapsed in hours.
  • Figure 6B A graph of pharmacokinetic data from Example 27, 15 ⁇ g/kg dose level group, showing serum IFN beta concentration measured in ng/mL over time elapsed in hours.
  • Figure 7A - A graph of pharmacokinetic data from Example 27, 50 ⁇ g/kg dose level group, showing serum IFN beta concentration measured in ng/mL over time elapsed in hours.
  • Figure 9B A graph of data from Example 27 showing Cmax of some IFN beta variants of the present invention against a control (Serum IFNbeta Cmax/Dose).
  • FIG. 1OA A graph of area under the curve (AUC) data from Example 27 showing AUC of some IFN beta variants of the present invention against a control (Serum IFN
  • Figure 1OB A bar graph of data from Example 27 showing serum IFN beta concentrations at 168 hours following dosing with two different IFN beta variants of the present invention at different dosage levels and a control.
  • FIG. 1 IA - A graph of data from Example 27 showing Neopterin response of some IFN beta variants of the present invention at the 3 ⁇ g/kg dose group against placebo and a control (Serum Neopterin/Time).
  • FIG. 11B A graph of data from Example 27 showing Neopterin response of some IFN beta variants of the present invention at the 15 ⁇ g/kg dose group against placebo and a control (Serum Neopterin/Time).
  • Figure 12A - A graph of data from Example 27 showing Neopterin response of some IFN beta variants of the present invention at the 50 ⁇ g/kg dose group against placebo and a control (Serum Neopterin/Time).
  • Figure 12B A graph of data from Example 27 showing area under the curve measurements for serum neopterin versus dose for some IFN beta variants of the present invention against placebo and a control (Serum Neopterin AUC/Dose).
  • Figure 13A Serum neopterin level data from Example 27 is shown at 168 hours post-dosing with placebo, control, and three different dosage levels of M36-30K.
  • Figure 13B Serum neopterin level data from Example 27 is shown at 168 hours post-dosing with placebo, control, and three different dosage levels of M36-40K.
  • Figure 13C Serum neopterin level data from Example 27 is shown at 168 hours post-dosing with placebo, control, and three different dosage levels of Fl 11-30K.
  • Figure 13D Serum neopterin level data from Example 27 is shown at 168 hours post-dosing with placebo, control, and three different dosage levels of Fl 11-40K.
  • Figure 14 A graph showing the Neopterin AUC/dose of Rebif from the protocol disclosed in Example 27 and the BLA.
  • Figure 15A A graph showing serum IFN beta al levels as measured by antiviral activity (filled symbols and solid lines on graph) and neopterin concentrations (open symbols and dashed lines on graph) in rhesus monkeys following intravenous administration.
  • Figure 15B A graph showing serum IFN beta al levels as measured by antiviral activity (filled symbols and solid lines on graph) and neopterin concentrations (open symbols and dashed lines on graph) in rhesus monkeys following subcutaneous administration.
  • Figure 15C A graph showing antiviral activity of unmodified and pegylated
  • IFN beta Ia assessed in antiviral assays using human lung carcinoma (A549) cells challenged with EMC virus. Following 2-day incubation with virus, viable cells were stained with XTT, the plates were read at 450 nm, and the absorbance, which is reflective of cell viability, is shown on the y-axis. Samples were analyzed in duplicate.
  • FIG. 16A A graph of data from Example 27 showing the fold induction vs. pre-dosing with vehicle, Rebif, and four different IFN beta variants at the 3 ⁇ g/kg dose level of the present invention of OASl gene expression against time.
  • Figure 16B A graph of data from Example 27 showing the fold induction vs. pre-dosing with vehicle, Rebif, and an average of the 3 different dose levels of four IFN beta variants of the present invention of OASl gene expression against time.
  • Figure 17A - A graph of data from Example 27 showing the fold induction vs. pre-dosing with vehicle, Rebif, and four different IFN beta variants at the 15 ⁇ g/kg dose level of the present invention of OASl gene expression against time.
  • Figure 17B - A graph of data from Example 27 showing the fold induction vs. pre-dosing with vehicle, Rebif, and four different IFN beta variants at the 50 ⁇ g/kg dose level of the present invention of OASl gene expression against time.
  • Figure 18 - A graph of data from Example 27 showing the area under the curve of the fold induction of OAS 1 gene expression against dose level for a vehicle, a control, and four different IFN beta Ia variants of the present invention.
  • Figure 19 A bar graph of the percentage of injection site reactions during the first twenty-four hours post dose administration.
  • Figure 20 - A table of data collected in Example 27 for each different treatment group - N. S. indicating not significant.
  • Figure 21 - A table of data collected in Example 27 for each different treatment group - N. S. indicating not significant.
  • FIG. 22 A model of the crystal structure of IFN beta with potential sites for methionine oxidation is shown. M36 and 117 are susceptible for oxidation, Ml is a bit susceptible however when produced in E. CoIi 1 the Ml should be cleaved (however may not be fully processed), and M62 is a site that is not susceptible for oxidation.
  • FIG. 23 - A model of the crystal structure of IFN beta with potential sites for deamidation is shown. M36 and N25 are both potential sites - N25 is a known site for deamidation.
  • Example 28 [123] Figure 26 - Overlay chromatogram of IFN M36pAF and PEG40-M36pAF using methods described in Example 28.
  • substantially purified refers to an IFN beta polypeptide that may be substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e. a native cell, or host cell in the case of recombinantly produced IFN beta polypeptides.
  • IFN beta polypeptide that may be substantially free of cellular material includes preparations of protein having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein.
  • the protein may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells.
  • the protein may be present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about lg/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about lmg/L or less of the dry weight of the cells.
  • substantially purified IFN beta polypeptide as produced by the methods of the present invention may have a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically, a purity level of at least about 75%, 80%, 85%, and more specifically, a purity level of at least about 90%, a purity level of at least about 95%, a purity level of at least about 99% or greater as determined by appropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
  • a "recombinant host cell” or “host cell” refers to a cell that includes an exogenous polynucleotide, regardless of the method used for insertion, for example, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells.
  • the exogenous polynucleotide may be maintained as a nonintegrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • the term “medium” or “media” includes any culture medium, solution, solid, semi-solid, or rigid support that may support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli, or Pseudomonas host cells, and cell contents.
  • the term may encompass medium in which the host cell has been grown, e.g., medium into which the IFN beta polypeptide has been secreted, including medium either before or after a proliferation step.
  • the term also may encompass buffers or reagents that contain host cell lysates, such as in the case where the IFN beta polypeptide is produced intracellularly and the host cells are lysed or disrupted to release the IFN beta polypeptide.
  • "Reducing agent” as used herein with respect to protein refolding, is defined as any compound or material which maintains sulfhydryl groups in the reduced state and reduces intra- or intermolecular disulfide bonds. Suitable reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2- aminoethanethiol), and reduced glutathione. It is readily apparent to those of ordinary skill in the art that a wide variety of reducing agents are suitable for use in the methods and compositions of the present invention.
  • Oxidizing agent as used herein with respect to protein refolding, is defined as any compound or material which is capable of removing an electron from a compound being oxidized. Suitable oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. It is readily apparent to those of ordinary skill in the art that a wide variety of oxidizing agents are suitable for use in the methods of the present invention.
  • Denaturing agent or "denaturant,” as used herein, is defined as any compound or material which will cause a reversible unfolding of a protein.
  • the strength of a denaturing agent or denaturant will be determined both by the properties and the concentration of the particular denaturing agent or denaturant.
  • Suitable denaturing agents or denaturants may be chaotropes, detergents, organic solvents, water miscible solvents, phospholipids, or a combination of two or more such agents. Suitable chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate.
  • Useful detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate, or polyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as N->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g.
  • zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3-(3-chlolamidopropyl)dimethylammonio-l-propane sulfate (CHAPS), and 3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-l -propane sulfonate (CHAPSO).
  • Organic, water miscible solvents such as acetonitrile, lower alkanols (especially C 2 - C 4 alkanols such as ethanol or isopropanol), or lower alkandiols (especially C 2 - C 4 alkandiols such as ethylene-glycol) may be used as denaturants.
  • lower alkanols especially C 2 - C 4 alkanols such as ethanol or isopropanol
  • lower alkandiols especially C 2 - C 4 alkandiols such as ethylene-glycol
  • Phospholipids useful in the present invention may be naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidy lcholine .
  • Refolding describes any process, reaction or method which transforms disulfide bond containing polypeptides from an improperly folded or unfolded state to a native or properly folded conformation with respect to disulfide bonds.
  • Cofolding refers specifically to refolding processes, reactions, or methods which employ at least two polypeptides which interact with each other and result in the transformation of unfolded or improperly folded polypeptides to native, properly folded polypeptides.
  • IFN beta polypeptide As used herein, "IFN beta polypeptide,” “IFN beta” or “IFN ⁇ ” and hyphenated and unhyphenated forms thereof shall include those polypeptides and proteins that have at least one biological activity of a IFN beta, as well as Ia and Ib forms, a and b forms, IFN beta analogs, IFN beta isoforms, IFN beta mimetics, IFN beta fragments, hybrid IFN beta proteins, fusion proteins, oligomers and multimers, homologues, glycosylation pattern variants, variants, splice variants, and muteins, thereof, regardless of the biological activity of same, and further regardless of the method of synthesis or manufacture thereof including, but not limited to, recombinant (whether produced from cDNA, genomic DNA, synthetic DNA or other form of nucleic acid), in vitro, in vivo, by microinjection of nucleic acid molecules, synthetic, transgenic, and gene activated methods.
  • IFN beta polypeptide As used herein
  • interferon or “IFN” shall include those polypeptides and proteins that have at least one biological activity of an interferon, including but not limited to IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , or IFN ⁇ or interferon-like cytokines such as limitin (such as those described in U.S.
  • IFN include, but are not limited to, IFN ⁇ - Ib (Actimmune ® ), IFN ⁇ -la (Avonex ® , and Rebif ® ), IFN ⁇ -lb (Betaseron ® ), consensus IFN, IFN alfacon-1 (Infergen ® ), IFN ⁇ -2 (Intron A ® ), IFN ⁇ -2a (Roferon-A ® ), Peginterferon alfa-2a (PEGASYS ® ), Peginterferon alfa-2b (PEG-Intron ), IFN analog, IFN mutants, altered glycosylated human IFN, and PEG conjugated IFN analogs.
  • IFN ⁇ - Ib Actimmune ®
  • IFN ⁇ -la Avonex ® , and Rebif ®
  • IFN ⁇ -lb Betaseron ®
  • consensus IFN IFN alfacon-1 (Infergen ® ), IFN ⁇ -2 (Intron A ®
  • IFN- ⁇ variants exhibiting modified immunogenicity comprising at least one modification at a position selected from the group consisting of 1, 2, 3, 4, 5, 6, 8, 9, 12, 15, 16, 22, 28, 30, 32, 36, 42, 43, 46, 47, 48, 49, 51, 92, 93, 96, 100, 101, 104, 111, 113, 116, 117, 120, 121, 124, 130, 148, and 155 were described.
  • the modifications to residues 5, 8, 15, 47, 111, 116, and 120 may be substitution mutations that are selected from the group consisting of alanine, arginine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, histidine, and lysine.
  • Modifications to residues 22, 28, 30, 32, 36, 92, 130, 148, and 155 may be selected from the group including alanine, arginine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, histidine, serine, threonine and lysine. These variants were described to have increased solubility leading to reduced immunogenicity.
  • One modification selected from the group consisting of: L5A, L5D, L5E, L5K, L5N, L5Q, L5R, L5S, L5T, F8A, F8D, F8E, F8K, F8N, F8Q, F8R, F8S, S12E, S12K, S12Q, S12R, W22E, L28Q, Y30H, L32A, E43K, E43R, L47K, Y92Q, E104R, E104K, E104H, E104Q, E104A, Fl I lN, R113D, R113E, R113Q, R113A, L116D, L116E, L116N, L116Q, L116R, M117R, L120D, L120R, L130R, V148A, and Y155S may be present in a polypeptide of the invention.
  • An IFN beta polypeptide of the invention may comprise one or more natural amino acid substitution.
  • the C17S substitution has been described previously.
  • U.S. Patent No. 2005/0054053 describes IFN beta variants with a L5Q substitution in addition to the C17S substitution (SEQ ID NO:19 of U.S. Patent No. 2005/0054053), with a L5Q and F8E substitution in addition to the C17S substitution (SEQ ID NO:20 of U.S. Patent No. 2005/0054053), with a L5Q, F8E, and Fl 1 IN substitution in addition to the C17S substitution (SEQ ID NO:21 of U.S. Patent No.
  • variant IFN beta proteins with reduced immunogenicity exhibit reduced binding at least one human class II MHC allele.
  • At least one amino acid modification may be made in at least one of the following positions: agretope 1 : residues 3-11; agretope 2: residues 5-13; agretope 3: residues 8-16; agretope 4: residues 9-17; agretope 5: residues 15-23; agretope 6: residues 22-30; agretope 7: residues 30-38; agretope 8: residues 36-44; agretope 9: residues 47- 55; agretope 10: residues 57-65; agretope 11 : residues 60-68; agretope 12: residues 63-71; agretope 13: residues 70-78; agretope 14: residues 79-87; agretope 15: residues 95-103; agretope 16: residues 122-130; agretope 17: residues 125-133
  • the invention provides recombinant nucleic acids encoding the variant proteins, expression vectors containing the variant nucleic acids, host cells comprising the variant nucleic acids and/or expression vectors, and methods for producing the variant proteins.
  • the invention provides treating an interferon responsive disorder by administering to a patient a variant protein, usually with a pharmaceutical carrier, in a therapeutically effective amount.
  • the invention provides methods for modulating immunogenicity (particularly reducing immunogenicity) of interferons (particularly IFN beta) by altering MHC Class II epitopes.
  • IFN beta mutants discussed in U.S. Patent No. 7,144,574 which is incorporated by reference in its entirety, include polypeptides comprising one or more of the following modifications: DI lOF; C17S; Q49N; Q51T; Fl I lN; R113T; K19R; K33R; K45R; Q51S; R113S; Q48F; Q48V; Q48W; Q48Y; DI lOV; DI lOW; DI lOY that are glycosylated or not glycosylated.
  • WO 2005/016371 which is incorporated by reference herein, describe a IFN beta- Ib polypeptide that is selectively oxidized.
  • mutants include, but are not limited to, an analog comprising native interferon-beta in which asparagine at position 25 is deamidated (see WO 2006/053134) and those with different glycosylation as described in WO 2006/049423, both of which are incorporated by reference herein. Codon optimization for E. coli expression of IFN beta is described in WO 2006/015165, which is incorporated by reference herein. [144] For sequences of IFN beta that lack a leader sequence, see SEQ ID NO: 1 and 3 herein. For a sequence of IFN beta with a leader sequence, see SEQ ID NO: 4 herein.
  • IFN beta polypeptides of the invention are substantially identical to SEQ ID NOs: 1, 3, 4, or any other sequence of a IFN beta polypeptide.
  • Nucleic acid molecules encoding IFN beta polypeptides including mutants and methods to express and purify IFN beta polypeptides are well known and include, but are not limited to, those disclosed in U.S. Patent No. 4,462,940; U.S. Patent No. 4,518,584; U.S. Patent No. 5,702,699; U.S. Patent No. 6,962,978; U.S. Patent No. 5,814,485; U.S. Patent No. 6,887,462; U.S. Patent No. 6,800,735; U.S. Patent No. 6,514,729; U.S. Publication Nos. US2002/0137895, US2004/0115169, and US2005/0054053, which are incorporated by reference in their entirety herein.
  • IFN beta polypeptide also includes the pharmaceutically acceptable salts and prodrugs, and prodrugs of the salts, polymorphs, hydrates, solvates, biologically-active fragments, biologically active variants and stereoisomers of the naturally-occurring IFN beta as well as agonist, mimetic, and antagonist variants of the naturally-occurring IFN beta and polypeptide fusions thereof.
  • Fusions comprising additional amino acids at the amino terminus, carboxyl terminus, or both, are encompassed by the term "IFN beta polypeptide.”
  • exemplary fusions include, but are not limited to, e.g., methionyl IFN beta in which a methionine is linked to the N-terminus of IFN beta resulting from the recombinant expression of the mature form of IFN beta lacking the leader or signal peptide or portion thereof (a methionine is linked to the N- terminus of IFN beta resulting from the recombinant expression), fusions for the purpose of purification (including, but not limited to, to poly-histidine or affinity epitopes), fusions with serum albumin binding peptides and fusions with serum proteins such as serum albumin.
  • U.S. Patent No. 5,750,373 which is incorporated by reference herein, describes a method for selecting novel proteins such as growth hormone and antibody fragment variants having altered binding properties for their respective receptor molecules.
  • the method comprises fusing a gene encoding a protein of interest to the carboxy terminal domain of the gene III coat protein of the filamentous phage Ml 3.
  • Chimeric molecules comprising IFN beta and one or more other molecules.
  • the chimeric molecule can contain specific regions or fragments of one or both of the IFN beta and the other molecule(s). Any such fragments can be prepared from the proteins by standard biochemical methods, or by expressing a polynucleotide encoding the fragment.
  • IFN beta, or a fragment thereof can be produced as a fusion protein comprising human serum albumin (HSA), Fc, or a portion thereof.
  • HSA human serum albumin
  • fusion constructs are suitable for enhancing expression of the IFN beta, or fragment thereof, in a eukaryotic host cell.
  • HSA portions include the N-terminal polypeptide (amino acids 1-369, 1-419, and intermediate lengths starting with amino acid 1), as disclosed in U.S. Pat. No. 5,766,883, and publication WO 97/24445, which are incorporated by reference herein.
  • Other chimeric polypeptides can include a HSA protein with IFN beta, or fragments thereof, attached to each of the C-terminal and N- terminal ends of the HSA.
  • HSA constructs are disclosed in U.S. Pat. No. 5,876,969, which is incorporated by reference herein.
  • Other fusions may be created by fusion of interferon beta with a) the Fc portion of an immunoglobulin; b) an analog of the Fc portion of an immunoglobulin; and c) fragments of the Fc portion of an immunoglobulin.
  • US Patent Publication No. 2005/0054053 describes circularly permuted interferon beta and fusions of IFN beta with an immunoglobulin or a region of an immunoglobulin.
  • IFN beta pol ypeptide includes polypeptides conjugated to a polymer such as PEG and may be comprised of one or more additional derivitizations of cysteine, lysine, or other residues.
  • the IFN beta polypeptide may comprise a linker or polymer, wherein the amino acid to which the linker or polymer is conjugated may be a non- natural amino acid according to the present invention, or may be conjugated to a naturally encoded amino acid utilizing techniques known in the art such as coupling to lysine or cysteine.
  • Polymer modification of native IFN ⁇ or a C17S variant thereof has been reported
  • Bioconjugate Chem 2006 17:618-630 evaluated the stability, solubility, aggregation, immunogenicity, and pharmacokinetic properties of over 20 site-selective mono-PEGylated or multi-PEGylated interferon-beta-lb polypeptides.
  • the conjugates were formed at lysine residues or at the N- terminus.
  • PEGylated IFN molecules include those disclosed in U.S. Patent
  • IFN ⁇ is mentioned as one example of a polypeptide belonging to the growth hormone superfamily.
  • WO 00/23114 discloses glycosylated and pegylated IFN ⁇ .
  • WO 00/23472 discloses IFN ⁇ fusion proteins.
  • U.S. Pat. No. 4,904,584 discloses PEGylated lysine depleted polypeptides, wherein at least one lysine residue has been deleted or replaced with any other amino acid residue.
  • WO 99/67291 discloses a process for conjugating a protein with PEG, wherein at least one amino acid residue on the protein is deleted and the protein is contacted with PEG under conditions sufficient to achieve conjugation to the protein.
  • WO 99/03887 discloses PEGylated variants of polypeptides belonging to the growth hormone superfamily, wherein a cysteine residue has been substituted with a non-essential amino acid residue located in a specified region of the polypeptide.
  • WO 00/26354 discloses a method of producing a glycosylated polypeptide variant with reduced allergenicity, which as compared to a corresponding parent polypeptide comprises at least one additional glycosylation site.
  • IFN ⁇ is disclosed as one example among many polypeptides that can be modified according to the technology described in U.S. Pat. No. 5,218,092, which is incorporated by reference herein.
  • G-CSF granulocyte colony stimulating factor
  • IFN beta polypeptide also includes glycosylated IFN beta, such as but not limited to, polypeptides glycosylated at any amino acid position, N-linked or O-linked glycosylated forms of the polypeptide. Variants containing single nucleotide changes are also considered as biologically active variants of IFN beta polypeptide. In addition, splice variants are also included.
  • IFN beta polypeptide also includes IFN beta polypeptide heterodimers, homodimers, heteromultimers, or homomultimers of any one or more IFN beta polypeptides or any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or other biologically active molecule of any type, linked by chemical means or expressed as a fusion protein, as well as polypeptide analogues containing, for example, specific deletions or other modifications yet maintain biological activity.
  • amino acid positions corresponding to positions in SEQ ID NO: 1, 3, 4, or any other IFN beta sequence can be readily identified in any other IFN beta molecule such as IFN beta fusions, variants, fragments, etc.
  • sequence alignment programs such as BLAST can be used to align and identify a particular position in a protein that corresponds with a position in SEQ ID NO: 1, 3, 4, or other IFN beta sequence.
  • substitutions, deletions or additions of amino acids described herein in reference to SEQ ID NO: 1, 3, 4, or other IFN beta sequence are intended to also refer to substitutions, deletions or additions in corresponding positions in IFN beta fusions, variants, fragments, etc. described herein or known in the art and are expressly encompassed by the present invention.
  • IFN beta polypeptide or "IFN beta” encompasses IFN beta polypeptides comprising one or more amino acid substitutions, additions or deletions.
  • IFN beta polypeptides of the present invention may be comprised of modifications with one or more natural amino acids in conjunction with one or more non-natural amino acid modification. Exemplary substitutions in a wide variety of amino acid positions in naturally-occurring IFN beta polypeptides have been described, including but not limited to substitutions that modulate pharmaceutical stability, that modulate one or more of the biological activities of the IFN beta polypeptide, such as but not limited to, increase agonist activity, increase solubility of the polypeptide, decrease protease susceptibility, convert the polypeptide into an antagonist, etc. and are encompassed by the term " IFN beta polypeptide.”
  • the IFN beta antagonist comprises a non-naturally encoded amino acid linked to a water soluble polymer that is present in a receptor binding region of the IFN beta molecule.
  • the IFN beta polypeptides further comprise an addition, substitution or deletion that modulates biological activity of the IFN beta polypeptide. In some embodiments, the IFN beta polypeptides further comprise an addition, substitution or deletion that modulates anti-viral activity of the IFN beta polypeptide. In some embodiments, the IFN beta polypeptides further comprise an addition, substitution or deletion that enhances anti-viral activity of the IFN beta polypeptide. For example, the additions, substitutions or deletions may modulate one or more properties or activities of IFN beta.
  • the additions, substitutions or deletions may modulate affinity for the IFN receptor, modulate circulating half- life, modulate therapeutic half-life, modulate stability of the polypeptide, modulate cleavage by proteases, modulate dose, modulate release or bio-availability, facilitate purification, or improve or alter a particular route of administration.
  • IFN beta polypeptides may comprise protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to, biotin) that improve detection (including but not limited to, GFP), purification or other traits of the polypeptide.
  • protease cleavage sequences including but not limited to, FLAG or poly-His
  • affinity based sequences including but not limited to, FLAG, poly-His, GST, etc.
  • linked molecules including but not limited to, biotin
  • IFN beta polypeptide also encompasses homodimers, heterodimers, homomultimers, and heteromultimers that are linked, including but not limited to those linked directly via non-naturally encoded amino acid side chains, either to the same or different non- naturally encoded amino acid side chains, to naturally-encoded amino acid side chains, or indirectly via a linker.
  • linkers including but are not limited to, small organic compounds, water soluble polymers of a variety of lengths such as poly(ethylene glycol) or polydextran, or polypeptides of various lengths.
  • non-naturally encoded amino acid refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine.
  • Other terms that may be used synonymously with the term “non-naturally encoded amino acid” are “non-natural amino acid,” “unnatural amino acid,” “non-naturally-occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof.
  • the term “non-naturally encoded amino acid” also includes, but is not limited to, amino acids that occur by modification (e.g.
  • a naturally encoded amino acid including but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine
  • non-naturally- occurring amino acids include, but are not limited to, JV-acetylglucosaminyl-L-serine, JV- acetylglucosaminyl-L-threonine, and O-phosphotyrosine.
  • amino terminus modification group refers to any molecule that can be attached to the amino terminus of a polypeptide.
  • a “carboxy terminus modification group” refers to any molecule that can be attached to the carboxy terminus of a polypeptide.
  • Terminus modification groups include, but are not limited to, various water soluble polymers, peptides or proteins such as serum albumin, or other moieties that increase serum half-life of peptides.
  • Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely.
  • Hydrolytically unstable or degradable linkages mean that the linkages are degradable in water or in aqueous solutions, including for example, blood.
  • Enzymatically unstable or degradable linkages mean that the linkage can be degraded by one or more enzymes.
  • PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule.
  • ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent generally hydrolyze under physiological conditions to release the agent.
  • Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end
  • biologically active agent when used herein means any substance which can affect any physical or biochemical properties of a biological system, pathway, molecule, or interaction relating to an organism, including but not limited to, viruses, bacteria, bacteriophage, transposon, prion, insects, fungi, plants, animals, and humans.
  • biologically active molecules include, but are not limited to, any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals.
  • biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, vaccines, immunogens, hard drugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxoids, toxins, prokaryotic and eukaryotic cells, viruses, polysaccharides, nucleic acids and portions thereof obtained or derived from viruses, bacteria, insects, animals or any other cell or cell type, liposomes, microparticles and micelles.
  • the IFN beta polypeptides may be added in a micellular formulation; see U.S. Pat. No. 5,833,948, which is incorporated by reference herein in its entirety.
  • Classes of biologically active agents that are suitable for use with the invention include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, microbially derived toxins, and the like.
  • a "bifunctional polymer” refers to a polymer comprising two discrete functional groups that are capable of reacting specifically with other moieties (including but not limited to, amino acid side groups) to form covalent or non-covalent linkages.
  • a bifunctional linker having one functional group reactive with a group on a particular biologically active component, and another group reactive with a group on a second biological component may be used to form a conjugate that includes the first biologically active component, the bifunctional linker and the second biologically active component.
  • Many procedures and linker molecules for attachment of various compounds to peptides are known. See, e.g., European Patent Application No. 188,256; U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789 which are incorporated by reference herein.
  • a "multi-functional polymer” refers to a polymer comprising two or more discrete functional groups that are capable of reacting specifically with other moieties (including but not limited to, amino acid side groups) to form covalent or non- covalent linkages.
  • a bi-functional polymer or multi-functional polymer may be any desired length or molecular weight, and may be selected to provide a particular desired spacing or conformation between one or more molecules linked to the IFN beta and its receptor or IFN beta.
  • non-interfering substituents include but is not limited to "non-interfering substituents".
  • Non-interfering substituents are those groups that yield stable compounds. Suitable non-interfering substituents or radicals include, but are not limited to, halo, C 1 -C] 0 alkyl, C 2 -Ci 0 alkenyl, C 2 -Ci 0 alkynyl, Ci-Ci 0 alkoxy, Ci-Ci 2 aralkyl, Ci-C) 2 alkaryl, C 3 -Ci 2 cycloalkyl, C 3 -Ci 2 cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl, C 2 -Ci 2 alkoxyalkyl, C 2 -Ci 2 alkoxyaryl, C 7 -Ci 2 aryloxyalkyl, C 7 -C 12 oxy
  • R as used herein is H, alkyl or substituted alkyl, aryl or substituted aryl, aralkyl, or alkaryl.
  • halogen includes fluorine, chlorine, iodine, and bromine.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C 1 -C 10 means one to ten carbons).
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n- hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • alkyl groups examples include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkyl unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl.”
  • Alkyl groups which are limited to hydrocarbon groups are termed "homoalkyl".
  • alkylene by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by the structures -CH 2 CH 2 - and -CH 2 CH 2 CH 2 CH 2 -, and further includes those groups described below as “heteroalkylene.”
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being a particular embodiment of the methods and compositions described herein.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 -CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
  • heteroalkylene groups the same or different heteroatoms can also occupy either or both of the chain termini (including but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula - C(O) 2 R'- represents both -C(O) 2 R'- and -R 5 C(O) 2 -.
  • cycloalkyl and heterocycloalkyl represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively.
  • a cycloalkyl or heterocycloalkyl include saturated, partially unsaturated and fully unsaturated ring linkages.
  • a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3- cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, l-(l,2,5,6-tetrahydropyridyl), 1 -piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. Additionally, the term encompasses bicyclic and tricyclic ring structures.
  • heterocycloalkylene by itself or as part of another substituent means a divalent radical derived from heterocycloalkyl
  • cycloalkylene by itself or as part of another substituent means a divalent radical derived from cycloalkyl
  • water soluble polymer refers to any polymer that is soluble in aqueous solvents. Linkage of water soluble polymers to IFN beta polypeptides can result in changes including, but not limited to, increased or modulated serum half-life, or increased or modulated therapeutic half-life relative to the unmodified form, modulated immunogenicity, modulated physical association characteristics such as aggregation and multimer formation, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization.
  • the water soluble polymer may or may not have its own biological activity, and may be utilized as a linker for attaching IFN beta to other substances, including but not limited to one or more IFN beta polypeptides, or one or more biologically active molecules.
  • Suitable polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, mono Cl-ClO alkoxy or aryloxy derivatives thereof (described in U.S. Patent No.
  • polyalkylene glycol or “poly(alkene glycol)” refers to polyethylene glycol (po ly(ethylene glycol)), polypropylene glycol, polybutylene glycol, and derivatives thereof.
  • polyalkylene glycol encompasses both linear and branched polymers and average molecular weights of between 0.1 kDa and 100 kDa.
  • Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog “Polyethylene Glycol and Derivatives for Biomedical Applications” (2001).
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (including but not limited to, from 1 to 3 rings) which are fused together or linked covalently.
  • heteroaryl refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
  • Non-limiting examples of aryl and heteroaryl groups include phenyl, 1 - naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, 1 -
  • arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (including but not limited to, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (including but not limited to, a methylene group) has been replaced by, for example, an oxygen atom (including but not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like).
  • alkyl and heteroalkyl radicals are meant to include both substituted and unsubstituted forms of the indicated radical. Exemplary substituents for each type of radical are provided below.
  • R', R", R'" and R" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
  • R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.
  • -NR'R is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (including but not limited to, -CF 3 and -CH 2 CF 3 ) and acyl (including but not limited to, -C(O)CH 3 , -C(O)CF 3 , - C(O)CH 2 OCH 3 , and the like).
  • the term "modulated serum half-life” means the positive or negative change in circulating half-life of a modified IFN beta relative to its non-modified form. Serum half-life is measured by taking blood samples at various time points after administration of IFN beta, and determining the concentration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum half-life. Increased serum half-life desirably has at least about two-fold, but a smaller increase may be useful, for example where it enables a satisfactory dosing regimen or avoids a toxic effect. In some embodiments, the increase is at least about three-fold, at least about five-fold, or at least about ten-fold.
  • modulated therapeutic half-life means the positive or negative change in the half-life of the therapeutically effective amount of IFN beta, relative to its non-modified form.
  • Therapeutic half-life is measured by measuring pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic half-life desirably enables a particular beneficial dosing regimen, a particular beneficial total dose, or avoids an undesired effect.
  • the increased therapeutic half-life results from increased potency, increased or decreased binding of the modified molecule to its target, increased or decreased breakdown of the molecule by enzymes such as proteases, or an increase or decrease in another parameter or mechanism of action of the non-modified molecule or an increase or decrease in receptor-mediated clearance of the molecule.
  • isolated when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is free of at least some of the cellular components with which it is associated in the natural state, or that the nucleic acid or protein has been concentrated to a level greater than the concentration of its in vivo or in vitro production. It can be in a homogeneous state. Isolated substances can be in either a dry or semi-dry state, or in solution, including but not limited to, an aqueous solution. It can be a component of a pharmaceutical composition that comprises additional pharmaceutically acceptable carriers and/or excipients. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • a protein which is the predominant species present in a preparation is substantially purified.
  • an isolated gene is separated from open reading frames which flank the gene and encode a protein other than the gene of interest.
  • the term "purified” denotes that a nucleic acid or protein gives rise to substantially one band in an electrophoretic gel. Particularly, it may mean that the nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or greater pure.
  • nucleic acid refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also refers to oligonucleotide analogs including PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like).
  • PNA peptidonucleic acid
  • analogs of DNA used in antisense technology phosphorothioates, phosphoroamidates, and the like.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., MoI. Cell. Probes 8:91-98 (1994)).
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • the terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid.
  • the terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • amino acid refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Reference to an amino acid includes, for example, naturally occurring proteogenic L-amino acids; D-amino acids, chemically modified amino acids such as amino acid variants and derivatives; naturally occurring non-proteogenic amino acids such as ⁇ -alanine, ornithine, etc.; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids.
  • non-naturally occurring amino acids include, but are not limited to, ⁇ -methyl amino acids (e.g., ⁇ -methyl alanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine and ⁇ -methyl- histidine), amino acids having an extra methylene in the side chain (“homo" amino acids), and amino acids in which a carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (e.g., cysteic acid).
  • ⁇ -methyl amino acids e.g., ⁇ -methyl alanine
  • D-amino acids e.g., D-amino acids
  • histidine-like amino acids e.g., 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine and ⁇ -
  • D- amino acid-containing peptides, etc. exhibit increased stability in vitro or in vivo compared to L-amino acid-containing counterparts.
  • the construction of peptides, etc., incorporating D- amino acids can be particularly useful when greater intracellular stability is desired or required.
  • D-peptides, etc. are resistant to endogenous peptidases and proteases, thereby providing improved bioavailability of the molecule, and prolonged lifetimes in vivo when such properties are desirable.
  • D-peptides, etc. cannot be processed efficiently for major histocompatibility complex class II-restricted presentation to T helper cells, and are therefore, less likely to induce humoral immune responses in the whole organism.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms (or other algorithms available to persons of ordinary skill in the art) or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence.
  • the identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 75-100 amino acids or nucleotides in length, or, where not specified, across the entire sequence of a polynucleotide or polypeptide.
  • a polynucleotide encoding a polypeptide of the present invention, including homologs from species other than human, may be obtained by a process comprising the steps of screening a library under stringent hybridization conditions with a labeled probe having a polynucleotide sequence of the invention or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to the skilled artisan.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are known to those of ordinary skill in the art.
  • Optimal alignment of sequences for comparison can be conducted, including but not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J MoI. Biol.
  • BLAST and BLAST 2.0 algorithms are described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J MoI. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information available at the World Wide Web at ncbi.nlm.nih.gov.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • the BLAST algorithm is typically performed with the "low complexity" filter turned off.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.
  • the phrase "selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (including but not limited to, total cellular or library DNA or RNA).
  • stringent hybridization conditions refers to hybridization of sequences of DNA, RNA, PNA, or other nucleic acid mimics, or combinations thereof under conditions of low ionic strength and high temperature as is known in the art.
  • a probe will hybridize to its target subsequence in a complex mixture of nucleic acid (including but not limited to, total cellular or library DNA or RNA) but does not hybridize to other sequences in the complex mixture.
  • Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • T m thermal melting point
  • Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O 0 C for short probes (including but not limited to, 10 to 50 nucleotides) and at least about 60° C for long probes (including but not limited to, greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least two times background, optionally 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 42 0 C, or 5X SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2X SSC, and 0.1% SDS at 65 0 C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes.
  • the term "eukaryote” refers to organisms belonging to the phylogenetic domain Eucarya such as animals (including but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to, monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.
  • non-eukaryote refers to non-eukaryotic organisms.
  • a non-eukaryotic organism can belong to the Eubacteria (including but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or the Archaea (including but not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-I, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic domain.
  • Eubacteria including but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomon
  • subject refers to an animal, in some embodiments a mammal, and in other embodiments a human, who is the object of treatment, observation or experiment.
  • An animal may be a companion animal (e.g., dogs, cats, and the like), farm animal (e.g., cows, sheep, pigs, horses, and the like) or a laboratory animal (e.g., rats, mice, guinea pigs, and the like).
  • compositions containing the modified non-natural amino acid polypeptide described herein can be administered for prophylactic, enhancing, and/or therapeutic treatments.
  • the terms “enhance” or “enhancing” means to increase or prolong either in potency or duration a desired effect.
  • the term “enhancing” refers to the ability to increase or prolong, either in potency or duration, the effect of other therapeutic agents on a system.
  • An “enhancing-effective amount,” as used herein, refers to an amount adequate to enhance the effect of another therapeutic agent in a desired system. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.
  • modified refers to any changes made to a given polypeptide, such as changes to the length of the polypeptide, the amino acid sequence, chemical structure, co-translational modification, or post-translational modification of a polypeptide.
  • the form "(modified)” term means that the polypeptides being discussed are optionally modified, that is, the polypeptides under discussion can be modified or unmodified.
  • post-translationally modified refers to any modification of a natural or non-natural amino acid that occurs to such an amino acid after it has been incorporated into a polypeptide chain.
  • compositions containing the IFN beta polypeptide are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined to be a "prophylactically effective amount.” In this use, the precise amounts also depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).
  • the term "protected” refers to the presence of a “protecting group” or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions.
  • the protecting group will vary depending on the type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or a hydrazide, the protecting group can be selected from the group of tert-butyloxycarbonyl (t-Boc) and 9- fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group can be orthopyridyldisulfide.
  • the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group
  • the protecting group can be benzyl or an alkyl group such as methyl, ethyl, or tert-butyl.
  • Other protecting groups known in the art may also be used in or with the methods and compositions described herein, including photolabile groups such as Nvoc and MeNvoc.
  • Other protecting groups known in the art may also be used in or with the methods and compositions described herein.
  • blocking/protecting groups may be selected from:
  • compositions containing the modified non-natural amino acid polypeptide are administered to a patient already suffering from a disease, condition or disorder, in an amount sufficient to cure or at least partially arrest the symptoms of the disease, disorder or condition.
  • an amount is defined to be a "therapeutically effective amount,” and will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. It is considered well within the skill of the art for one to determine such therapeutically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).
  • treating is used to refer to either prophylactic and/or therapeutic treatments.
  • Non-naturally encoded amino acid polypeptides presented herein may include isotopically-labelled compounds with one or more atoms replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that can be incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36 Cl, respectively.
  • Certain isotopically-labelled compounds described herein, for example those into which radioactive isotopes such as 3 H and C are incorporated, may be useful in drug and/or substrate tissue distribution assays.
  • non-naturally encoded amino acid polypeptides are metabolized upon administration to an organism in need to produce a metabolite that is then used to produce a desired effect, including a desired therapeutic effect.
  • active metabolites of non-naturally encoded amino acid polypeptides are active metabolites of non-naturally encoded amino acid polypeptides.
  • non-naturally encoded amino acid polypeptides may exist as tautomers.
  • the non-naturally encoded amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • the solvated forms are also considered to be disclosed herein.
  • Those of ordinary skill in the art will recognize that some of the compounds herein can exist in several tautomeric forms. All such tautomeric forms are considered as part of the compositions described herein.
  • HPLC protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art are employed.
  • IFN beta molecules comprising at least one unnatural amino acid are provided in the invention.
  • the IFN beta polypeptide with at least one unnatural amino acid includes at least one post-translational modification.
  • the at least one post-translational modification comprises attachment of a molecule including but not limited to, a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cycl
  • the first reactive group is an alkynyl moiety (including but not limited to, in the unnatural amino acid />-propargyloxyphenylalanine, where the propargyl group is also sometimes referred to as an acetylene moiety) and the second reactive group is an azido moiety, and [3+2] cycloaddition chemistry methodologies are utilized.
  • the first reactive group is the azido moiety (including but not limited to, in the unnatural amino acid p- azido-L-phenylalanine) and the second reactive group is the alkynyl moiety.
  • At least one unnatural amino acid comprising at least one post-translational modification
  • the at least one post-translational modification comprises a saccharide moiety.
  • the post-translational modification is made in vivo in a eukaryotic cell or in a non-eukaryotic cell.
  • a linker, polymer, water soluble polymer, or other molecule may attach the molecule to the polypeptide. The molecule may be linked directly to the polypeptide.
  • the protein includes at least one post-translational modification that is made in vivo by one host cell, where the post-translational modification is not normally made by another host cell type.
  • the protein includes at least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-translational modification is not normally made by a non-eukaryotic cell.
  • post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid- linkage modification, and the like.
  • the IFN beta polypeptide comprises one or more non- naturally encoded amino acids for glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linkage modification of the polypeptide. In some embodiments, the IFN beta polypeptide comprises one or more non- naturally encoded amino acids for glycosylation of the polypeptide. In some embodiments, the IFN beta polypeptide comprises one or more naturally encoded amino acids for glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linkage modification of the polypeptide. In some embodiments, the IFN beta polypeptide comprises one or more naturally encoded amino acids for glycosylation of the polypeptide.
  • the IFN beta polypeptide comprises one or more non- naturally encoded amino acid additions and/or substitutions that enhance glycosylation of the polypeptide. In some embodiments, the IFN beta polypeptide comprises one or more deletions that enhance glycosylation of the polypeptide. In some embodiments, the IFN beta polypeptide comprises one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation at a different amino acid in the polypeptide. In some embodiments, the IFN beta polypeptide comprises one or more deletions that enhance glycosylation at a different amino acid in the polypeptide.
  • the IFN beta polypeptide comprises one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation at a non-naturally encoded amino acid in the polypeptide. In some embodiments, the IFN beta polypeptide comprises one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation at a naturally encoded amino acid in the polypeptide. In some embodiments, the IFN beta polypeptide comprises one or more naturally encoded amino acid additions and/or substitutions that enhance glycosylation at a different amino acid in the polypeptide.
  • the IFN beta polypeptide comprises one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation at a naturally encoded amino acid in the polypeptide. In some embodiments, the IFN beta polypeptide comprises one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation at a non-naturally encoded amino acid in the polypeptide.
  • the post-translational modification comprises attachment of an oligosaccharide to an asparagine by a GlcNAc-asparagine linkage (including but not limited to, where the oligosaccharide comprises (GlcNAc-Manh-Man-GlcNAc-GlcNAc, and the like).
  • the post-translational modification comprises attachment of an oligosaccharide (including but not limited to, GaI-GaINAc, GaI-GIcNAc, etc.) to a serine or threonine by a GalNAc-serine, a GalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage.
  • a protein or polypeptide of the invention can comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or the like.
  • secretion signal sequences include, but are not limited to, a prokaryotic secretion signal sequence, a eukaryotic secretion signal sequence, a eukaryotic secretion signal sequence 5 '-optimized for bacterial expression, a novel secretion signal sequence, pectate lyase secretion signal sequence, Omp A secretion signal sequence, and a phage secretion signal sequence.
  • secretion signal sequences include, but are not limited to, STII (prokaryotic), Fd GUI and M 13 (phage), Bgl2 (yeast), and the signal sequence bla derived from a transposon.
  • the protein or polypeptide of interest can contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more unnatural amino acids.
  • the unnatural amino acids can be the same or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids.
  • At least one, but fewer than all, of a particular amino acid present in a naturally occurring version of the protein is substituted with an unnatural amino acid.
  • the present invention provides methods and compositions based on IFN beta comprising at least one non-naturally encoded amino acid. Introduction of at least one non- naturally encoded amino acid into IFN beta can allow for the application of conjugation chemistries that involve specific chemical reactions, including, but not limited to, with one or more non-naturally encoded amino acids while not reacting with the commonly occurring 20 amino acids.
  • IFN beta comprising the non-naturally encoded amino acid is linked to a water soluble polymer, such as polyethylene glycol (PEG), via the side chain of the non-naturally encoded amino acid.
  • PEG polyethylene glycol
  • This invention provides a highly efficient method for the selective modification of proteins with PEG derivatives, which involves the selective incorporation of non-genetically encoded amino acids, including but not limited to, those amino acids containing functional groups or substituents not found in the 20 naturally incorporated amino acids, including but not limited to a ketone, an azide or acetylene moiety, into proteins in response to a selector codon and the subsequent modification of those amino acids with a suitably reactive PEG derivative.
  • the amino acid side chains can then be modified by utilizing chemistry methodologies known to those of ordinary skill in the art to be suitable for the particular functional groups or substituents present in the non-naturally encoded amino acid.
  • Known chemistry methodologies of a wide variety are suitable for use in the present invention to incorporate a water soluble polymer into the protein.
  • Such methodologies include but are not limited to a Huisgen [3+2] cycloaddition reaction ⁇ see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p. 1069- 1109; and, Huisgen, R. in 1.3-Dipolar Cvcloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, including but not limited to, acetylene or azide derivatives, respectively.
  • the Huisgen [3+2] cycloaddition method involves a cycloaddition rather than a nucleophilic substitution reaction, proteins can be modified with extremely high selectivity.
  • the reaction can be carried out at room temperature in aqueous conditions with excellent regioselectivity (1,4 > 1,5) by the addition of catalytic amounts of Cu(I) salts to the reaction mixture. See, e.g., Tornoe, et al., (2002) J. Org. Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599; and WO 03/101972.
  • a molecule that can be added to a protein of the invention through a [3+2] cycloaddition includes virtually any molecule with a suitable functional group or substituent including but not limited to an azido or acetylene derivative. These molecules can be added to an unnatural amino acid with an acetylene group, including but not limited to, p-propargyloxyphenylalanine, or azido group, including but not limited to p-azido-phenylalanine, respectively.
  • the invention also provides water soluble and hydrolytically stable derivatives of
  • PEG derivatives and related hydrophilic polymers having one or more acetylene or azide moieties.
  • the PEG polymer derivatives that contain acetylene moieties are highly selective for coupling with azide moieties that have been introduced selectively into proteins in response to a selector codon.
  • PEG polymer derivatives that contain azide moieties are highly selective for coupling with acetylene moieties that have been introduced selectively into proteins in response to a selector codon.
  • the azide moieties comprise, but are not limited to, alk yl azides, aryl azides and derivatives of these azides.
  • the derivatives of the alkyl and aryl azides can include other substituents so long as the acetylene-specific reactivity is maintained.
  • the acetylene moieties comprise alkyl and aryl acetylenes and derivatives of each.
  • the derivatives of the alkyl and aryl acetylenes can include other substituents so long as the azide-specific reactivity is maintained.
  • the present invention provides conjugates of substances having a wide variety of functional groups, substituents or moieties, with other substances including but not limited to a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin; an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing mofetil
  • the present invention also includes conjugates of substances having azide or acetylene moieties with PEG polymer derivatives having the corresponding acetylene or azide moieties.
  • a PEG polymer containing an azide moiety can be coupled to a biologically active molecule at a position in the protein that contains a non- genetically encoded amino acid bearing an acetylene functionality.
  • the linkage by which the PEG and the biologically active molecule are coupled includes but is not limited to the Huisgen [3+2] cycloaddition product.
  • the invention also includes biomaterials comprising a surface having one or more reactive azide or acetylene sites and one or more of the azide- or acetylene-containing polymers of the invention coupled to the surface via the Huisgen [3+2] cycloaddition linkage.
  • Biomaterials and other substances can also be coupled to the azide- or acetylene-activated polymer derivatives through a linkage other than the azide or acetylene linkage, such as through a linkage comprising a carboxylic acid, amine, alcohol or thiol moiety, to leave the azide or acetylene moiety available for subsequent reactions.
  • the invention includes a method of synthesizing the azide- and acetylene- containing polymers of the invention.
  • the azide can be bonded directly to a carbon atom of the polymer.
  • the azide- containing PEG derivative can be prepared by attaching a linking agent that has the azide moiety at one terminus to a conventional activated polymer so that the resulting polymer has the azide moiety at its terminus.
  • the acetylene-containing PEG derivative the acetylene can be bonded directly to a carbon atom of the polymer.
  • the acetylene-containing PEG derivative can be prepared by attaching a linking agent that has the acetylene moiety at one terminus to a conventional activated polymer so that the resulting polymer has the acetylene moiety at its terminus.
  • a water soluble polymer having at least one active hydroxyl moiety undergoes a reaction to produce a substituted polymer having a more reactive moiety, such as a mesylate, tresylate, tosylate or halogen leaving group, thereon.
  • PEG derivatives containing sulfonyl acid halides, halogen atoms and other leaving groups are known to those of ordinary skill in the art.
  • the resulting substituted polymer then undergoes a reaction to substitute for the more reactive moiety an azide moiety at the terminus of the polymer.
  • a water soluble polymer having at least one active nucleophilic or electrophilic moiety undergoes a reaction with a linking agent that has an azide at one terminus so that a covalent bond is formed between the PEG polymer and the linking agent and the azide moiety is positioned at the terminus of the polymer.
  • Nucleophilic and electrophilic moieties including amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters and the like, are known to those of ordinary skill in the art.
  • a water soluble polymer having at least one active hydroxyl moiety undergoes a reaction to displace a halogen or other activated leaving group from a precursor that contains an acetylene moiety.
  • a water soluble polymer having at least one active nucleophilic or electrophilic moiety undergoes a reaction with a linking agent that has an acetylene at one terminus so that a covalent bond is formed between the PEG polymer and the linking agent and the acetylene moiety is positioned at the terminus of the polymer.
  • the invention also provides a method for the selective modification of proteins to add other substances to the modified protein, including but not limited to water soluble polymers such as PEG and PEG derivatives containing an azide or acetylene moiety.
  • the azide- and acetylene-containing PEG derivatives can be used to modify the properties of surfaces and molecules where biocompatibility, stability, solubility and lack of immunogenicity are important, while at the same time providing a more selective means of attaching the PEG derivatives to proteins than was previously known in the art.
  • New members of the IFN family can be identified through computer-aided secondary and tertiary structure analyses of the predicted protein sequences, and by selection techniques designed to identify molecules that bind to a particular target.
  • IFN family is provided for illustrative purposes and by way of example only and not as a limit on the scope of the methods, compositions, strategies and techniques described herein.
  • reference to IFN beta in this application is intended to use the generic term as an example of any member of the IFN family.
  • modifications and chemistries described herein with reference to IFN beta polypeptides or protein can be equally applied to any member of the IFN family, including those specifically listed herein.
  • nucleic acids encoding a IFN beta polypeptide of interest will be isolated, cloned and often altered using recombinant methods. Such embodiments are used, including but not limited to, for protein expression or during the generation of variants, derivatives, expression cassettes, or other sequences derived from a IFN beta polypeptide.
  • sequences encoding the polypeptides of the invention are operably linked to a heterologous promoter.
  • a nucleotide sequence encoding a IFN beta polypeptide comprising a non- naturally encoded amino acid may be synthesized on the basis of the amino acid sequence of the parent polypeptide, including but not limited to, having the amino acid sequence shown in SEQ ID NO: 1, 3, 4 and then changing the nucleotide sequence so as to effect introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residue(s).
  • the nucleotide sequence may be conveniently modified by site-directed mutagenesis in accordance with conventional methods.
  • the nucleotide sequence may be prepared by chemical synthesis, including but not limited to, 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. See, e.g., Barany, et ah, Proc. Natl. Acad. ScL 88: 189-193 (1991); U.S. Patent 6,521,427 which are incorporated by reference herein.
  • This invention utilizes routine techniques in the field of recombinant genetics.
  • mutagenesis Various types are used in the invention for a variety of purposes, including but not limited to, to produce novel synthetases or tRNAs, to mutate tRNA molecules, to mutate polynucleotides encoding synthetases, to produce libraries of tRNAs, to produce libraries of synthetases, to produce selector codons, to insert selector codons that encode unnatural amino acids in a protein or polypeptide of interest.
  • mutagenesis include but are not limited to site-directed, random point mutagenesis, homologous recombination, DNA shuffling or other recursive mutagenesis methods, chimeric construction, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like, PCT-mediated mutagenesis, or any combination thereof.
  • Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like.
  • Mutagenesis including but not limited to, involving chimeric constructs, are also included in the present invention.
  • mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, including but not limited to, sequence, sequence comparisons, physical properties, secondary, tertiary, or quaternary structure, crystal structure or the like.
  • Kunkel The efficiency of oligonucleotide directed mutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D.M.J, eds., Springer Verlag, Berlin) (1987); Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol.
  • Oligonucleotides e.g., for use in mutagenesis of the present invention, e.g., mutating libraries of synthetases, or altering tRNAs, are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts. 22(20): 1859-1862, (1981) e.g., using an automated synthesizer, as described in Needham-VanDevanter et al., Nucleic Acids Res., 12:6159-6168 (1984).
  • the invention also relates to eukaryotic host cells, non-eukaryotic host cells, and organisms for the in vivo incorporation of an unnatural amino acid via orthogonal tRNA/RS pairs.
  • Host cells are genetically engineered (including but not limited to, transformed, transduced or transfected) with the polynucleotides of the invention or constructs which include a polynucleotide of the invention, including but not limited to, a vector of the invention, which can be, for example, a cloning vector or an expression vector.
  • the coding regions for the orthogonal tRNA, the orthogonal tRNA synthetase, and the protein to be derivatized are operably linked to gene expression control elements that are functional in the desired host cell.
  • the vector can be, for example, in the form of a plasmid, a cosmid, a phage, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide.
  • the vectors are introduced into cells and/or microorganisms by standard methods including electroporation (Fromm et al., Proc. Natl. Acad. Sci.
  • nucleic acid in vitro includes the use of liposomes, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • in vivo gene transfer techniques include, but are not limited to, transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection [Dzau et al., Trends in Biotechnology 11 :205-210 (1993)].
  • the nucleic acid source may be desirable to provide with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, screening steps, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic organisms.
  • Other useful references including but not limited to for cell isolation and culture (e.g., for subsequent nucleic acid isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley- Liss, New York and the references cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc.
  • the bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook).
  • kits are commercially available for the purification of plasmids from bacteria, (see, e.g., EasyPrepTM, FlexiPrepTM, both from Pharmacia Biotech; StrataCleanTM from Stratagene; and, QIAprepTM from Qiagen).
  • the isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect cells or incorporated into related vectors to infect organisms.
  • Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (including but not limited to, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. See, Gillam & Smith, Gene 8:81 (1979); Roberts, et al, Nature, 328:731 (1987); Schneider, E., et al, Protein Expr. Purif.
  • nucleic acid and virtually any labeled nucleic acid, whether standard or non-standard
  • Selector codons of the invention expand the genetic codon framework of protein biosynthetic machinery.
  • a selector codon includes, but is not limited to, a unique three base codon, a nonsense codon, such as a stop codon, including but not limited to, an amber codon (UAG), an ochre codon, or an opal codon (UGA), an unnatural codon, a four or more base codon, a rare codon, or the like.
  • selector codons that can be introduced into a desired gene or polynucleotide, including but not limited to, one or more, two or more, three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotide encoding at least a portion of the IFN beta polypeptide.
  • the methods involve the use of a selector codon that is a stop codon for the incorporation of one or more unnatural amino acids in vivo.
  • a selector codon that is a stop codon for the incorporation of one or more unnatural amino acids in vivo.
  • an O- tRNA is produced that recognizes the stop codon, including but not limited to, UAG, and is aminoacylated by an O-RS with a desired unnatural amino acid.
  • This O-tRNA is not recognized by the naturally occurring host's aminoacyl-tRNA synthetases.
  • Conventional site-directed mutagenesis can be used to introduce the stop codon, including but not limited to, TAG, at the site of interest in a polypeptide of interest. See, e.g., Sayers, J.R., et al.
  • the suppression efficiency for the UAG codon depends upon the competition between the O-tRNA, including but not limited to, the amber suppressor tRNA, and a eukaryotic release factor (including but not limited to, eRF) (which binds to a stop codon and initiates release of the growing peptide from the ribosome)
  • the suppression efficiency can be modulated by, including but not limited to, increasing the expression level of O-tRNA, and/or the suppressor tRNA.
  • Unnatural amino acids can also be encoded with rare codons.
  • the rare arginine codon, AGG has proven to be efficient for insertion of Ala by a synthetic tRNA acylated with alanine.
  • the synthetic tRNA competes with the naturally occurring tRNAArg, which exists as a minor species in Escherichia coli. Some organisms do not use all triplet codons.
  • An unassigned codon AGA in Micrococcus luteus has been utilized for insertion of amino acids in an in vitro transcription/translation extract.
  • Selector codons also comprise extended codons, including but not limited to, four or more base codons, such as, four, five, six or more base codons. Examples of four base codons include, but are not limited to, AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like.
  • a feature of the invention includes using extended codons based on frameshift suppression.
  • Four or more base codons can insert, including but not limited to, one or multiple unnatural amino acids into the same protein.
  • mutated O-tRNAs including but not limited to, a special frameshift suppressor tRNAs, with anticodon loops, for example, with at least 8-10 nt anticodon loops
  • the four or more base codon is read as single amino acid.
  • the anticodon loops can decode, including but not limited to, at least a four-base codon, at least a five-base codon, or at least a six-base codon or more. Since there are 256 possible four-base codons, multiple unnatural amino acids can be encoded in the same cell using a four or more base codon.
  • Moore et al. examined the ability of tRNALeu derivatives with NCUA anticodons to suppress UAGN codons (N can be U, A, G, or C), and found that the quadruplet UAGA can be decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13 to 26% with little decoding in the 0 or -1 frame. See, Moore et al., (2000) J. MoI. Biol., 298:195.
  • extended codons based on rare codons or nonsense codons can be used in the present invention, which can reduce missense readthrough and frameshift suppression at other unwanted sites.
  • a selector codon can also include one of the natural three base codons, where the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes a system that is lacking a tRNA that recognizes the natural three base codon, and/or a system where the three base codon is a rare codon.
  • Selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125.
  • Third base pairs include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by a polymerase, and the efficient continued primer extension after synthesis of the nascent unnatural base pair.
  • Descriptions of unnatural base pairs which can be adapted for methods and compositions include, e.g., Hirao, et al., (2002) An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20:177-182. See, also, Wu, Y., et al., (2002) J. Am. Chem. Soc. 124: 14626-14630. Other relevant publications are listed below.
  • the unnatural nucleoside is membrane permeable and is phosphorylated to form the corresponding triphosphate.
  • the increased genetic information is stable and not destroyed by cellular enzymes.
  • Previous efforts by Benner and others took advantage of hydrogen bonding patterns that are different from those in canonical Watson-Crick pairs, the most noteworthy example of which is the iso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc. 111:8322; and Piccirilli et al., (1990) Nature. 343:33; Kool, (2000) Curr. Opin. Chem. Biol., 4:602.
  • a PICS:PICS self-pair is found to be more stable than natural base pairs, and can be efficiently incorporated into DNA by Klenow fragment of Escherichia coli DNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem. Soc. 121 :11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc, 122:3274.
  • a 3MN:3MN self-pair can be synthesized by KF with efficiency and selectivity sufficient for biological function. See, e.g., Ogawa et al., (2000) J. Am. Chem. Soc, 122:8803.
  • both bases act as a chain terminator for further replication.
  • a mutant DNA polymerase has been recently evolved that can be used to replicate the PICS self pair.
  • a 7AI self pair can be replicated. See, e.g., Tae et al., (2001) J. Am. Chem. Soc, 123:7439.
  • a novel metallobase pair, DipicPy has also been developed, which forms a stable pair upon binding Cu(II). See, Meggers et al., (2000) J. Am. Chem. Soc. 122:10714. Because extended codons and unnatural codons are intrinsically orthogonal to natural codons, the methods of the invention can take advantage of this property to generate orthogonal tRNAs for them.
  • a translational bypassing system can also be used to incorporate an unnatural amino acid in a desired polypeptide.
  • a large sequence is incorporated into a gene but is not translated into protein.
  • the sequence contains a structure that serves as a cue to induce the ribosome to hop over the sequence and resume translation downstream of the insertion.
  • the protein or polypeptide of interest (or portion thereof) in the methods and/or compositions of the invention is encoded by a nucleic acid.
  • the nucleic acid comprises at least one selector codon, at least two selector codons, at least three selector codons, at least four selector codons, at least five selector codons, at least six selector codons, at least seven selector codons, at least eight selector codons, at least nine selector codons, ten or more selector codons.
  • Genes coding for proteins or polypeptides of interest can be mutagenized using methods known to one of ordinary skill in the art and described herein to include, for example, one or more selector codon for the incorporation of an unnatural amino acid.
  • a nucleic acid for a protein of interest is mutagenized to include one or more selector codon, providing for the incorporation of one or more unnatural amino acids.
  • the invention includes any such variant, including but not limited to, mutant, versions of any protein, for example, including at least one unnatural amino acid.
  • the invention also includes corresponding nucleic acids, i.e., any nucleic acid with one or more selector codon that encodes one or more unnatural amino acid.
  • Nucleic acid molecules encoding a protein of interest such as a IFN beta polypeptide may be readily mutated to introduce a cysteine at any desired position of the polypeptide.
  • Cysteine is widely used to introduce reactive molecules, water soluble polymers, proteins, or a wide variety of other molecules, onto a protein of interest.
  • Methods suitable for the incorporation of cysteine into a desired position of a polypeptide are known to those of ordinary skill in the art, such as those described in U.S. Patent No. 6,608,183, which is incorporated by reference herein, and standard mutagenesis techniques.
  • non-naturally encoded amino acids are suitable for use in the present invention. Any number of non-naturally encoded amino acids can be introduced into a IFN beta polypeptide. In general, the introduced non-naturally encoded amino acids are substantially chemically inert toward the 20 common, genetically-encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).
  • alanine arginine
  • asparagine aspartic acid
  • cysteine glutamine
  • glutamic acid glutamic acid
  • histidine isoleucine
  • leucine leucine
  • lysine methionine
  • phenylalanine proline
  • serine
  • the non-naturally encoded amino acids include side chain functional groups that react efficiently and selectively with functional groups not found in the 20 common amino acids (including but not limited to, azido, ketone, aldehyde and aminooxy groups) to form stable conjugates.
  • a IFN beta polypeptide that includes a non-naturally encoded amino acid containing an azido functional group can be reacted with a polymer (including but not limited to, poly(ethylene glycol) or, alternatively, a second polypeptide containing an alkyne moiety to form a stable conjugate resulting for the selective reaction of the azide and the alkyne functional groups to form a Huisgen [3+2] cycloaddition product.
  • a polymer including but not limited to, poly(ethylene glycol) or, alternatively, a second polypeptide containing an alkyne moiety to form a stable conjugate resulting for the selective reaction of the azide and the alkyne functional groups to form a Huisgen [3+2
  • a non-naturally encoded amino acid is typically any structure having the above- listed formula wherein the R group is any substituent other than one used in the twenty natural amino acids, and may be suitable for use in the present invention. Because the non-naturally encoded amino acids of the invention typically differ from the natural amino acids only in the structure of the side chain, the non-naturally encoded amino acids form amide bonds with other amino acids, including but not limited to, natural or non-naturally encoded, in the same manner in which they are formed in naturally occurring polypeptides. However, the non-naturally encoded amino acids have side chain groups that distinguish them from the natural amino acids.
  • R optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group, or the like or any combination thereof.
  • Non-naturally occurring amino acids of interest include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids,
  • non-naturally encoded amino acids that may be suitable for use in the present invention and that are useful for reactions with water soluble polymers include, but are not limited to, those with carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactive groups.
  • non-naturally encoded amino acids comprise a saccharide moiety.
  • amino acids examples include iV-acetyl-L-glucosaminyl-L-serine, N- acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L- glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.
  • amino acids also include examples where the naturally-occuring N- or O- linkage between the amino acid and the saccharide is replaced by a covalent linkage not commonly found in nature - including but not limited to, an alkene, an oxime, a thioether, an amide and the like.
  • amino acids also include saccharides that are not commonly found in naturally-occuring proteins such as 2-deoxy-glucose, 2-deoxygalactose and the like.
  • unnatural amino acids that may be suitable for use in the present invention also optionally comprise modified backbone structures, including but not limited to, as illustrated by the structures of Formula II and III: II
  • Z typically comprises OH, NH 2 , SH, NH-R', or S-R';
  • X and Y which can be the same or different, typically comprise S or O, and
  • R and R' which are optionally the same or different, are typically selected from the same list of constituents for the R group described above for the unnatural amino acids having Formula I as well as hydrogen.
  • unnatural amino acids of the invention optionally comprise substitutions in the amino or carboxyl group as illustrated by Formulas II and III.
  • Unnatural amino acids of this type include, but are not limited to, ⁇ -hydroxy acids, ⁇ -thioacids, ⁇ -aminothiocarboxylates, including but not limited to, with side chains corresponding to the common twenty natural amino acids or unnatural side chains.
  • substitutions at the ⁇ -carbon optionally include, but are not limited to, L, D, or ⁇ - ⁇ - disubstituted amino acids such as D-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like.
  • cyclic amino acids such as proline analogues as well as 3, 4 ,6, 7, 8, and 9 membered ring proline analogues, ⁇ and ⁇ amino acids such as substituted ⁇ -alanine and ⁇ -amino butyric acid.
  • Tyrosine analogs include, but are not limited to, para-substituted tyrosines, ortho-substituted tyrosines, and meta substituted tyrosines, where the substituted tyrosine comprises, including but not limited to, a keto group (including but not limited to, an acetyl group), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C 6 - C 20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, a polyether group, a nitro group, an alkynyl group or the like.
  • a keto group including but not limited to, an acetyl group
  • benzoyl group an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group
  • Glutamine analogs that may be suitable for use in the present invention include, but are not limited to, ⁇ -hydroxy derivatives, ⁇ -substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives.
  • Example phenylalanine analogs that may be suitable for use in the present invention include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenyalanines, and meta- substituted phenylalanines, where the substituent comprises, including but not limited to, a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group), a benzoyl, an alkynyl group, or the like.
  • unnatural amino acids include, but are not limited to, a/7-acetyl-L- phenylalanine, an O-methyl-L- tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4- propyl-L-tyrosine, a tri-O-acetyl-GlcNAc ⁇ -serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a /?-azido-L-phenylalanine, a j9-acyl-L-phenylalanine, a /7-benzoyl-L- phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a />-
  • compositions of an IFN beta polypeptide that include an unnatural amino acid are provided.
  • an unnatural amino acid such as />-(propargyloxy)-phenyalanine
  • compositions comprising />(propargyloxy)-phenyalanine and, including but not limited to, proteins and/or cells, are also provided.
  • a composition that includes the p- (propargyloxy)-phenyalanine unnatural amino acid further includes an orthogonal tRNA.
  • the unnatural amino acid can be bonded (including but not limited to, covalently) to the orthogonal tRNA, including but not limited to, covalently bonded to the orthogonal tRNA though an amino- acyl bond, covalently bonded to a 3 'OH or a 2 'OH of a terminal ribose sugar of the orthogonal tRNA, etc.
  • the chemical moieties via unnatural amino acids that can be incorporated into proteins offer a variety of advantages and manipulations of the protein.
  • the unique reactivity of a keto functional group allows selective modification of proteins with any of a number of hydrazine- or hydroxylamine-containing reagents in vitro and in vivo.
  • a heavy atom unnatural amino acid for example, can be useful for phasing X-ray structure data.
  • the site- specific introduction of heavy atoms using unnatural amino acids also provides selectivity and flexibility in choosing positions for heavy atoms.
  • Photoreactive unnatural amino acids include but not limited to, amino acids with benzophenone and arylazides (including but not limited to, phenylazide) side chains), for example, allow for efficient in vivo and in vitro photocrosslinking of protein.
  • photoreactive unnatural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine.
  • the protein with the photoreactive unnatural amino acids can then be crosslinked at will by excitation of the photoreactive group-providing temporal control.
  • the methyl group of an unnatural amino can be substituted with an isotopically labeled, including but not limited to, methyl group, as a probe of local structure and dynamics, including but not limited to, with the use of nuclear magnetic resonance and vibrational spectroscopy.
  • Alkynyl or azido functional groups allow the selective modification of proteins with molecules through a [3+2] cycloaddition reaction.
  • a non-natural amino acid incorporated into a polypeptide at the amino terminus can be composed of an R group that is any substituent other than one used in the twenty natural amino acids and a 2 nd reactive group different from the NH 2 group normally present in ⁇ -amino acids (see Formula I).
  • a similar non-natural amino acid can be incorporated at the carboxyl terminus with a 2 nd reactive group different from the COOH group normally present in ⁇ -amino acids (see Formula I).
  • the unnatural amino acids of the invention may be selected or designed to provide additional characteristics unavailable in the twenty natural amino acids.
  • unnatural amino acid may be optionally designed or selected to modify the biological properties of a protein, e.g., into which they are incorporated.
  • the following properties may be optionally modified by inclusion of an unnatural amino acid into a protein: toxicity, biodistribution, solubility, stability, e.g., thermal, hydrolytic, oxidative, resistance to enzymatic degradation, and the like, facility of purification and processing, structural properties, spectroscopic properties, chemical and/or photochemical properties, catalytic activity, redox potential, half-life, ability to react with other molecules, e.g., covalently or noncovalently, and the like.
  • STRUCTURE AND SYNTHESIS OF NON-NATURAL AMINO ACIDS CARBONYL. CARBONYL-LIKE, MASKED CARBONYL. PROTECTED CARBONYL GROUPS, AND HYDROXYLAMINE GROUPS
  • the present invention provides IFN beta linked to a water soluble polymer, e.g., a PEG, by an oxime bond.
  • a water soluble polymer e.g., a PEG
  • non-naturally encoded amino acids are suitable for formation of oxime bonds. These include, but are not limited to, non-naturally encoded amino acids containing a carbonyl, dicarbonyl, or hydroxylamine group. Such amino acids are described in U.S. Patent Publication Nos. 2006/0194256, 2006/0217532, and 2006/0217289 and WO 2006/069246 entitled "Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides," which are incorporated herein by reference in their entirety. Non-naturally encoded amino acids are also described in U.S. Patent No. 7,083,970 and U.S. Patent No.
  • Some embodiments of the invention utilize IFN beta polypeptides that are substituted at one or more positions with a para-acetylphenylalanine amino acid.
  • the synthesis of p-acetyl-(+/-)-phenylalanine and m-acetyl-(+/-)-phenylalanine are described in Zhang, Z., et al., Biochemistry 42: 6735-6746 (2003), incorporated by reference.
  • Other carbonyl- or dicarbonyl-containing amino acids can be similarly prepared by one of ordinary skill in the art.
  • non-limiting examplary syntheses of non-natural amino acid that are included herein are presented in FIGS. 4, 24-34 and 36-39 of U.S. Patent No. 7,083,970, which is incorporated by reference herein in its entirety.
  • Amino acids with an electrophilic reactive group allow for a variety of reactions to link molecules via nucleophilic addition reactions among others.
  • electrophilic reactive groups include a carbonyl group (including a keto group and a dicarbonyl group), a carbonyl- like group (which has reactivity similar to a carbonyl group (including a keto group and a dicarbonyl group) and is structurally similar to a carbonyl group), a masked carbonyl group (which can be readily converted into a carbonyl group (including a keto group and a dicarbonyl group)), or a protected carbonyl group (which has reactivity similar to a carbonyl group (including a keto group and a dicarbonyl group) upon deprotection).
  • Such amino acids include amino acids having the structure of Formula (IV): wherein:
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - where k is 1, 2, or 3, -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR' -(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R' )-(alkylene or substitute
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; each R" is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R" optionally form a heterocycloalkyl;
  • R 1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide
  • each of R 3 and R 4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R 3 groups optionally form a cycloalkyl or a heterocycloalkyl
  • the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group
  • the -J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycl
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • R 1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; with a proviso that when A is phenylene, B is present; and that when A is -(CH 2 ) 4 -, B is not - NHC(O)(CH 2 CH 2 )-; and that when A and B are absent, R is not methyl.
  • amino acids having the structure of Formula (VI) are included:
  • B is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, - N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN
  • R 1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(0) k R' where k is 1, 2, or 3, -C(O)N(R') 2 , -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl.
  • any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.
  • B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR' -(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alky
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R' where k is 1, 2, or 3, -C(O)N(R') 2 , -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl; and n is O to 8; with a proviso that when A is -(CH 2 ) 4 -, B is not -NHC(O)(CH 2 CH 2 )-.
  • the following amino acids are included:
  • non-natural amino acids are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof.
  • these non-natural amino acids and any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - where k is 1, 2, or 3, -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR' -(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.
  • B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - where k is 1, 2, or 3, -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR' -(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R' where k is 1, 2, or 3, -C(0)N(R') 2 , -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl.
  • amino acids are included: compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof.
  • these non-natural amino acids and any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.
  • B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR' -(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide
  • each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R' where k is 1, 2, or 3, -C(0)N(R') 2 , -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8.
  • non-natural amino acids and any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.
  • non-natural amino acids described herein may include groups such as dicarbonyl, dicarbonyl like, masked dicarbonyl and protected dicarbonyl groups.
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1,
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • R 1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.
  • B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - where k is 1, 2, or 3, -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR' -(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted al
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • R 1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R' where k is 1, 2, or 3, -C(O)N(R') 2 , -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl.
  • non-natural amino acids and any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.
  • B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - where k is 1, 2, or 3, -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR' -(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(0) k R' where k is 1, 2, or 3, -C(0)N(R') 2 , -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8.
  • non-natural amino acids and any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • X 1 is C, S, or S(O); and L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • R 1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • R 1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • X] is C, S, or S(O); and L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • R 1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl
  • R 1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
  • amino acids having the structure of Formula (XVII) are included:
  • A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
  • M is -C(R 3 )-, W
  • R 3 and R 4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R 3 and R 4 or two R 3 groups or two R 4 groups optionally form a cycloalkyl or a heterocycloalkyl;
  • R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
  • T 3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.
  • amino acids having the structure of Formula (XVIII) are included:
  • M is -C(R 3 )-
  • R 3 and R 4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R 3 and R 4 or two R 3 groups or two R 4 groups optionally form a cycloalkyl or a heterocycloalkyl;
  • R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
  • T 3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
  • Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;
  • R 2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(0) k R' where k is 1, 2, or 3, -C(0)N(R') 2 , -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl.
  • amino acids having the structure of Formula (XIX) are included:
  • R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; and T 3 is O, or S.
  • amino acids having the structure of Formula (XX) are included:
  • R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
  • a polypeptide comprising a non-natural amino acid is chemically modified to generate a reactive carbonyl or dicarbonyl functional group.
  • an aldehyde functionality useful for conjugation reactions can be generated from a functionality having adjacent amino and hydroxyl groups.
  • an N-terminal serine or threonine which may be normally present or may be exposed via chemical or enzymatic digestion
  • an aldehyde functionality under mild oxidative cleavage conditions using periodate. See, e.g., Gaertner, et. al., Bioconjug. Chem. 3: 262-268 (1992); Geoghegan, K.
  • a non-natural amino acid bearing adjacent hydroxy 1 and amino groups can be incorporated into the polypeptide as a "masked" aldehyde functionality.
  • 5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.
  • Reaction conditions for generating the aldehyde typically involve addition of molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide.
  • the pH of the oxidation reaction is typically about 7.0.
  • a typical reaction involves the addition of about 1.5 molar excess of sodium meta periodate to a buffered solution of the polypeptide, followed by incubation for about 10 minutes in the dark. See, e.g. U.S. Patent No.
  • the carbonyl or dicarbonyl functionality can be reacted selectively with a hydroxylamine-containing reagent under mild conditions in aqueous solution to form the corresponding oxime linkage that is stable under physiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tarn, J. P., J. Am. Chem. Soc. 117:3893-3899 (1995). Moreover, the unique reactivity of the carbonyl or dicarbonyl group allows for selective modification in the presence of the other amino acid side chains. See, e.g., Cornish, V.
  • Amino acids with a carbonyl reactive group allow for a variety of reactions to link molecules (including but not limited to, PEG or other water soluble molecules) via nucleophilic addition or aldol condensation reactions among others.
  • n is 0-10; R] is an alkyl, aryl, substituted alkyl, or substituted aryl; R 2 is H, alkyl, aryl, substituted alkyl, and substituted aryl; and R 3 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R 4 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.
  • n is 1, R 1 is phenyl and R 2 is a simple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety is positioned in the para position relative to the alkyl side chain.
  • n is 1, R 1 is phenyl and R 2 is a simple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety is positioned in the meta position relative to the alkyl side chain.
  • a polypeptide comprising a non-naturally encoded amino acid is chemically modified to generate a reactive carbonyl functional group.
  • an aldehyde functionality useful for conjugation reactions can be generated from a functionality having adjacent amino and hydroxyl g roups.
  • an iV-terminal serine or threonine which may be normally present or may be exposed via chemical or enzymatic digestion
  • an aldehyde functionality under mild oxidative cleavage conditions using periodate. See, e.g., Gaertner, et al, Bioconjug. Chem.
  • Reaction conditions for generating the aldehyde typically involve addition of molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide.
  • the pH of the oxidation reaction is typically about 7.0.
  • a typical reaction involves the addition of about 1.5 molar excess of sodium meta periodate to a buffered solution of the polypeptide, followed by incubation for about 10 minutes in the dark. See, e.g. U.S. Patent No. 6,423,685, which is incorporated by reference herein.
  • the carbonyl functionality can be reacted selectively with a hydrazine-, hydrazide-, hydroxylamine-, or semicarbazide-containing reagent under mild conditions in aqueous solution to form the corresponding hydrazone, oxime, or semicarbazone linkages, respectively, that are stable under physiological conditions.
  • a hydrazine-, hydrazide-, hydroxylamine-, or semicarbazide-containing reagent under mild conditions in aqueous solution to form the corresponding hydrazone, oxime, or semicarbazone linkages, respectively, that are stable under physiological conditions.
  • a hydrazine-, hydrazide-, hydroxylamine-, or semicarbazide-containing reagent under mild conditions in aqueous solution to form the corresponding hydrazone, oxime, or semicarbazone linkages, respectively, that are stable under physiological conditions.
  • Non-naturally encoded amino acids containing a nucleophilic group such as a hydrazine, hydrazide or semicarbazide, allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water soluble polymers).
  • a nucleophilic group such as a hydrazine, hydrazide or semicarbazide
  • electrophilic groups such as PEG or other water soluble polymers.
  • Exemplary hydrazine, hydrazide or semicarbazide -containing amino acids can be represented as follows:
  • n is 4, R 1 is not present, and X is N. In some embodiments, n is 2, R 1 is not present, and X is not present. In some embodiments, n is 1 , R 1 is phenyl, X is O, and the oxygen atom is positioned para to the alphatic group on the aryl ring.
  • Hydrazide-, hydrazine-, and semicarbazide-containing amino acids are available from commercial sources. For instance, L-glutamate- ⁇ -hydrazide is available from Sigma Chemical (St. Louis, MO). Other amino acids not available commercially can be prepared by one of ordinary skill in the art See, e.g., U.S. Pat. No. 6,281,211, which is incorporated by reference herein.
  • Polypeptides containing non-naturally encoded amino acids that bear hydrazide, hydrazine or semicarbazide functionalities can be reacted efficiently and selectively with a variety of molecules that contain aldehydes or other functional groups with similar chemical reactivity. See, e.g., Shao, J. and Tarn, J., J. Am. Chem. Soc. 117:3893-3899 (1995).
  • hydrazide, hydrazine and semicarbazide functional groups make them significantly more reactive toward aldehydes, ketones and other electrophilic groups as compared to the nucleophilic groups present on the 20 common amino acids (including but not limited to, the hydroxy 1 group of serine or threonine or the amino groups of lysine and the N-terminus).
  • Non-naturally encoded amino acids containing an aminooxy (also called a hydroxylamine) group allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water soluble polymers).
  • an aminooxy (also called a hydroxylamine) group allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water soluble polymers).
  • the enhanced nucleophilicity of the aminooxy group permits it to react efficiently and selectively with a variety of molecules that contain aldehydes or other functional groups with similar chemical reactivity. See, e.g., Shao, J. and Tarn, J., J. Am. Chem. Soc. 117:3893-3899 (1995); H. Hang and C. Bertozzi, A cc. Chem. Res.
  • n is 1, R 1 is phenyl, X is O, m is 1, and Y is present.
  • n is 2, R 1 and X are not present, m is 0, and Y is not present.
  • Aminooxy-containing amino acids can be prepared from readily available amino acid precursors (homoserine, serine and threonine). See, e.g., M. Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certain aminooxy-containing amino acids, such as L-2- amino-4-(aminooxy)butyric acid), have been isolated from natural sources (Rosenthal, G., Life Sci. 60: 1635-1641 (1997). Other aminooxy-containing amino acids can be prepared by one of ordinary skill in the art.
  • azide and alkyne functional groups make them extremely useful for the selective modification of polypeptides and other biological molecules.
  • Organic azides, particularly alphatic azides, and alkynes are generally stable toward common reactive chemical conditions.
  • both the azide and the alkyne functional groups are inert toward the side chains (i.e., R groups) of the 20 common amino acids found in naturally- occuring polypeptides.
  • R groups side chains
  • the Huisgen cycloaddition reaction involves a selective cycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANIC SYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R. in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984) , p. 1-176 ) rather than a nucleophilic substitution, the incorporation of non-naturally encoded amino acids bearing azide and alkyne-containing side chains permits the resultant polypeptides to be modified selectively at the position of the non-naturally encoded amino acid.
  • Cycloaddition reaction involving azide or alkyne-containing IFN beta polypeptide can be carried out at room temperature under aqueous conditions by the addition of Cu(II) (including but not limited to, in the form of a catalytic amount of CuSO 4 ) in the presence of a reducing agent for reducing Cu(II) to Cu(I), in situ, in catalytic amount.
  • Cu(II) including but not limited to, in the form of a catalytic amount of CuSO 4
  • a reducing agent for reducing Cu(II) to Cu(I) in situ, in catalytic amount.
  • Exemplary reducing agents include, including but not limited to, ascorbate, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe , Co , and an applied electric potential.
  • the IFN beta polypeptide comprises a non-naturally encoded amino acid comprising an alkyne moiety and the water soluble polymer to be attached to the amino acid comprises an azide moiety.
  • the converse reaction i.e., with the azide moiety on the amino acid and the alkyne moiety present on the water soluble polymer can also be performed.
  • the azide functional group can also be reacted selectively with a water soluble polymer containing an aryl ester and appropriately functionalized with an aryl phosphine moiety to generate an amide linkage.
  • the aryl phosphine group reduces the azide in situ and the resulting amine then reacts efficiently with a proximal ester linkage to generate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi, Science 287, 2007-2010 (2000).
  • the azide-containing amino acid can be either an alkyl azide (including but not limited to, 2-amino- 6-azido-l-hexanoic acid) or an aryl azide (p-azido-phenylalanine).
  • Exemplary water soluble polymers containing an aryl ester and a phosphine moiety can be represented as follows:
  • R can be H, alkyl, aryl, substituted alkyl and substituted aryl groups.
  • R groups include but are not limited to -CH 2 , -C(CH 3 ) 3 , -OR', -NR'R", -SR', -halogen, -C(O)R', -CONR'R", - S(O) 2 R', -S(O) 2 NR 5 R", -CN and -NO 2 .
  • R', R", R'" and R" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
  • R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.
  • -NR'R is meant to include, but not be limited to, 1- pyrrolidinyl and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (including but not limited to, -CF 3 and - CH 2 CF 3 ) and acyl (including but not limited to, -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like).
  • the azide functional group can also be reacted selectively with a water soluble polymer containing a thioester and appropriately functionalized with an aryl phosphine moiety to generate an amide linkage.
  • the aryl phosphine group reduces the azide in situ and the resulting amine then reacts efficiently with the thioester linkage to generate the corresponding amide.
  • Exemplary water soluble polymers containing a thioester and a phosphine moiety can be represented as follows: wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and W is a water soluble polymer.
  • alkyne-containing amino acids can be represented as follows:
  • X is O, N, S or not present;
  • m is 0-10, R 2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R 3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.
  • n is 1, R 1 is phenyl, X is not present, m is 0 and the acetylene moiety is positioned in the para position relative to the alkyl side chain.
  • n is 1
  • R] is phenyl
  • X is O
  • m is 1
  • the propargyloxy group is positioned in the para position relative to the alkyl side chain (i.e., O-propargyl-tyrosine).
  • n is 1, R 1 and X are not present and m is 0 (i.e., proparylglycine).
  • alkyne-containing amino acids are commercially available.
  • propargylglycine is commercially available from Peptech (Burlington, MA).
  • alkyne-containing amino acids can be prepared according to standard methods.
  • p- propargyloxyphenylalanine can be synthesized, for example, as described in Deiters, A., et ah, J. Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalanine can be synthesized as described in Kayser, B., et ah, Tetrahedron 53(7): 2475-2484 (1997).
  • Other alkyne-containing amino acids can be prepared by one of ordinary skill in the art.
  • n is 1, R 1 is phenyl, X is not present, m is 0 and the azide moiety is positioned para to the alkyl side chain.
  • n is 1, R 1 is phenyl, X is O, m is 2 and the ⁇ -azidoethoxy moiety is positioned in the para position relative to the alkyl side chain.
  • Azide-containing amino acids are available from commercial sources. For instance, 4-azidophenylalanine can be obtained from Chem-Impex International, Inc. (Wood Dale, IL).
  • the azide group can be prepared relatively readily using standard methods known to those of ordinary skill in the art, including but not limited to, via displacement of a suitable leaving group (including but not limited to, halide, mesylate, tosylate) or via opening of a suitably protected lactone. See, e.g., Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York).
  • beta-substituted aminothiol functional groups make them extremely useful for the selective modification of polypeptides and other biological molecules that contain aldehyde groups via formation of the thiazolidine. See, e.g., J. Shao and J. Tarn, J. Am. Chem. Soc. 1995, 117 (14) 3893-3899.
  • beta-substituted aminothiol amino acids can be incorporated into IFN beta polypeptides and then reacted with water soluble polymers comprising an aldehyde functionality.
  • a water soluble polymer, drug conjugate or other payload can be coupled to a IFN beta polypeptide comprising a beta-substituted aminothiol amino acid via formation of the thiazolidine.
  • Provisional Patent No. 60/743,041 U.S. Provisional Patent No. 60/743,040; International Patent Application No. PCT/US06/47822; U.S. Provisional Patent No. 60/882,819; U.S. Provisional Patent No. 60/882,500; and U.S. Provisional Patent No. 60/870,594.
  • These applications also discuss reactive groups that may be present on PEG or other polymers, including but not limited to, hydroxylamine (aminooxy) groups for conjugation.
  • Unnatural amino acid uptake by a cell is one issue that is typically considered when designing and selecting unnatural amino acids, including but not limited to, for incorporation into a protein.
  • the high charge density of ⁇ -amino acids suggests that these compounds are unlikely to be cell permeable.
  • Natural amino acids are taken up into the eukaryotic cell via a collection of protein-based transport systems. A rapid screen can be done which assesses which unnatural amino acids, if any, are taken up by cells. See, e.g., the toxicity assays in, e.g., U.S. Patent Publication No. US 2004/0198637 entitled "Protein Arrays" which is incorporated by reference herein; and Liu, D.R.
  • biosynthetic pathways already exist in cells for the production of amino acids and other compounds. While a biosynthetic method for a particular unnatural amino acid may not exist in nature, including but not limited to, in a cell, the invention provides such methods.
  • biosynthetic pathways for unnatural amino acids are optionally generated in host cell by adding new enzymes or modifying existing host cell pathways. Additional new enzymes are optionally naturally occurring enzymes or artificially evolved enzymes.
  • the biosynthesis of /?-aminophenylalanine (as presented in an example in WO 2002/085923 entitled "In vivo incorporation of unnatural amino acids") relies on the addition of a combination of known enzymes from other organisms.
  • the genes for these enzymes can be introduced into a eukaryotic cell by transforming the cell with a plasmid comprising the genes.
  • the genes when expressed in the cell, provide an enzymatic pathway to synthesize the desired compound. Examples of the types of enzymes that are optionally added are provided in the examples below. Additional enzymes sequences are found, for example, in Genbank. Artificially evolved enzymes are also optionally added into a cell in the same manner. In this manner, the cellular machinery and resources of a cell are manipulated to produce unnatural amino acids.
  • DesignPathTM developed by Genencor (available on the World Wide Web at genencor.com) is optionally used for metabolic pathway engineering, including but not limited to, to engineer a pathway to create 0-methyl-L-tyrosine in a cell.
  • This technology reconstructs existing pathways in host organisms using a combination of new genes, including but not limited to, those identified through functional genomics, and molecular evolution and design.
  • Di versa Corporation available on the World Wide Web at diversa.com
  • the unnatural amino acid produced with an engineered biosynthetic pathway of the invention is produced in a concentration sufficient for efficient protein biosynthesis, including but not limited to, a natural cellular amount, but not to such a degree as to affect the concentration of the other amino acids or exhaust cellular resources.
  • concentrations produced in vivo in this manner are about 10 mM to about 0.05 mM.
  • an unnatural amino acid can be done for a variety of purposes, including but not limited to, tailoring changes in protein structure and/or function, changing size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, accessibility of protease target sites, targeting to a moiety (including but not limited to, for a protein array), adding a biologically active molecule, attaching a polymer, attaching a radionuclide, modulating serum half-life, modulating tissue penetration (e.g. tumors), modulating active transport, modulating tissue, cell or organ specificity or distribution, modulating immunogenicity, modulating protease resistance, etc. Proteins that include an unnatural amino acid can have enhanced or even entirely new catalytic or biophysical properties.
  • compositions including proteins that include at least one unnatural amino acid are useful for, including but not limited to, novel therapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to, antibodies), and including but not limited to, the study of protein structure and function. See, e.g., Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4:645-652.
  • a composition includes at least one protein with at least one, including but not limited to, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more unnatural amino acids.
  • the unnatural amino acids can be the same or different, including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnatural amino acids.
  • a composition includes a protein with at least one, but fewer than all, of a particular amino acid present in the protein is substituted with the unnatural amino acid.
  • the unnatural amino acids can be identical or different (including but not limited to, the protein can include two or more different types of unnatural amino acids, or can include two of the same unnatural amino acid).
  • the unnatural amino acids can be the same, different or a combination of a multiple unnatural amino acid of the same kind with at least one different unnatural amino acid.
  • Proteins or polypeptides of interest with at least one unnatural amino acid are a feature of the invention.
  • the invention also includes polypeptides or proteins with at least one unnatural amino acid produced using the compositions and methods of the invention.
  • An excipient (including but not limited to, a pharmaceutically acceptable excipient) can also be present with the protein.
  • proteins or polypeptides of interest will typically include eukaryotic post- translational modifications.
  • a protein includes at least one unnatural amino acid and at least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-translational modification is not made by a prokaryotic cell.
  • the post-translation modification includes, including but not limited to, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, glycosylation, and the like.
  • the post-translational modification includes attachment of an oligosaccharide (including but not limited to, (GlcNAc-Man) 2 -Man- GlcNAc-GlcNAc)) to an asparagine by a GlcNAc-asparagine linkage.
  • an oligosaccharide including but not limited to, (GlcNAc-Man) 2 -Man- GlcNAc-GlcNAc)
  • GlcNAc-asparagine linkage See Table 1 which lists some examples of N-linked oligosaccharides of eukaryotic proteins (additional residues can also be present, which are not shown).
  • the post-translational modification includes attachment of an oligosaccharide (including but not limited to, GaI-GaINAc, GaI-GIcNAc, etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threonine linkage, or a GlcNAc-serine or a GlcNAc-threonine linkage.
  • an oligosaccharide including but not limited to, GaI-GaINAc, GaI-GIcNAc, etc.
  • the post-translation modification includes proteolytic processing of precursors (including but not limited to, calcitonin precursor, calcitonin gene- related peptide precursor, preproparathyroid hormone, preproinsulin, proinsulin, prepro- opiomelanocortin, pro-opiomelanocortin and the like), assembly into a multisubunit protein or macromolecular assembly, translation to another site in the cell (including but not limited to, to organelles, such as the endoplasmic reticulum, the Golgi apparatus, the nucleus, lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., or through the secretory pathway).
  • the protein comprises a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, or the like.
  • the post- translational modification is through the unnatural amino acid.
  • the post- translational modification can be through a nucleophilic-electrophilic reaction.
  • Most reactions currently used for the selective modification of proteins involve covalent bond formation between nucleophilic and electrophilic reaction partners, including but not limited to the reaction of ⁇ -haloketones with histidine or cysteine side chains. Selectivity in these cases is determined by the number and accessibility of the nucleophilic residues in the protein.
  • Post- translational modifications including but not limited to, through an azido amino acid, can also made through the Staudinger ligation (including but not limited to, with triarylphosphine reagents). See, e.g., Kiick et al., (2002) Incorporation ofazides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24.
  • This invention provides another highly efficient method for the selective modification of proteins, which involves the genetic incorporation of unnatural amino acids, including but not limited to, containing an azide or alkynyl moiety into proteins in response to a selector codon.
  • These amino acid side chains can then be modified by, including but not limited to, a Huisgen [3+2] cycloaddition reaction ⁇ see, e.g., Padwa, A. in Comprehensive Organic Synthesis. Vol. 4. (1991) Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in 1.3-Dipolar Cycloaddition Chemistry. (1984) Ed. Padwa, A., Wiley, New York, p.
  • a molecule that can be added to a protein of the invention through a [3+2] cycloaddition includes virtually any molecule with an azide or alkynyl derivative.
  • Molecules include, but are not limited to, dyes, fluorophores, crosslinking agents, saccharide derivatives, polymers (including but not limited to, derivatives of polyethylene glycol), photocrosslinkers, cytotoxic compounds, affinity labels, derivatives of biotin, resins, beads, a second protein or polypeptide (or more), polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metal chelators, cofactors, fatty acids, carbohydrates, and the like.
  • These molecules can be added to an unnatural amino acid with an alkynyl group, including but not limited to, p- propargyloxyphenylalanine, or azido group, including but not limited to, p-azido-phenylalanine, respectively.
  • alkynyl group including but not limited to, p- propargyloxyphenylalanine, or azido group, including but not limited to, p-azido-phenylalanine, respectively.
  • the IFN beta polypeptides of the invention can be generated in vivo using modified tRNA and tRNA synthetases to add to or substitute amino acids that are not encoded in naturally-occurring systems.
  • the ORS preferentially aminoacylates the O-tRNA with at least one non-naturally occurring amino acid in the translation system and the O-tRNA recognizes at least one selector codon that is not recognized by other tRNAs in the system.
  • the translation system thus inserts the non-naturally-encoded amino acid into a protein produced in the system, in response to an encoded selector codon, thereby "substituting" an amino acid into a position in the encoded polypeptide.
  • orthogonal tRNAs and aminoacyl tRNA synthetases have been described in the art for inserting particular synthetic amino acids into polypeptides, and are generally suitable for use in the present invention.
  • keto-specif ⁇ c O- tRNA/aminoacyl-tRNA synthetases are described in Wang, L., et al, Proc. Natl. Acad. Sci. USA 100:56-61 (2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003).
  • Exemplary O-RS, or portions thereof are encoded by polynucleotide sequences and include amino acid sequences disclosed in U.S. Patent Nos.
  • O-RS sequences for/?-azido-L-Phe include, but are not limited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and amino acid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. Patent No. 7,083,970 which is incorporated by reference herein.
  • O-tRNA sequences suitable for use in the present invention include, but are not limited to, nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. Patent No. 7,083,970, which is incorporated by reference herein.
  • Other examples of 0-tRNA/aminoacyl-tRNA synthetase pairs specific to particular non- naturally encoded amino acids are described in U.S. Patent No. 7,045,337 which is incorporated by reference herein.
  • O-RS and O-tRNA that incorporate both keto- and azide-containing amino acids in S. cerevisiae are described in Chin, J. W., et al, Science 301:964-967 (2003). [343]
  • Glutaminyl see, e.g., Liu, D.
  • O-tRNA/aminoacyl-tRNA synthetases involves selection of a specific codon which encodes the non-naturally encoded amino acid. While any codon can be used, it is generally desirable to select a codon that is rarely or never used in the cell in which the O- tRNA/aminoacyl-tRNA synthetase is expressed.
  • exemplary codons include nonsense codon such as stop codons (amber, ochre, and opal), four or more base codons and other natural three-base codons that are rarely or unused.
  • Specific selector codon(s) can be introduced into appropriate positions in the IFN beta polynucleotide coding sequence using mutagenesis methods known in the art (including but not limited to, site-specific mutagenesis, cassette mutagenesis, restriction selection mutagenesis, etc.).
  • WO 04/094593 entitled “Expanding the Eukaryotic Genetic Code,” which is incorporated by reference herein in its entirety, describes orthogonal RS and tRNA pairs for the incorporation of non-naturally encoded amino acids in eukaryotic host cells.
  • Methods for producing at least one recombinant orthogonal aminoacyl-tRNA synthetase comprise: (a) generating a library of (optionally mutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS) from a first organism, including but not limited to, a prokaryotic organism, such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, Escherichia colt, A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T.
  • a prokaryotic organism such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, Escherichia colt, A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T.
  • thermophilus or the like, or a eukaryotic organism; (b) selecting (and/or screening) the library of RSs (optionally mutant RSs) for members that aminoacylate an orthogonal tRNA (O-tRNA) in the presence of a non-naturally encoded amino acid and a natural amino acid, thereby providing a pool of active (optionally mutant) RSs; and/or, (c) selecting (optionally through negative selection) the pool for active RSs (including but not limited to, mutant RSs) that preferentially aminoacylate the O-tRNA in the absence of the non-naturally encoded amino acid, thereby providing the at least one recombinant O-RS; wherein the at least one recombinant O-RS preferentially aminoacylates the O-tRNA with the non-naturally encoded amino acid.
  • O-tRNA orthogonal tRNA
  • the RS is an inactive RS.
  • the inactive RS can be generated by mutating an active RS.
  • the inactive RS can be generated by mutating at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, or at least about 10 or more amino acids to different amino acids, including but not limited to, alanine.
  • mutant RSs can be generated using various techniques known in the art, including but not limited to rational design based on protein three dimensional RS structure, or mutagenesis of RS nucleotides in a random or rational design technique.
  • the mutant RSs can be generated by site-specific mutations, random mutations, diversity generating recombination mutations, chimeric constructs, rational design and by other methods described herein or known in the art.
  • selecting (and/or screening) the library of RSs (optionally mutant RSs) for members that are active, including but not limited to, that aminoacylate an orthogonal tRNA (O-tRNA) in the presence of a non-naturally encoded amino acid and a natural amino acid includes: introducing a positive selection or screening marker, including but not limited to, an antibiotic resistance gene, or the like, and the library of (optionally mutant) RSs into a plurality of cells, wherein the positive selection and/or screening marker comprises at least one selector codon, including but not limited to, an amber, ochre, or opal codon; growing the plurality of cells in the presence of a selection agent; identifying cells that survive (or show a specific response) in the presence of the selection and/or screening agent by suppressing the at least one selector codon in the positive selection or screening marker, thereby providing a subset of positively selected cells that contains the pool of active (optionally mutant) RSs.
  • the selection and/or screening marker comprises at least one
  • the positive selection marker is a chloramphenicol acetyltransferase (CAT) gene and the selector codon is an amber stop codon in the CAT gene.
  • the positive selection marker is a ⁇ -lactamase gene and the selector codon is an amber stop codon in the ⁇ -lactamase gene.
  • the positive screening marker comprises a fluorescent or luminescent screening marker or an affinity based screening marker (including but not limited to, a cell surface marker).
  • a negative selection or screening marker with the pool of active (optionally mutant) RSs from the positive selection or screening into a plurality of cells of a second organism, wherein the negative selection or screening marker comprises at least one selector codon (including but not limited to, an antibiotic resistance gene, including but not limited to, a chloramphenicol acetyltransferase (CAT) gene); and, identifying cells that survive or show a specific screening response in a first medium supplemented with the non-naturally encoded amino acid and a screening or selection agent, but fail to survive or to show the specific response in a second medium not supplemented with the non-naturally encoded amino acid and the selection or screening agent, thereby providing surviving cells or screened cells with the at least one recombinant O-RS.
  • the negative selection or screening marker comprises at least one selector codon (including but not limited to, an antibiotic resistance gene, including but not limited to, a chloramphenicol acetyltransferase (CAT) gene)
  • a CAT identification protocol optionally acts as a positive selection and/or a negative screening in determination of appropriate O-RS recombinants.
  • a pool of clones is optionally replicated on growth plates containing CAT (which comprises at least one selector codon) either with or without one or more non-naturally encoded amino acid. Colonies growing exclusively on the plates containing non-naturally encoded amino acids are thus regarded as containing recombinant O-RS.
  • the concentration of the selection (and/or screening) agent is varied.
  • the first and second organisms are different.
  • the first and/or second organism optionally comprises: a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, a yeast, an archaebacterium, a eubacterium, a plant, an insect, a protist, etc.
  • the screening marker comprises a fluorescent or luminescent screening marker or an affinity based screening marker.
  • screening or selecting (including but not limited to, negatively selecting) the pool for active (optionally mutant) RSs includes: isolating the pool of active mutant RSs from the positive selection step (b); introducing a negative selection or screening marker, wherein the negative selection or screening marker comprises at least one selector codon (including but not limited to, a toxic marker gene, including but not limited to, a ribonuclease barnase gene, comprising at least one selector codon), and the pool of active (optionally mutant) RSs into a plurality of cells of a second organism; and identifying cells that survive or show a specific screening response in a first medium not supplemented with the non- naturally encoded amino acid, but fail to survive or show a specific screening response in a second medium supplemented with the non-naturally encoded amino acid, thereby providing surviving or screened cells with the at least one recombinant O-RS, wherein the at least one recombinant O-RS is specific for the
  • the at least one selector codon comprises about two or more selector codons.
  • Such embodiments optionally can include wherein the at least one selector codon comprises two or more selector codons, and wherein the first and second organism are different (including but not limited to, each organism is optionally, including but not limited to, a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, a yeast, an archaebacteria, a eubacteria, a plant, an insect, a protist, etc.).
  • the negative selection marker comprises a ribonuclease barnase gene (which comprises at least one selector codon).
  • the screening marker optionally comprises a fluorescent or luminescent screening marker or an affinity based screening marker.
  • the screenings and/or selections optionally include variation of the screening and/or selection stringency.
  • the methods for producing at least one recombinant orthogonal aminoacyl-tRNA synthetase can further comprise: (d) isolating the at least one recombinant O-RS; (e) generating a second set of O-RS (optionally mutated) derived from the at least one recombinant O-RS; and, (f) repeating steps (b) and (c) until a mutated O-RS is obtained that comprises an ability to preferentially aminoacylate the O-tRNA.
  • steps (d)-(f) are repeated, including but not limited to, at least about two times.
  • the second set of mutated O-RS derived from at least one recombinant O-RS can be generated by mutagenesis, including but not limited to, random mutagenesis, site-specific mutagenesis, recombination or a combination thereof.
  • the stringency of the selection/screening steps optionally includes varying the selection/screening stringency.
  • the positive selection/screening step (b), the negative selection/screening step (c) or both the positive and negative selection/screening steps (b) and (c) comprise using a reporter, wherein the reporter is detected by fluorescence-activated cell sorting (FACS) or wherein the reporter is detected by luminescence.
  • FACS fluorescence-activated cell sorting
  • the reporter is displayed on a cell surface, on a phage display or the like and selected based upon affinity or catalytic activity involving the non- naturally encoded amino acid or an analogue.
  • the mutated synthetase is displayed on a cell surface, on a phage display or the like.
  • Methods for producing a recombinant orthogonal tRNA include: (a) generating a library of mutant tRNAs derived from at least one tRNA, including but not limited to, a suppressor tRNA, from a first organism; (b) selecting (including but not limited to, negatively selecting) or screening the library for (optionally mutant) tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of a RS from the first organism, thereby providing a pool of tRNAs (optionally mutant); and, (c) selecting or screening the pool of tRNAs (optionally mutant) for members that are aminoacylated by an introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA; wherein the at least one recombinant O-tRNA recognizes a selector codon and is not efficiency recognized by the RS from the second organism and is preferential
  • the at least one tRNA is a suppressor tRNA and/or comprises a unique three base codon of natural and/or unnatural bases, or is a nonsense codon, a rare codon, an unnatural codon, a codon comprising at least 4 bases, an amber codon, an ochre codon, or an opal stop codon.
  • the recombinant O-tRNA possesses an improvement of orthogonality. It will be appreciated that in some embodiments, O-tRNA is optionally imported into a first organism from a second organism without the need for modification.
  • the first and second organisms are either the same or different and are optionally chosen from, including but not limited to, prokaryotes (including but not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Escherichia coli, Halobacterium, etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria, plants, insects, protists, etc.
  • the recombinant tRNA is optionally aminoacylated by a non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is biosynthesized in vivo either naturally or through genetic manipulation.
  • the non- naturally encoded amino acid is optionally added to a growth medium for at least the first or second organism.
  • selecting (including but not limited to, negatively selecting) or screening the library for (optionally mutant) tRNAs that are aminoacylated by an aminoacyl- tRNA synthetase includes: introducing a toxic marker gene, wherein the toxic marker gene comprises at least one of the selector codons (or a gene that leads to the production of a toxic or static agent or a gene essential to the organism wherein such marker gene comprises at least one selector codon) and the library of (optionally mutant) tRNAs into a plurality of cells from the second organism; and, selecting surviving cells, wherein the surviving cells contain the pool of (optionally mutant) tRNAs comprising at least one orthogonal tRNA or nonfunctional tRNA. For example, surviving cells can be selected by using a comparison ratio cell density assay.
  • the toxic marker gene can include two or more selector codons.
  • the toxic marker gene is a ribonuclease barnase gene, where the ribonuclease barnase gene comprises at least one amber codon.
  • the ribonuclease barnase gene can include two or more amber codons.
  • selecting or screening the pool of (optionally mutant) tRNAs for members that are aminoacylated by an introduced orthogonal RS can include: introducing a positive selection or screening marker gene, wherein the positive marker gene comprises a drug resistance gene (including but not limited to, ⁇ -lactamase gene, comprising at least one of the selector codons, such as at least one amber stop codon) or a gene essential to the organism, or a gene that leads to detoxification of a toxic agent, along with the O-RS, and the pool of (optionally mutant) tRNAs into a plurality of cells from the second organism; and, identifying surviving or screened cells grown in the presence of a selection or screening agent, including but not limited to, an antibiotic, thereby providing a pool of cells possessing the at least one recombinant tRNA, where the at least one recombinant tRNA is aminoacylated by the O-RS and inserts an amino acid into a translation product encode
  • a drug resistance gene including but not limited to
  • Methods for generating specific O-tRNA/O-RS pairs include: (a) generating a library of mutant tRNAs derived from at least one tRNA from a first organism; (b) negatively selecting or screening the library for (optionally mutant) tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of a RS from the first organism, thereby providing a pool of (optionally mutant) tRNAs; (c) selecting or screening the pool of (optionally mutant) tRNAs for members that are aminoacylated by an introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA.
  • RS aminoacyl-tRNA synthetase
  • the at least one recombinant O-tRNA recognizes a selector codon and is not efficiency recognized by the RS from the second organism and is preferentially aminoacylated by the O-RS.
  • the method also includes (d) generating a library of (optionally mutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS) from a third organism; (e) selecting or screening the library of mutant RSs for members that preferentially aminoacylate the at least one recombinant O-tRNA in the presence of a non-naturally encoded amino acid and a natural amino acid, thereby providing a pool of active (optionally mutant) RSs; and, (f) negatively selecting or screening the pool for active (optionally mutant) RSs that preferentially aminoacylate the at least one recombinant O-tRNA in the absence of the non-naturally encoded amino acid, thereby providing the at least one specific O-tRNA/O-RS pair, wherein the at least one specific O-
  • the specific O-tRNA/O-RS pair can include, including but not limited to, a mutRNATyr-mutTyrRS pair, such as a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, a mutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.
  • a mutRNATyr-mutTyrRS pair such as a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, a mutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.
  • such methods include wherein the first and third organism are the same (including but not limited to, Methanococcus jannaschii).
  • Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use in an in vivo translation system of a second organism are also included in the present invention.
  • the methods include: introducing a marker gene, a tRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from a first organism into a first set of cells from the second organism; introducing the marker gene and the tRNA into a duplicate cell set from a second organism; and, selecting for surviving cells in the first set that fail to survive in the duplicate cell set or screening for cells showing a specific screening response that fail to give such response in the duplicate cell set, wherein the first set and the duplicate cell set are grown in the presence of a selection or screening agent, wherein the surviving or screened cells comprise the orthogonal tRNA-tRNA synthetase pair for use in the in the in vivo translation system of the second organism.
  • RS aminoacyl-tRNA synthetase
  • comparing and selecting or screening includes an in vivo complementation assay.
  • concentration of the selection or screening agent can be varied.
  • the organisms of the present invention comprise a variety of organism and a variety of combinations.
  • the first and the second organisms of the methods of the present invention can be the same or different.
  • the organisms are optionally a prokaryotic organism, including but not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like.
  • the organisms optionally comprise a eukaryotic organism, including but not limited to, plants (including but not limited to, complex plants such as monocots, or dicots), algae, protists, fungi (including but not limited to, yeast, etc), animals (including but not limited to, mammals, insects, arthropods, etc.), or the like.
  • the second organism is a prokaryotic organism, including but not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like.
  • the second organism can be a eukaryotic organism, including but not limited to, a yeast, a animal cell, a plant cell, a fungus, a mammalian cell, or the like.
  • the first and second organisms are different.
  • the present invention contemplates incorporation of one or more non-naturally- occurring amino acids into IFN beta polypeptides.
  • One or more non-naturally-occurring amino acids may be incorporated at a particular position which does not disrupt activity of the polypeptide. This can be achieved by making "conservative" substitutions, including but not limited to, substituting hydrophobic amino acids with hydrophobic amino acids, bulky amino acids for bulky amino acids, hydrophilic amino acids for hydrophilic amino acids and/or inserting the non-naturally-occurring amino acid in a location that is not required for activity.
  • Selection of desired sites may be for producing a IFN beta molecule having any desired property or activity, including but not limited to, agonists, super-agonists, inverse agonists, antagonists, receptor binding modulators, receptor activity modulators, dimer or multimer formation, no change to activity or property compared to the native molecule, or manipulating any physical or chemical property of the polypeptide such as solubility, aggregation, or stability.
  • locations in the polypeptide required for biological activity of IFN beta polypeptides can be identified using point mutation analysis, alanine scanning, saturation mutagenesis and screening for biological activity, or homolog scanning methods known in the art. Other methods can be used to identify residues for modification of
  • IFN beta polypeptides include, but are not limited to, sequence profiling (Bowie and Eisenberg,
  • Residues that are critical for IFN beta bioactivity residues that are involved with pharmaceutical stability, antibody epitopes, or receptor binding residues may be mutated.
  • the sites identified as critical to biological activity may also be good candidates for substitution with a non-naturally encoded amino acid, again depending on the desired activity sought for the polypeptide.
  • Another alternative would be to simply make serial substitutions in each position on the polypeptide chain with a non-naturally encoded amino acid and observe the effect on the activities of the polypeptide. It is readily apparent to those of ordinary skill in the art that any means, technique, or method for selecting a position for substitution with a non-natural amino acid into any polypeptide is suitable for use in the present invention.
  • mutants of IFN beta polypeptides that contain deletions can also be examined to determine regions of the protein that are likely to be tolerant of substitution with a non-naturally encoded amino acid.
  • protease digestion and monoclonal antibodies can be used to identify regions of IFN beta that are responsible for binding the IFN receptor. Once residues that are likely to be intolerant to substitution with non- naturally encoded amino acids have been eliminated, the impact of proposed substitutions at each of the remaining positions can be examined. Models may be generated from the three- dimensional crystal structures of other IFN family members and IFN receptors.
  • Protein Data Bank (PDB, available on the World Wide Web at rcsb.org) is a centralized database containing three-dimensional structural data of large molecules of proteins and nucleic acids. Models may be made investigating the secondary and tertiary structure of polypeptides, if three-dimensional structural data is not available. Thus, those of ordinary skill in the art can readily identify amino acid positions that can be substituted with non-naturally encoded amino acids.
  • the IFN beta polypeptides of the invention comprise one or more non-naturally occurring amino acids positioned in a region of the protein that does not disrupt the structure of the polypeptide.
  • Exemplary residues of incorporation of a non-naturally encoded amino acid may be those that are excluded from potential receptor binding regions, may be fully or partially solvent exposed, have minimal or no hydrogen-bonding interactions with nearby residues, may be minimally exposed to nearby reactive residues, may be on one or more of the exposed faces, may be a site or sites that are juxtaposed to a second IFN beta, or other molecule or fragment thereof, may be in regions that are highly flexible, or structurally rigid, as predicted by the three- dimensional, secondary, tertiary, or quaternary structure of IFN beta, bound or unbound to its receptor, or coupled or not coupled to another biologically active molecule, or may modulate the conformation of the IFN beta itself or a dimer or multimer comprising one or more IFN beta, by altering the flexibility or rigidity of the complete structure as desired.
  • one or more non-naturally encoded amino acids are incorporated in one or more of the following positions in IFN beta: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,
  • one or more non-naturally encoded amino acids are incorporated at any position in one or more of the following regions corresponding to secondary structures in interferon beta as follows: Helix A (2-22); Helix B (51-71); Helix C (80-107); Helix D (118-136); helix E (139-162); AB loop: ABl (23-35); AB2 (36-40); AB3 (41-50) from SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 3, 4.
  • the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 25-35, 80-100, and 121-135 of interferon beta (SEQ ID NO: 1 or the corresponding amino acids from SEQ ID NO: 3, 4). In other embodiments, the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 41-49 from interferon beta from SEQ ID NO: 1 or the corresponding amino acids from SEQ ID NO: 3, 4.
  • one or more non-naturally encoded amino acids are incorporated at one or more of the following positions of IFN beta: 28, 36, 76, 80, 107, 108, 111, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4.
  • one or more non-naturally encoded amino acids are incorporated at one or more of the following positions of IFN beta: 8, 15, 19, 36, 42, 46, 48, 49, 80, 108, 111, 113, 155, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4.
  • one or more non-naturally encoded amino acids are incorporated at one or more of the following positions of IFN beta: 15, 42, 80, 108, 111, 155, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4).
  • the polypeptide of the invention comprises one or more natural amino acid substitution, addition, or deletion.
  • the polypeptide of the invention comprises a C17S substitution (serine for a cysteine at position 17) of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NO: 3, 4.
  • the polypeptide of the invention comprises a C17S substitution (serine for a cysteine at position 17) and one or more natural amino acid substitution, addition, or deletion. In some embodiments, the polypeptide of the invention comprises one or more non-naturally encoded amino acid substitution, addition, or deletion in the signal sequence. In some embodiments, the polypeptide of the invention comprises one or more non-naturally encoded amino acid substitution, addition, or deletion in the signal sequence of SEQ ID NO: 4. In some embodiments, the polypeptide of the invention comprises one ore more naturally encoded amino acid substitution, addition, or deletion in the signal sequence of SEQ ID NO: 4. In some embodiments, one or more non-natural amino acids are incorporated in the leader or signal sequence of SEQ ID NOs: 4 or other IFN beta sequence.
  • the non-naturally occurring amino acid in one or more of these regions is linked to a water soluble polymer, including but not limited to: Helix A (2-22); Helix B (51-71); Helix C (80-107); Helix D (118-136); Helix E (139-162); AB loop: ABl (23-35); AB2 (36-40); AB3 (41-50) from SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 3, 4.
  • a water soluble polymer including but not limited to: Helix A (2-22); Helix B (51-71); Helix C (80-107); Helix D (118-136); Helix E (139-162); AB loop: ABl (23-35); AB2 (36-40); AB3 (41-50) from SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 3, 4.
  • the non-naturally occurring amino acid in one or more of these regions is linked to a water soluble polymer, including but not limited to, residues 25-35, 80-100, and 121- 135 of interferon beta (SEQ ID NO: 1 or the corresponding amino acids from SEQ ID NO: 3, 4).
  • the non-naturally occurring amino acid in one or more of these regions is linked to a water soluble polymer, including but not limited to, residues 41-49 from interferon beta from SEQ ID NO: 1 or the corresponding amino acids from SEQ ID NO: 3, 4.
  • the non-naturally occurring amino acid at one or more of these positions is linked to a water soluble polymer, including but not limited to, positions: 28, 36, 76, 80, 107, 108, 111, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4.
  • the non-naturally occurring amino acid at one or more of these positions is linked to a water soluble polymer, including but not limited to, positions: 8, 15, 19, 36, 42, 46, 48, 49, 80, 108, 111, 113, 155, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4).
  • the non- naturally occurring amino acid at one or more of these positions is linked to a water soluble polymer, including but not limited to, positions: 15, 42, 80, 108, 111, 155, and any combination thereof of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 3, 4).
  • the non-naturally occurring amino acid in the signal or leader sequence is linked to a water soluble polymer (SEQ ID NO: 4 or other IFN beta sequence).
  • non-naturally encoded amino acids can be substituted for, or incorporated into, a given position in a IFN beta polypeptide.
  • a particular non- naturally encoded amino acid is selected for incorporation based on an examination of the three dimensional crystal structure of a IFN beta polypeptide or other IFN family member with its receptor, a preference for conservative substitutions (i.e., aryl-based non-naturally encoded amino acids, such as p-acetylphenylalanine or O-propargyltyrosine substituting for Phe, Tyr or Trp), and the specific conjugation chemistry that one desires to introduce into the IFN beta polypeptide (e.g., the introduction of 4-azidophenylalanine if one wants to effect a Huisgen [3+2] cycloaddition with a water soluble polymer bearing an alkyne moiety or a amide bond formation with a water soluble polymer that bears an aryl ester that
  • the method further includes incorporating into the protein the unnatural amino acid, where the unnatural amino acid comprises a first reactive group; and contacting the protein with a molecule (including but not limited to, a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin
  • a molecule including
  • the first reactive group reacts with the second reactive group to attach the molecule to the unnatural amino acid through a [3+2] cycloaddition.
  • the first reactive group is an alkynyl or azido moiety and the second reactive group is an azido or alkynyl moiety.
  • the first reactive group is the alkynyl moiety (including but not limited to, in unnatural amino acid p-propargyloxyphenylalanine) and the second reactive group is the azido moiety.
  • the first reactive group is the azido moiety (including but not limited to, in the unnatural amino acid p-azido-L-phenylalanine) and the second reactive group is the alkynyl moiety.
  • the non-naturally encoded amino acid substitution(s) will be combined with other additions, substitutions or deletions within the IFN beta polypeptide to affect other biological traits of the IFN beta polypeptide.
  • the other additions, substitutions or deletions may increase the stability (including but not limited to, resistance to proteolytic degradation) of the IFN beta polypeptide or increase affinity of the IFN beta polypeptide for its receptor.
  • the other additions, substitutions or deletions may increase the pharmaceutical stability of the IFN beta polypeptide.
  • the other additions, substitutions or deletions may enhance the anti-viral activity of the IFN beta polypeptide.
  • the other additions, substitutions or deletions may increase the solubility (including but not limited to, when expressed in E. coli or other host cells) of the IFN beta polypeptide.
  • additions, substitutions or deletions may increase the IFN beta polypeptide solubility following expression in E. coli or other recombinant host cells.
  • sites are selected for substitution with a naturally encoded or non-natural amino acid in addition to another site for incorporation of a non-natural amino acid that results in increasing the polypeptide solubility following expression in K coli or other recombinant host cells.
  • the IFN beta polypeptides comprise another addition, substitution or deletion that modulates affinity for the IFN beta polypeptide receptor, binding proteins, or associated ligand, modulates signal transduction after binding to the IFN receptor, modulates circulating half-life, modulates release or bio-availability, facilitates purification, or improves or alters a particular route of administration.
  • the IFN beta polypeptides comprise an addition, substitution or deletion that increases the affinity of the IFN beta variant for its receptor.
  • IFN beta polypeptides can comprise chemical or enzyme cleavage sequences, protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including, but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including, but not limited to, biotin) that improve detection (including, but not limited to, G FP), purification, transport through tissues or cell membranes, prodrug release or activation, IFN beta size reduction, or other traits of the polypeptide.
  • antibody-binding domains including but not limited to, FLAG or poly-His
  • affinity based sequences including, but not limited to, FLAG, poly-His, GST, etc.
  • linked molecules including, but not limited to, biotin
  • the substitution of a non-naturally encoded amino acid generates an IFN beta antagonist.
  • a non-naturally encoded amino acid is substituted or added in a region involved with receptor binding.
  • IFN beta antagonists comprise at least one substitution that cause IFN beta to act as an antagonist.
  • the IFN beta antagonist comprises a non-naturally encoded amino acid linked to a water soluble polymer that is present in a receptor binding region of the IFN beta molecule.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are substituted with one or more non-naturally-encoded amino acids.
  • the IFN beta polypeptide further includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions of one or more non-naturally encoded amino acids for naturally-occurring amino acids.
  • one or more residues in IFN beta are substituted with one or more non-naturally encoded amino acids.
  • the one or more non-naturally encoded residues are linked to one or more lower molecular weight linear or branched PEGs, thereby enhancing binding affinity and comparable serum half-life relative to the species attached to a single, higher molecular weight PEG.
  • up to two of the following residues of IFN beta are substituted with one or more non-naturally-encoded amino acids.
  • Suitable bacterial promoters are known to those of ordinary skill in the art and described, e.g., in Sambrook et al. and Ausubel et al.
  • Bacterial expression systems for expressing IFN beta polypeptides of the invention are available in, including but not limited to, E. coli, Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are known to those of ordinary skill in the art and are also commercially available.
  • host cells for expression are selected based on their ability to use the orthogonal components.
  • Exemplary host cells include Gram-positive bacteria (including but not limited to B. brevis, B. subtilis, or Streptomyces) and Gram-negative bacteria (E. coli, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida), as well as yeast and other eukaryotic cells.
  • Cells comprising O-tRNA/O- RS pairs can be used as described herein.
  • a eukaryotic host cell or non-eukaryotic host cell of the present invention provides the ability to synthesize proteins that comprise unnatural amino acids in large useful quantities.
  • the composition optionally includes, including but not limited to, at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least one gram, or more of the protein that comprises an unnatural amino acid, or an amount that can be achieved with in vivo protein production methods (details on recombinant protein production and purification are provided herein).
  • the protein is optionally present in the composition at a concentration of, including but not limited to, at least 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75 micrograms of protein per liter, at least 100 micrograms of protein per liter, at least 200 micrograms of protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of protein per liter, at least 1 milligram of protein per liter, or at least 10 milligrams of protein per liter or more, in, including but not limited to, a cell lysate, a buffer, a pharmaceutical buffer, or other liquid suspension (including but not limited to, in a volume of, including but not limited to, anywhere from about 1 nl to about 100 L or more).
  • the production of large quantities (including but not limited to, greater that that typically possible with other methods, including but not limited to, in vitro translation) of a protein in a eukaryotic cell including at least one unnatural amino acid is a concentration
  • a eukaryotic host cell or non-eukaryotic host cell of the present invention provides the ability to biosynthesize proteins that comprise unnatural amino acids in large useful quantities.
  • proteins comprising an unnatural amino acid can be produced at a concentration of, including but not limited to, at least 10 ⁇ g/liter, at least 50 ⁇ g/liter, at least 75 ⁇ g/liter, at least 100 ⁇ g/liter, at least 200 ⁇ g/liter, at least 250 ⁇ g/liter, or at least 500 ⁇ g/liter, at least lmg/liter, at least 2mg/liter, at least 3 mg/liter, at least 4 mg/liter, at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least 8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg/liter, 1 g/liter, 5
  • vectors suitable for expression of IFN beta are commercially available.
  • Useful expression vectors for eukaryotic hosts include but are not limited to, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus.
  • vectors include pCDNA3.1(+) ⁇ Hyg (Invitrogen, Carlsbad, Calif., USA) and pCI-neo (Stratagene, La Jolla, Calif, USA).
  • Bacterial plasmids such as plasmids from E.
  • coli including pBR322, pET3a and pET12a, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M 13 and filamentous single stranded DNA phages may be used.
  • phage DNAs e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M 13 and filamentous single stranded DNA phages
  • the vectors include but are not limited to, pVL941, pBG311 (Cate 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 (Invitrogen, Carlsbad, CA).
  • the nucleotide sequence encoding an IFNB polypeptide may or may not also include sequence that encodes 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 may be any sequence.
  • the signal peptide may be prokaryotic or eukaryotic. Coloma, M (1992) J. Imm. Methods 152:89 104) describe a signal peptide for use in mammalian cells (murine Ig kappa light chain signal peptide).
  • Other signal peptides include but are not limited to, the ⁇ -factor signal peptide from S. cerevisiae (U.S. Patent No.
  • Suitable mammalian host cells are known to those of ordinary skill in the art.
  • Such host cells may be Chinese hamster ovary (CHO) cells, (e.g. CHO-Kl; ATCC CCL-61), Green Monkey cells (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-IO), and human cells (e.g. HEK 293 (ATCC CRL-1573)), as well as plant cells in tissue culture.
  • COS Green Monkey cells
  • BHK Baby Hamster Kidney
  • BHK Baby Hamster Kidney
  • human cells e.g. ATCC CRL-1632 or ATCC CCL-IO
  • human cells e.g. HEK 293 (ATCC CRL-1573)
  • a mammalian host cell may be modified to express sialyltransferase, e.g. 1 ,6-sialyltransferase, e.g. as described in U.S. Pat. No. 5,047,335, which is incorporated by reference herein.
  • Methods for the introduction of exogenous DNA into mammalian host cells include but are not limited to, calcium phosphare-mediated transfection, electroporation, DEAE- dextran mediated transfection, liposome-mediated transfection, viral vectors and the transfection methods described by Life Technologies Ltd, Paisley, UK using Lipofectamin 2000 and Roche Diagnostics Corporation, Indianapolis, USA using FuGENE 6. These methods are well known in the art and are described by Ausbel et al. (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. The cultivation of mammalian cells may be performed according to established methods, e.g. as disclosed in (Animal Cell Biotechnology, Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc. Totowa, N. J., USA and Harrison Mass. and Rae IF, General Techniques of Cell Culture, Cambridge University Press 1997).
  • IFN beta polypeptides may be expressed in any number of suitable expression systems including, for example, yeast, insect cells, mammalian cells, and bacteria. A description of exemplary expression systems is provided below.
  • yeast includes any of the various yeasts capable of expressing a gene encoding a IFN beta polypeptide.
  • Such yeasts include, but are not limited to, ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts and yeasts belonging to the Fungi imperfecti (Blastomycetes) group.
  • the ascosporogenous yeasts are divided into two families, Spermophthoraceae and Saccharomycetaceae.
  • the latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saccharomyces).
  • the basidiosporogenous yeasts include the genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, a nd Filobasidiella.
  • Yeasts belonging to the Fungi Imperfecti ⁇ Blastomycetes) group are divided into two families, Sporobolomycetaceae (e.g., genera Sporobolomyces and Buller ⁇ ) and Cryptococcaceae (e.g., genus Candida).
  • Sporobolomycetaceae e.g., genera Sporobolomyces and Buller ⁇
  • Cryptococcaceae e.g., genus Candida
  • Candida Of particular interest for use with the present invention are species within the genera Pichia, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Hansenula, Torulopsis, and Candida, including, but not limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S. carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S, norbensis, S. oviformis, K. lac
  • yeast host or "yeast host cell” includes yeast that can be, or has been, used as a recipient for recombinant vectors or other transfer DNA.
  • the term includes the progeny of the original yeast host cell that has received the recombinant vectors or other transfer DNA. It is understood that the progeny of a single par ental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell that are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a IFN beta polypeptide, are included in the progeny intended by this definition.
  • Expression and transformation vectors including extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeast hosts.
  • expression vectors have been developed for S. cerevisiae (Sikorski et al., GENETICS (1989) 122:19; Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL. ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL. BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL.
  • Control sequences for yeast vectors are known to those of ordinary skill in the art and include, but are not limited to, promoter regions from genes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase; glucokinase; glucose-6-phosphate isomerase; glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase; phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase (PyK) (EP 0 329 203).
  • ADH alcohol dehydrogenase
  • GAP glyceraldehyde-3-phosphate-dehydrogenase
  • hexokinase phosphofructokinase
  • 3-phosphoglycerate mutase 3-phosphoglycerate mutase
  • pyruvate kinase PyK
  • the yeast PHO5 gene encoding acid phosphatase, also may provide useful promoter sequences (Miyanohara et al., PROC. NATL. ACAD. SCI. USA (1983) 80:1).
  • Other suitable promoter sequences for use with yeast hosts may include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. BiOL. CHEM. (1980) 255:12073); and other glycolytic enzymes, such as pyruvate decarboxylase, triosephosphate isomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY (1978) 17:4900; Hess et al., J. ADV.
  • Inducible yeast promoters having the additional advantage of transcription controlled by growth conditions may include the promoter regions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase; metallothionein; glyceraldehyde-3 -phosphate dehydrogenase; degradative enzymes associated with nitrogen metabolism; and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 0 073 657.
  • Yeast enhancers also may be used with yeast promoters.
  • synthetic promoters may also function as yeast promoters.
  • the upstream activating sequences (UAS) of a yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.
  • hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region. See U.S. Patent Nos. 4,880,734 and 4,876,197, which are incorporated by reference herein.
  • Other examples of hybrid promoters include promoters that consist of the regulatory sequences of the ADH2, GAL4, GALlO, or PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK.
  • a yeast promoter may include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.
  • Other control elements that may comprise part of the yeast expression vectors include terminators, for example, from GAPDH or the enolase genes (Holland et al., J. BIOL. CHEM. (1981) 256:1385).
  • the origin of replication from the 2 ⁇ plasmid origin is suitable for yeast.
  • a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid. See Tschumper et al., GENE (1980) 10:157; Kingsman et al., GENE (1979) 7:141.
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan.
  • Leu2-deficient yeast strains ATCC 20,622 or 38,626 are complemented by known plasmids bearing the Leu2 gene.
  • Methods of introducing exogenous DNA into yeast hosts are known to those of ordinary skill in the art, and typically include, but are not limited to, either the transformation of spheroplasts or of intact yeast host cells treated with alkali cations.
  • transformation of yeast can be carried out according to the method described in Hsiao et al., PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al., J. BACT. (1977) 130:946.
  • other methods for introducing DNA into cells such as by nuclear injection, electroporation, or protoplast fusion may also be used as described generally in SAMBROOK ET AL., MOLECULAR CLONING: A LAB. MANUAL (2001).
  • Yeast host cells may then be cultured using standard techniques known to those of ordinary skill in the art.
  • yeast host strains may be grown in fermentors during the amplification stage using standard feed batch fermentation methods known to those of ordinary skill in the art. The fermentation methods may be adapted to account for differences in a particular yeast host's carbon utilization pathway or mode of expression control.
  • fermentation of a Saccharomyces yeast host may require a single glucose feed, complex nitrogen source (e.g., casein hydrolysates), and multiple vitamin supplementation.
  • complex nitrogen source e.g., casein hydrolysates
  • methylotrophic yeast P. pastoris may require glycerol, methanol, and trace mineral feeds, but only simple ammonium (nitrogen) salts for optimal growth and expression.
  • simple ammonium (nitrogen) salts for optimal growth and expression.
  • Such fermentation methods may have certain common features independent of the yeast host strain employed.
  • a growth limiting nutrient typically carbon
  • fermentation methods generally employ a fermentation medium designed to contain adequate amounts of carbon, nitrogen, basal salts, phosphorus, and other minor nutrients (vitamins, trace minerals and salts, etc.). Examples of fermentation media suitable for use with Pichia are described in U.S. Patent Nos. 5,324,639 and 5,231,178, which are incorporated by reference herein.
  • insect host or “insect host cell” refers to a insect that can be, or has been, used as a recipient for recombinant vectors or other transfer DNA.
  • the term includes the progeny of the original insect host cell that has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell that are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a IFN beta polypeptide, are included in the progeny intended by this definition. Baculovirus expression of IFN beta polypeptides has been described in U.S. Patent No. 7,144,574, which is incorporated by reference herein.
  • IFN beta polypeptides The selection of suitable insect cells for expression of IFN beta polypeptides is known to those of ordinary skill in the art. Several insect species are well described in the art and are commercially available including Aedes aegypti, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni. In selecting insect hosts for expression, suitable hosts may include those shown to have, inter alia, good secretion capacity, low proteolytic activity, and overall robustness.
  • Insect are generally available from a variety of sources including, but not limited to, the Insect Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); and the American Type Culture Collection (“ATCC”) (Manassas, VA).
  • ATCC American Type Culture Collection
  • the components of a baculovirus-infected insect expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene to be expressed; a wild type baculovirus with sequences homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.
  • the materials, methods and techniques used in constructing vectors, transfecting cells, picking plaques, growing cells in culture, and the like are known in the art and manuals are available describing these techniques.
  • the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome recombine.
  • the packaged recombinant virus is expressed and recombinant plaques are identified and purified.
  • Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, for example, Invitrogen Corp. (Carlsbad, CA). These techniques are generally known to those of ordinary skill in the art and fully described in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987), herein incorporated by reference.
  • Vectors that are useful in baculovirus/insect cell expression systems include, for example, insect expression and transfer vectors derived from the baculovirus Autographacalifornica nuclear polyhedrosis virus (AcNPV), which is a helper- independent, viral expression vector.
  • AdNPV baculovirus Autographacalifornica nuclear polyhedrosis virus
  • Viral expression vectors derived from this system usually use the strong viral polyhedrin gene promoter to drive expression of heterologous genes. See generally, O 'Reilly ET AL., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).
  • the above- described components comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are typically assembled into an intermediate transplacement construct (transfer vector).
  • Intermediate transplacement constructs are often maintained in a replicon, such as an extra chromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria.
  • the replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.
  • the plasmid may contain the polyhedrin polyadenylation signal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryotic ampicillin-resi stance (amp) gene and origin of replication for selection and propagation in E. coli.
  • One commonly used transfer vector for introducing foreign genes into AcNPV is pAc373.
  • Many other vectors known to those of skill in the art, have also been designed including, for example, pVL985, which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 base pairs downstream from the ATT. See Luckow and Summers, VIROLOGY 170:31 (1989).
  • Other commercially available vectors include, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac; pBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).
  • the transfer vector and wild type baculoviral genome are co-transfected into an insect cell host.
  • Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. See SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al., M ⁇ L. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989) 170:31.
  • the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene.
  • a gene such as the polyhedrin gene
  • insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene.
  • Transfection may be accomplished by electroporation. See TROTTER AND WOOD,
  • liposomes may be used to transfect the insect cells with the recombinant expression vector and the baculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36; Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL. CHEM.
  • Baculovirus expression vectors usually contain a baculovirus promoter.
  • a baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g., structural gene) into mRNA.
  • a promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence.
  • This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site.
  • a baculovirus promoter may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Moreover, expression may be either regulated or constitutive. [411] Structural genes, abundantly transcribed at late times in the infection cycle, provide particularly useful promoter sequences.
  • baculovirus expression vector is packaged into an infectious recombinant baculovirus and subsequently grown plaques may be purified by techniques known to those of ordinary skill in the art.
  • Recombinant baculovirus expression vectors have been developed for infection into several insect cells.
  • recombinant baculoviruses have been developed for, inter alia, Aedes aegypti (ATCC No. CCL- 125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster (ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni.
  • the cell lines used for baculovirus expression vector systems commonly include, but are not limited to, Sf9 ⁇ Spodoptera frugiperda) (ATCC No. CRL-1711), Sf21 ⁇ Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 11497-013 (Carlsbad, CA)), Tri-368 ⁇ Trichopulsia ni), and High-FiveTM BTI-TN-5B1-4 ⁇ Trichopulsia ni).
  • E. CoIi, Pseudomonas species, and other Prokaryotes Bacterial expression techniques are known to those of ordinary skill in the art.
  • a wide variety of vectors are available for use in bacterial hosts.
  • the vectors may be single copy or low or high multicopy vectors.
  • Vectors may serve for cloning and/or expression.
  • the vectors normally involve markers allowing for selection, which markers may provide for cytotoxic agent resistance, prototrophy or immunity. Frequently, a plurality of markers is present, which provide for different characteristics.
  • a bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA.
  • a promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site.
  • a bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene.
  • Constitutive expression may occur in the absence of negative regulatory elements, such as the operator.
  • positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5') to the RNA polymerase binding sequence.
  • An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984) 18:173].
  • Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al., NATURE (1977) 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al., Nuc. ACIDS RES. (1980) 8:4057; Yelverton et al., NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EP Pub. Nos.
  • Such vectors are known to those of ordinary skill in the art and include the pET29 series from Novagen, and the pPOP vectors described in WO99/05297, which is incorporated by reference herein. Such expression systems produce high levels of IFN beta polypeptides in the host without compromising host cell viability or growth parameters.
  • pET19 Novagen
  • synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No.
  • the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al., GENE (1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].
  • a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.
  • the bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al., J. M ⁇ L. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985) 82:1074].
  • a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EP Pub. No. 267 851).
  • an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes.
  • the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al., NATURE (1975) 254:34].
  • SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3' and of E. coli 16S rRNA [Steitz et al.
  • bacterial host or "bacterial host cell” refers to a bacterial that can be, or has been, used as a recipient for recombinant vectors or other transfer DNA.
  • the term includes the progeny of the original bacterial host cell that has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell that are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a IFN beta polypeptide, are included in the progeny intended by this definition.
  • suitable host bacteria for expression of IFN beta polypeptides is known to those of ordinary skill in the art.
  • suitable hosts may include those shown to have, inter alia, good inclusion body formation capacity, low proteolytic activity, and overall robustness.
  • Bacterial hosts are generally available from a variety of sources including, but not limited to, the Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); and the American Type Culture Collection ("ATCC”) (Manassas, VA).
  • Industrial/pharmaceutical fermentation generally use bacterial derived from K strains (e.g. W3110) or from bacteria derived from B strains (e.g. BL21).
  • E. coli hosts include, but are not limited to, strains of BL21, DHlOB, or derivatives thereof.
  • the E. coli host is a protease minus strain including, but not limited to, OMP- and LON-.
  • the host cell strain may be a species of Pseudomonas, including but not limited to, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida.
  • Pseudomonas fluorescens biovar 1 designated strain MBlOl
  • strain MBlOl is known to be useful for recombinant production and is available for therapeutic protein production processes.
  • Examples of a Pseudomonas expression system include the system available from The Dow Chemical Company as a host strain (Midland, MI available on the World Wide Web at dow.com).
  • the recombinant host cell strain is cultured under conditions appropriate for production of IFN beta polypeptides.
  • the method of culture of the recombinant host cell strain will be dependent on the nature of the expression construct utilized and the identity of the host cell.
  • Recombinant host strains are normally cultured using methods that are known to those of ordinary skill in the art.
  • Recombinant host cells are typically cultured in liquid medium containing assimilatable sources of carbon, nitrogen, and inorganic salts and, optionally, containing vitamins, amino acids, growth factors, and other proteinaceous culture supplements known to those of ordinary skill in the art.
  • Liquid media for culture of host cells may optionally contain antibiotics or anti-fungals to prevent the growth of undesirable microorganisms and/or compounds including, but not limited to, antibiotics to select for host cells containing the expression vector.
  • Recombinant host cells may be cultured in batch or continuous formats, with either cell harvesting (in the case where the IFN beta polypeptide accumulates intracellularly) or harvesting of culture supernatant in either batch or continuous formats. For production in prokaryotic host cells, batch culture and cell harvest are preferred.
  • the IFN beta polypeptides of the present invention are normally purified after expression in recombinant systems.
  • the IFN beta polypeptide may be purified from host cells or culture medium by a variety of methods known to the art.
  • IFN beta polypeptides produced in bacterial host cells may be poorly soluble or insoluble (in the form of inclusion bodies).
  • amino acid substitutions may readily be made in the IFN beta polypeptide that are selected for the purpose of increasing the solubility of the recombinantly produced protein utilizing the methods disclosed herein as well as those known in the art.
  • the protein may be collected from host cell lysates by centrifugation and may further be followed by homogenization of the cells.
  • PKI polyethylene imine
  • Recombinant host cells may be disrupted or homogenized to release the inclusion bodies from within the cells using a variety of methods known to those of ordinary skill in the art. Host cell disruption or homogenization may be performed using well known techniques including, but not limited to, enzymatic cell disruption, sonication, dounce homogenization, or high pressure release disruption. In one embodiment of the method of the present invention, the high pressure release technique is used to disrupt the E. coli host cells to release the inclusion bodies of the IFN beta polypeptides.
  • IFN beta polypeptide When handling inclusion bodies of IFN beta polypeptide, it may be advantageous to minimize the homogenization time on repetitions in order to maximize the yield of inclusion bodies without loss due to factors such as solubilization, mechanical shearing or proteolysis.
  • IFN beta polypeptide may then be solubilized using any of a number of suitable solubilization agents known to the art.
  • the IFN beta polyeptide may be solubilized with urea or guanidine hydrochloride.
  • the volume of the solubilized IFN beta polypeptide should be minimized so that large batches may be produced using conveniently manageable batch sizes. This factor may be significant in a large-scale commercial setting where the recombinant host may be grown in batches that are thousands of liters in volume.
  • the milder denaturing agent urea can be used to solubilize the IFN beta polypeptide inclusion bodies in place of the harsher denaturing agent guanidine hydrochloride.
  • the use of urea significantly reduces the risk of damage to stainless steel equipment utilized in the manufacturing and purification process of IFN beta polypeptide while efficiently solubilizing the IFN beta polypeptide inclusion bodies.
  • the IFN beta may be secreted into the periplasmic space or into the culture medium.
  • soluble IFN beta may be present in the cytoplasm of the host cells. It may be desired to concentrate soluble IFN beta prior to performing purification steps. Standard techniques known to those of ordinary skill in the art may be used to concentrate soluble IFN beta from, for example, cell lysates or culture medium. In addition, standard techniques known to those of ordinary skill in the art may be used to disrupt host cells and release soluble IFN beta from the cytoplasm or periplasmic space of the host cells.
  • the fusion sequence may be removed. Removal of a fusion sequence may be accomplished by enzymatic or chemical cleavage. Enzymatic removal of fusion sequences may be accomplished using methods known to those of ordinary skill in the art. The choice of enzyme for removal of the fusion sequence will be determined by the identity of the fusion, and the reaction conditions will be specified by the choice of enzyme as will be apparent to one of ordinary skill in the art. Chemical cleavage may be accomplished using reagents known to those of ordinary skill in the art, including but not limited to, cyanogen bromide, TEV protease, and other reagents.
  • the cleaved IFN beta polypeptide may be purified from the cleaved fusion sequence by methods known to those of ordinary skill in the art. Such methods will be determined by the identity and properties of the fusion sequence and the IFN beta polypeptide, as will be apparent to one of ordinary skill in the art. Methods for purification may include, but are not limited to, size-exclusion chromatography, hydrophobic interaction chromatography, ion-exchange chromatography or dialysis or any combination thereof.
  • the IFN beta polypeptide may also be purified to remove DNA from the protein solution.
  • DNA may be removed by any suitable method known to the art, such as precipitation or ion exchange chromatography, but may be removed by precipitation with a nucleic acid precipitating agent, such as, but not limited to, protamine sulfate.
  • the IFN beta polypeptide may be separated from the precipitated DNA using standard well known methods including, but not limited to, centrifugation or filtration. Removal of host nucleic acid molecules is an important factor in a setting where the IFN beta polypeptide is to be used to treat humans and the methods of the present invention reduce host cell DNA to pharmaceutically acceptable levels.
  • Methods for small-scale or large-scale fermentation can also be used in protein expression, including but not limited to, fermentors, shake flasks, fluidized bed bioreactors, hollow fiber bioreactors, roller bottle culture systems, and stirred tank bioreactor systems. Each of these methods can be performed in a batch, fed-batch, or continuous mode process.
  • Human IFN beta polypeptides of the invention can generally be recovered using methods standard in the art. For example, culture medium or cell lysate can be centrifuged or filtered to remove cellular debris. The supernatant may be concentrated or diluted to a desired volume or diafiltered into a suitable buffer to condition the preparation for further purification. Further purification of the IFN beta polypeptide of the present invention includes separating deamidated and clipped forms of the IFN beta polypeptide variant from the intact form. [431] Any of the following exemplary procedures can be employed for purification of
  • IFN beta polypeptides of the invention affinity chromatography; anion- or cation-exchange chromatography (using, including but not limited to, DEAE SEPHAROSE); chromatography on silica; high performance liquid chromatography (HPLC); reverse phase HPLC; gel filtration (using, including but not limited to, SEPHADEX G-75); hydrophobic interaction chromatography; size-exclusion chromatography; metal-chelate chromatography; ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfate precipitation; chromatofocusing; displacement chromatography; electrophoretic procedures (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonium sulfate precipitation), SDS-PAGE, or extraction.
  • affinity chromatography anion- or cation-exchange chromatography (using, including but not limited to, DEAE SEPHAROSE); chromatography on silica; high performance liquid chromatography (HPLC); reverse phase HPLC; gel filtration
  • Proteins of the present invention including but not limited to, proteins comprising unnatural amino acids, peptides comprising unnatural amino acids, antibodies to proteins comprising unnatural amino acids, binding partners for proteins comprising unnatural amino acids, etc., can be purified, either partially or substantially to homogeneity, according to standard procedures known to and used by those of skill in the art.
  • polypeptides of the invention can be recovered and purified by any of a number of methods known to those of ordinary skill in the art, including but not limited to, ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography, gel electrophoresis and the like. Protein refolding steps can be used, as desired, in making correctly folded mature proteins. High performance liquid chromatography (HPLC), affinity chromatography or other suitable methods can be employed in final purification steps where high purity is desired.
  • HPLC high performance liquid chromatography
  • affinity chromatography affinity chromatography or other suitable methods can be employed in final purification steps where high purity is desired.
  • antibodies made against unnatural amino acids are used as purification reagents, including but not limited to, for affinity-based purification of proteins or peptides comprising one or more unnatural amino acid(s).
  • the polypeptides are optionally used for a wide variety of utilities, including but not limited to, as assay components, therapeutics, prophylaxis, diagnostics, research reagents, and/or as immunogens for antibody production.
  • Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, or cells to an animal, preferably a non-human animal, using routine protocols.
  • an animal preferably a non-human animal
  • One of ordinary skill in the art could generate antibodies using a variety of known techniques.
  • transgenic mice, or other organisms, including other mammals may be used to express humanized antibodies.
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides.
  • Antibodies against polypeptides of the present invention may also be employed to treat diseases.
  • polypeptides and polynucleotides of the present invention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established within the individual or not.
  • An immunological response in a mammal may also be induced by a method comprises delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention.
  • One way of administering the vector is by accelerating it into the desired cells as a coating on particles or otherwise.
  • Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.
  • a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition).
  • the formulation may further comprise a suitable carrier.
  • a polypeptide may be broken down in the stomach, it may be administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intra-dermal injection).
  • parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents or thickening agents.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation which are known to those of ordinary skill in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • proteins or polypeptides of interest are produced with an unnatural amino acid in a eukaryotic host cell or non-eukaryotic host cell.
  • proteins or polypeptides will be folded in their native conformations.
  • those of skill in the art will recognize that, after synthesis, expression and/or purification, proteins or peptides can possess a conformation different from the desired conformations of the relevant polypeptides.
  • the expressed protein or polypeptide is optionally denatured and then renatured.
  • guanidine, urea, DTT, DTE, and/or a chaperonin can be added to a translation product of interest.
  • Methods of reducing, denaturing and renaturing proteins are known to those of ordinary skill in the art (see, the references above, and Debinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconiug. Chem.. 4: 581-585; and Buchner, et al., (1992) Anal. Biochem..
  • Debinski, et al. describe the denaturation and reduction of inclusion body proteins in guanidine-DTE.
  • the proteins can be refolded in a redox buffer containing, including but not limited to, oxidized glutathione and L- arginine.
  • Refolding reagents can be flowed or otherwise moved into contact with the one or more polypeptide or other expression product, or vice-versa.
  • the IFN beta polypeptide thus produced may be misfolded and thus lacks or has reduced biological activity.
  • the bioactivity of the protein may be restored by "refolding".
  • misfolded IFN beta polypeptide is refolded by solubilizing (where the IFN beta polypeptide is also insoluble), unfolding and reducing the polypeptide chain using, for example, one or more chaotropic agents (e.g. urea and/or guanidine) and a reducing agent capable of reducing disulfide bonds (e.g. dithiothreitol, DTT or 2-mercaptoethanol, 2-ME).
  • chaotropic agents e.g. urea and/or guanidine
  • a reducing agent capable of reducing disulfide bonds e.g. dithiothreitol, DTT or 2-mercaptoethanol, 2-ME
  • IFN beta polypeptide may be refolded using standard methods known in the art, such as those described in U.S. Pat. Nos. 4,511,502, 4,511,503, and 4,512,922, which are incorporated by reference herein.
  • the IFN beta polypeptide may also be cofolded with other proteins to form heterodimers or heteromultimers.
  • the IFN beta may be further purified.
  • Purification of IFN beta may be accomplished using a variety of techniques known to those of ordinary skill in the art, including hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, reverse-phase high performance liquid chromatography, affinity chromatography, and the like or any combination thereof. Additional purification may also include a step of drying or precipitation of the purified protein.
  • IFN beta may be exchanged into different buffers and/or concentrated by any of a variety of methods known to the art, including, but not limited to, diafiltration and dialysis. IFN beta that is provided as a single purified protein may be subject to aggregation and precipitation.
  • the purified IFN beta may be at least 90% pure (as measured by reverse phase high performance liquid chromatography, RP-HPLC, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or at least 95% pure, or at least 98% pure, or at least 99% or greater pure. Regardless of the exact numerical value of the purity of the IFN beta, the IFN beta is sufficiently pure for use as a pharmaceutical product or for further processing, such as conjugation with a water soluble polymer such as PEG.
  • pharmacokinetically the IFN beta polypeptides of the present invention have dose linear Cmax and AUC. In some embodiments, the pharmacokinetics of the IFN beta polypeptides of the present invention have a Cmax not less than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of Rebif at the equivalent dose. In some embodiments, the pharmacokinetics of the IFN beta polypeptides of the present invention have a Cmax not less than 100% of Rebif at the equivalent dose.
  • the pharmacokinetics of the IFN beta polypeptides of the present invention have a Cmax not less than 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200% or more of Rebif at the equivalent dose.
  • the pharmacokinetics of the IFN beta polypeptides of the present invention have a Cmax not less than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of Avonex or another control administered at the equivalent dose.
  • the pharmacokinetics of the IFN beta polypeptides of the present invention have a Cmax not less than 100% of Avonex or another control administered at the equivalent dose. In some embodiments, the pharmacokinetics of the IFN beta polypeptides of the present invention have a Cmax not less than 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200% or more of Avonex or another control administered at the equivalent dose. In some embodiments, the pharmacokinetics of the IFN beta polypeptides of the present invention have PK trough levels sufficient to support once per week dosing allometrically.
  • the pharmacodynamics of the IFN beta polypeptides of the present invention induce neopterin and sustain neopterin levels for one week.
  • the IFN beta polypeptides of the present invention induce sustained neopterin levels for 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days,
  • the pharmacodynamics of the IFN beta polypeptides of the present invention induce OASl gene expression not less than a positive control (a currently marketed IFN beta therapeutic) and sustains levels for one week.
  • the IFN beta polypeptides of the present invention induce OAS 1 gene expression not less than a positive control (a currently marketed IFN beta therapeutic) and sustains levels for 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days,
  • injection site reactions are no worse than the positive controls in Example 27 at equivalent dose. In some embodiments of the present invention, injection site reactions are better than the positive controls in Example 27 at equivalent dose. In other embodiments, there are 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% fewer injection site reactions caused by the IFN beta polypeptides of the present invention.
  • IFN beta molecules may be used as therapeutic agents in the absence of other active ingredients or proteins (other than excipients, carriers, and stabilizers, serum albumin and the like), or they may be complexed with another protein or a polymer.
  • isolation steps may be performed on the cell lysate, extract, culture medium, inclusion bodies, periplasmic space of the host cells, cytoplasm of the host cells, or other material, comprising IFN beta polypeptide or on any IFN beta polypeptide mixtures resulting from any isolation steps including, but not limited to, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, high performance liquid chromatography
  • HPLC reversed phase-HPLC
  • RP-HPLC reversed phase-HPLC
  • expanded bed adsorption or any combination and/or repetition thereof and in any appropriate order.
  • Chromatographic materials including, but not limited to, exchange matrix materials, media, and buffers are also available from such companies.
  • fraction collectors include RediFrac Fraction Collector, FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® Fraction Collector (Amersham
  • Mixers are also available to form pH and linear concentration gradients.
  • Commercially available mixers include Gradient Mixer GM-I and In-Line Mixers
  • the chromatographic process may be monitored using any commercially available monitor. Such monitors may be used to gather information like UV, pH, and conductivity. Examples of detectors include Monitor UV-I, UVICORD® S II, Monitor UV-M
  • the IFN beta polypeptide may be reduced and denatured by first denaturing the resultant purified IFN beta polypeptide in urea, followed by dilution into TRIS buffer containing a reducing agent (such as DTT) at a suitable pH.
  • a reducing agent such as DTT
  • the IFN beta polypeptide is denatured in urea in a concentration range of between about 2 M to about 9 M, followed by dilution in TRIS buffer at a pH in the range of about 5.0 to about 8.0.
  • the refolding mixture of this embodiment may then be incubated.
  • the refolding mixture is incubated at room temperature for four to twenty-four hours.
  • the reduced and denatured IFN beta polypeptide mixture may then be further isolated or purified.
  • the pH of the first IFN beta polypeptide mixture may be adjusted prior to performing any subsequent isolation steps.
  • the first IFN beta polypeptide mixture or any subsequent mixture thereof may be concentrated using techniques known in the art.
  • the elution buffer comprising the first IFN beta polypeptide mixture or any subsequent mixture thereof may be exchanged for a buffer suitable for the next isolation step using techniques known to those of ordinary skill in the art.
  • Ion Exchange Chromatography In one embodiment, and as an optional, additional step, ion exchange chromatography may be performed on the first IFN beta polypeptide mixture. See generally ION EXCHANGE CHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No.
  • Such columns utilize strong anion exchangers such as Q SEPHAROSE ® Fast Flow, Q SEPHAROSE ® High Performance, and Q SEPHAROSE ® XL; strong cation exchangers such as SP SEPHAROSE ® High Performance, SP SEPHAROSE ® Fast Flow, and SP SEPHAROSE ® XL; weak anion exchangers such as DEAE SEPHAROSE ® Fast Flow; and weak cation exchangers such as CM SEPHAROSE ® Fast Flow (Amersham Biosciences, Piscataway, NJ).
  • Anion or cation exchange column chromatography may be performed on the IFN beta polypeptide at any stage of the purification process to isolate substantially purified IFN beta polypeptide.
  • the cation exchange chromatography step may be performed using any suitable cation exchange matrix.
  • Useful cation exchange matrices include, but are not limited to, fibrous, porous, non-porous, microgranular, beaded, or cross-linked cation exchange matrix materials.
  • Such cation exchange matrix materials include, but are not limited to, cellulose, agarose, dextran, polyacrylate, polyvinyl, polystyrene, silica, polyether, or composites of any of the foregoing.
  • the cation exchange matrix may be any suitable cation exchanger including strong and weak cation exchangers. Strong cation exchangers may remain ionized over a wide pH range and thus, may be capable of binding IFN beta over a wide pH range. Weak cation exchangers, however, may lose ionization as a function of pH. For example, a weak cation exchanger may lose charge when the pH drops below about pH 4 or pH 5. Suitable strong cation exchangers include, but are not limited to, charged functional groups such as sulfopropyl (SP), methyl sulfonate (S), or sulfoethyl (SE).
  • SP sulfopropyl
  • S methyl sulfonate
  • SE sulfoethyl
  • the cation exchange matrix may be a strong cation exchanger, preferably having an IFN beta binding pH range of about 2.5 to about 6.0. Alternatively, the strong cation exchanger may have an IFN beta binding pH range of about pH 2.5 to about pH 5.5.
  • the cation exchange matrix may be a strong cation exchanger having an IFN beta binding pH of about 3.0.
  • the cation exchange matrix may be a strong cation exchanger, preferably having an IFN beta binding pH range of about 6.0 to about 8.0.
  • the cation exchange matrix may be a strong cation exchanger preferably having an IFN beta binding pH range of about 8.0 to about 12.5. Alternatively, the strong cation exchanger may have an IFN beta binding pH range of about pH 8.0 to about pH 12.0.
  • the cation exchange matrix Prior to loading the IFN beta, the cation exchange matrix may be equilibrated, for example, using several column volumes of a dilute, weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3. Following equilibration, the IFN beta may be added and the column may be washed one to several times, prior to elution of substantially purified IFN beta, also using a weak acid solution such as a weak acetic acid or phosphoric acid solution. For example, approximately 2-4 column volumes of 20 mM acetic acid, pH 3, may be used to wash the column.
  • a weak acid solution such as a weak acetic acid or phosphoric acid solution.
  • substantially purified IFN beta may be eluted by contacting the cation exchanger matrix with a buffer having a sufficiently low pH or ionic strength to displace the IFN beta from the matrix.
  • the pH of the elution buffer may range from about pH 2.5 to about pH 6.0. More specifically, the pH of the elution buffer may range from about pH 2.5 to about pH 5.5, about pH 2.5 to about pH 5.0.
  • the elution buffer may have a pH of about 3.0.
  • the quantity of elution buffer may vary widely and will generally be in the range of about 2 to about 10 column volumes.
  • substantially purified IFN beta polypeptide may be eluted by contacting the matrix with a buffer having a sufficiently high pH or ionic strength to displace the IFN beta polypeptide from the matrix.
  • Suitable buffers for use in high pH elution of substantially purified IFN beta polypeptide may include, but not limited to, citrate, phosphate, formate, acetate, HEPES, and MES buffers ranging in concentration from at least about 5 mM to at least about 100 mM.
  • Reverse-Phase Chromatography RP-HPLC may be performed to purify proteins following suitable protocols that are known to those of ordinary skill in the art.
  • RP-HPLC may be performed on the IFN beta polypeptide to isolate substantially purified IFN beta polypeptide.
  • silica derivatized resins with alkyl functionalities with a wide variety of lengths including, but not limited to, at least about C 3 to at least about C 30 , at least about C 3 to at least about C 20 , or at least about C 3 to at least about C 18 , resins may be used.
  • a polymeric resin may be used.
  • TosoHaas Amberchrome CGlOOOsd resin may be used, which is a styrene polymer resin. Cyano or polymeric resins with a wide variety of alkyl chain lengths may also be used.
  • the RP-HPLC column may be washed with a solvent such as ethanol.
  • the Source RP column is another example of a RP-HPLC column.
  • a suitable elution buffer containing an ion pairing agent and an organic modifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile or ethanol may be used to elute the IFN beta polypeptide from the RP-HPLC column.
  • the most commonly used ion pairing agents include, but are not limited to, acetic acid, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, heptafluorobutyric acid, triethylamine, tetramethylammonium, tetrabutylammonium, and triethylammonium acetate.
  • Elution may be performed using one or more gradients or isocratic conditions, with gradient conditions preferred to reduce the separation time and to decrease peak width. Another method involves the use of two gradients with different solvent concentration ranges. Examples of suitable elution buffers for use herein may include, but are not limited to, ammonium acetate and acetonitrile solutions.
  • Hydrophobic Interaction Chromatography Purification Techniques Hydrophobic interaction chromatography (HIC) may be performed on the IFN beta polypeptide. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90, Amersham Biosciences (Piscataway, NJ) which is incorporated by reference herein.
  • Suitable HIC matrices may include, but are not limited to, alkyl- or aryl- substituted matrices, such as butyl-, hexyl-, octyl- or phenyl-substituted matrices including agarose, cross-linked agarose, sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate) matrices, and mixed mode resins, including but not limited to, a polyethyleneamine resin or a butyl- or phenyl-substituted poly(methacrylate) matrix.
  • Commercially available sources for hydrophobic interaction column chromatography include, but are not limited to, HITRAP ® , HIPREP ® , and HILO AD ® columns (Amersham Biosciences, Piscataway, NJ).
  • the HIC column may be equilibrated using standard buffers known to those of ordinary skill in the art, such as an acetic acid/sodium chloride solution or HEPES containing ammonium sulfate. Ammonium sulfate may be used as the buffer for loading the HIC column. After loading the IFN beta polypeptide, the column may then washed using standard buffers and conditions to remove unwanted materials but retaining the IFN beta polypeptide on the HIC column.
  • standard buffers known to those of ordinary skill in the art, such as an acetic acid/sodium chloride solution or HEPES containing ammonium sulfate. Ammonium sulfate may be used as the buffer for loading the HIC column.
  • the column may then washed using standard buffers and conditions to remove unwanted materials but retaining the IFN beta polypeptide on the HIC column.
  • the IFN beta polypeptide may be eluted with about 3 to about 10 column volumes of a standard buffer, such as a HEPES buffer containing EDTA and lower ammonium sulfate concentration than the equilibrating buffer, or an acetic acid/sodium chloride buffer, among others.
  • a standard buffer such as a HEPES buffer containing EDTA and lower ammonium sulfate concentration than the equilibrating buffer, or an acetic acid/sodium chloride buffer, among others.
  • a decreasing linear salt gradient using, for example, a gradient of potassium phosphate, may also be used to elute the IFN beta molecules.
  • the eluant may then be concentrated, for example, by filtration such as diafiltration or ultrafiltration. Diafiltration may be utilized to remove the salt used to elute the IFN beta polypeptide.
  • the yield of IFN beta pol ypeptide, including substantially purified IFN beta polypeptide, may be monitored at each step described herein using techniques known to those of ordinary skill in the art. Such techniques may also be used to assess the yield of substantially purified IFN beta polypeptide following the last isolation step. For example, the yield of IFN beta polypeptide may be monitored using any of several reverse phase high pressure liquid chromatography columns, having a variety of alkyl chain lengths such as cyano RP-HPLC, C 18 RP-HPLC; as well as cation exchange HPLC and gel filtration HPLC.
  • the yield of IFN beta after each purification step may be at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 99.99%, of the IFN beta in the starting material for each purification step.
  • Purity may be determined using standard techniques, such as SDS-PAGE, or by measuring IFN beta polypeptide using Western blot and ELISA assays.
  • polyclonal antibodies may be generated against proteins isolated from negative control yeast fermentation and the cation exchange recovery. The antibodies may also be used to probe for the presence of contaminating host cell proteins.
  • Vydac C4 RP-HPLC material
  • Vydac C4 RP-HPLC material
  • RP-HPLC material Vydac C4 consists of silica gel particles, the surfaces of which carry C4-alkyl chains. The separation of IFN beta polypeptide from the proteinaceous impurities is based on differences in the strength of hydrophobic interactions. Elution is performed with an acetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLC is performed using a stainless steel column (filled with 2.8 to 3.2 liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluate is acidified by adding trifluoroacetic acid and loaded onto the Vydac C4 column.
  • DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl (DEAE)- groups which are covalently bound to the surface of Sepharose beads.
  • the binding of IFN beta polypeptide to the DEAE groups is mediated by ionic interactions. Acetonitrile and trifluoroacetic acid pass through the column without being retained. After these substances have been washed off, trace impurities are removed by washing the column with acetate buffer at a low pH. Then the column is washed with neutral phosphate buffer and IFN beta polypeptide is eluted with a buffer with increased ionic strength. The column is packed with DEAE Sepharose fast flow.
  • the column volume is adjusted to assure a IFN beta polypeptide load in the range of 3-10 mg IFN beta polypeptide/ml gel.
  • the column is washed with water and equilibration buffer (sodium/potassium phosphate).
  • the pooled fractions of the HPLC eluate are loaded and the column is washed with equilibration buffer.
  • the column is washed with washing buffer (sodium acetate buffer) followed by washing with equilibration buffer.
  • IFN beta polypeptide is eluted from the column with elution buffer (sodium chloride, sodium/potassium phosphate) and collected in a single fraction in accordance with the master elution profile.
  • the eluate of the DEAE Sepharose column is adjusted to the specified conductivity.
  • the resulting drug substance is sterile filtered into Teflon bottles and stored at -7O 0 C.
  • Endotoxins are lipopoly-saccharides (LPSs) which are located on the outer membrane of Gram-negative host cells, such as, for example, Escherichia coli.
  • LPSs lipopoly-saccharides
  • Methods for reducing endotoxin levels are known to one of ordinary skill in the art and include, but are not limited to, purification techniques using silica supports, glass powder or hydroxyapatite, reverse- phase, affinity, size-exclusion, anion-exchange chromatography, hydrophobic interaction chromatography, a combination of these methods, and the like. Modifications or additional methods may be required to remove contaminants such as co-migrating proteins from the polypeptide of interest.
  • Methods for measuring endotoxin levels include, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.
  • LAL Limulus Amebocyte Lysate
  • the EndosafeTM-PTS assay is a colorimetric, single tube system that utilizes cartridges preloaded with LAL reagent, chromogenic substrate, and control standard endotoxin along with a handheld spectrophotometer.
  • Alternate methods include, but are not limited to, a Kinetic LAL method that is turbidmetric and uses a 96 well format.
  • IFN beta protein comprising one or more non-naturally encoded amino acids
  • methods and procedures can be used to assess the yield and purity of a IFN beta protein comprising one or more non-naturally encoded amino acids, including but not limited to, the Bradford assay, SDS-PAGE, silver stained SDS-PAGE, coomassie stained SDS-PAGE, mass spectrometry (including but not limited to, MALDI-TOF) and other methods for characterizing proteins known to one of ordinary skill in the art.
  • Additional methods include, but are not limited to: SDS-PAGE coupled with protein staining methods, immunoblotting, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), liquid chromatography/mass spectrometry, isoelectric focusing, analytical anion exchange, chromatofocusing, and circular dichroism. VIII. Expression in Alternate Systems
  • Induction of expression of the recombinant protein results in the accumulation of a protein containing the unnatural analog.
  • o, m and p-fluorophenylalanines have been incorporated into proteins, and exhibit two characteristic shoulders in the UV spectrum which can be easily identified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa, Anal. Biochem., 284:29 (2000); trifluoromethionine has been used to replace methionine in bacteriophage T4 lysozyme to study its interaction with chitooligosaccharide ligands by 19 F NMR, see, e.g., H. Duewel, E. Daub, V.
  • VaIRS valyl-tRNA synthetase
  • VaIRS can misaminoacylate tRNAVal with Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids are subsequently hydrolyzed by the editing domain.
  • a mutant Escherichia coli strain was selected that has a mutation in the editing site of VaIRS. This edit-defective VaIRS incorrectly charges tRNAVal with Cys.
  • the mutant VaIRS also incorporates Abu into proteins when this mutant Escherichia coli strain is grown in the presence of Abu. Mass spectrometric analysis shows that about 24% of valines are replaced by Abu at each valine position in the native protein.
  • a tRNA may be aminoacylated with a desired amino acid by any method or technique, including but not limited to, chemical or enzymatic aminoacylation.
  • Aminoacylation may be accomplished by aminoacyl tRNA synthetases or by other enzymatic molecules, including but not limited to, ribozymes.
  • the term "ribozyme” is interchangeable with "catalytic RNA.” Cech and coworkers (Cech, 1987, Science, 236:1532- 1539; McCorkle et al., 1987, Concepts Biochem. 64:221-226) demonstrated the presence of naturally occurring RNAs that can act as catalysts (ribozymes).
  • RNA molecules that can catalyze aminoacyl-RNA bonds on their own (2')3'-termini Illangakekare et al., 1995 Science 267:643-647
  • an RNA molecule which can transfer an amino acid from one RNA molecule to another Lihse et al., 1996, Nature 381 :442-444.
  • U.S. Patent Application Publication 2003/0228593 which is incorporated by reference herein, describes methods to construct ribozymes and their use in aminoacylation of tRNAs with naturally encoded and non-naturally encoded amino acids.
  • Substrate-immobilized forms of enzymatic molecules that can aminoacylate tRNAs may enable efficient affinity purification of the aminoacylated products.
  • suitable substrates include agarose, sepharose, and magnetic beads.
  • the production and use of a substrate-immobilized form of ribozyme for aminoacylation is described in Chemistry and Biology 2003, 10:1077-1084 and U.S. Patent Application Publication 2003/0228593, which are incorporated by reference herein.
  • Chemical aminoacylation methods include, but are not limited to, those introduced by Hecht and coworkers (Hecht, S. M. Ace. Chem. Res. 1992, 25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C; Chang, P.; Hecht, S. M. Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin, Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew. Chem. Int. Ed. Engl.
  • Methods for generating catalytic RNA may involve generating separate pools of randomized ribozyme sequences, performing directed evolution on the pools, screening the pools for desirable aminoacylation activity, and selecting sequences of those ribozymes exhibiting desired aminoacylation activity.
  • Ribozymes can comprise motifs and/or regions that facilitate acylation activity, such as a GGU motif and a U-rich region.
  • a GGU motif can facilitate recognition of an amino acid substrate
  • a GGU-motif can form base pairs with the 3' termini of a tRNA.
  • the GGU and motif and U-rich region facilitate simultaneous recognition of both the amino acid and tRNA simultaneously, and thereby facilitate aminoacylation of the 3 1 terminus of the tRNA.
  • Ribozymes can be generated by in vitro selection using a partially randomized r24mini conjugated with tRNA Asn ccc G , followed by systematic engineering of a consensus sequence found in the active clones.
  • An exemplary ribozyme obtained by this method is termed "Fx3 ribozyme" and is described in U.S. Pub. App. No. 2003/0228593, the contents of which is incorporated by reference herein, acts as a versatile catalyst for the synthesis of various aminoacyl-tRNAs charged with cognate non-natural amino acids.
  • Immobilization on a substrate may be used to enable efficient affinity purification of the aminoacylated tRNAs.
  • suitable substrates include, but are not limited to, agarose, sepharose, and magnetic beads.
  • Ribozymes can be immobilized on resins by taking advantage of the chemical structure of RNA, such as the 3'-cis-diol on the ribose of RNA can be oxidized with periodate to yield the corresponding dialdehyde to facilitate immobilization of the RNA on the resin.
  • Various types of resins can be used including inexpensive hydrazide resins wherein reductive amination makes the interaction between the resin and the ribozyme an irreversible linkage. Synthesis of aminoacyl-tRNAs can be significantly facilitated by this on- column aminoacylation technique. Kourouklis et al. Methods 2005; 36:239-4 describe a column-based aminoacylation system.
  • One suitable method is to elute the aminoacylated tRNAs from a column with a buffer such as a sodium acetate solution with 10 mM EDTA, a buffer containing 50 mM N-(2- hydroxyethyl)pi ⁇ erazine-N'-(3-propanesulfonic acid), 12.5 mM KCl, pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).
  • a buffer such as a sodium acetate solution with 10 mM EDTA, a buffer containing 50 mM N-(2- hydroxyethyl)pi ⁇ erazine-N'-(3-propanesulfonic acid), 12.5 mM KCl, pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).
  • the aminoacylated tRNAs can be added to translation reactions in order to incorporate the amino acid with which the tRNA was aminoacylated in a position of choice in a polypeptide made by the translation reaction.
  • Examples of translation systems in which the aminoacylated tRNAs of the present invention may be used include, but are not limited to cell lysates.
  • Cell lysates provide reaction components necessary for in vitro translation of a polypeptide from an input mRNA. Examples of such reaction components include but are not limited to ribosomal proteins, rRNA, amino acids, tRNAs, GTP, ATP, translation initiation and elongation factors and additional factors associated with translation.
  • translation systems may be batch translations or compartmentalized translation.
  • a coupled transcription/translation system may be used. Coupled transcription/translation systems allow for both transcription of an input DNA into a corresponding mRNA, which is in turn translated by the reaction components.
  • An example of a commercially available coupled transcription/translation is the Rapid Translation System (RTS, Roche Inc.).
  • the system includes a mixture containing E. coli lysate for providing translational components such as ribosomes and translation factors.
  • an RNA polymerase is included for the transcription of the input DNA into an mRNA template for use in translation.
  • RTS can use compartmentalization of the reaction components by way of a membrane interposed between reaction compartments, including a supply/waste compartment and a transcription/translation compartment.
  • Aminoacylation of tRNA may be performed by other agents, including but not limited to, transferases, polymerases, catalytic antibodies, multi-functional proteins, and the like.
  • Stephan in Principle 2005 Oct 10; pages 30-33 describes additional methods to incorporate non-naturally encoded amino acids into proteins.
  • Lu et al. in MoI Cell. 2001 Oct;8(4):759-69 describe a method in which a protein is chemically ligated to a synthetic peptide containing unnatural amino acids (expressed protein ligation).
  • Microinjection techniques have also been use incorporate unnatural amino acids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R. Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J. Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty and H. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin. Chem. Biol., 4:645 (2000).
  • a Xenopus oocyte was coinjected with two RNA species made in vitro: an mRNA encoding the target protein with a UAG stop codon at the amino acid position of interest and an amber suppressor tRNA aminoacylated with the desired unnatural amino acid.
  • the translational machinery of the oocyte then inserts the unnatural amino acid at the position specified by UAG.
  • This method has allowed in vivo structure-function studies of integral membrane proteins, which are generally not amenable to in vitro expression systems. Examples include the incorporation of a fluorescent amino acid into tachykinin neurokinin-2 receptor to measure distances by fluorescence resonance energy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.
  • a yeast amber suppressor tRNAPheCUA /phenylalanyl-tRNA synthetase pair was used in a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See, e.g., R. Furter, Protein ScL, 7:419 (1998).
  • Translation systems may be cellular or cell-free, and may be prokaryotic or eukaryotic.
  • Cellular translation systems include, but are not limited to, whole cell preparations such as permeabilized cells or cell cultures wherein a desired nucleic acid sequence can be transcribed to mRNA and the mRNA translated.
  • Cell-free translation systems are commercially available and many different types and systems are well-known. Examples of cell-free systems include, but are not limited to, prokaryotic lysates such as Escherichia coli lysates, and eukaryotic lysates such as wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates and human cell lysates.
  • prokaryotic lysates such as Escherichia coli lysates
  • eukaryotic lysates such as wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates and human cell lysates.
  • Eukaryotic extracts or lysates may be preferred when the resulting protein is glycosylated, phosphorylated or otherwise modified because many such modifications are only possible in eukaryotic systems.
  • Some of these extracts and lysates are available commercially (Promega;
  • Membranous extracts such as the canine pancreatic extracts containing microsomal membranes, are also available which are useful for translating secretory proteins.
  • IFN beta polypeptides comprising a non-naturally encoded amino acid
  • Another approach that may be applied to the expression of IFN beta polypeptides comprising a non-naturally encoded amino acid includes the mRNA-peptide fusion technique. See, e.g., R.
  • an mRNA template linked to puromycin is translated into peptide on the ribosome. If one or more tRNA molecules has been modified, non-natural amino acids can be incorporated into the peptide as well. After the last mRNA codon has been read, puromycin captures the C-terminus of the peptide. If the resulting mRNA-peptide conjugate is found to have interesting properties in an in vitro assay, its identity can be easily revealed from the mRNA sequence. In this way, one may screen libraries of IFN beta polypeptides comprising one or more non-naturally encoded amino acids to identify polypeptides having desired properties.
  • Reconstituted translation systems may also be used. Mixtures of purified translation factors have also been used successfully to translate mRNA into protein as well as combinations of lysates or lysates supplemented with purified translation factors such as initiation factor- 1 (IF-I), IF-2, IF-3 ( ⁇ or ⁇ ), elongation factor T (EF-Tu), or termination factors. Cell-free systems may also be coupled transcription/translation systems wherein DNA is introduced to the system, transcribed into mRNA and the mRNA translated as described in Current Protocols in Molecular Biology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), which is hereby specifically incorporated by reference.
  • IF-I initiation factor- 1
  • IF-2 IF-2
  • IF-3 ⁇ or ⁇
  • EF-Tu elongation factor T
  • RNA transcribed in eukaryotic transcription system may be in the form of heteronuclear RNA (hnRNA) or 5 '-end caps (J- methyl guanosine) and 3 '-end poly A tailed mature mRNA, which can be an advantage in certain translation systems.
  • capped mRNAs are translated with high efficiency in the reticulocyte lysate system.
  • non-natural amino acid polypeptides described herein can be effected using the compositions, methods, techniques and strategies described herein. These modifications include the incorporation of further functionality onto the non- natural amino acid component of the polypeptide, including but not limited to, a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin; an inhibitory ribonucleic
  • compositions, methods, techniques and strategies described herein will focus on adding macromolecular polymers to the non-natural amino acid polypeptide with the understanding that the compositions, methods, techniques and strategies described thereto are also applicable (with appropriate modifications, if necessary and for which one of skill in the art could make with the disclosures herein) to adding other functionalities, including but not limited to those listed above.
  • IFN beta polypeptides of the present invention to modulate biological properties of the IFN beta polypeptide, and/or provide new biological properties to the IFN beta molecule.
  • These macromolecular polymers can be linked to the IFN beta polypeptide via a naturally encoded amino acid, via a non-naturally encoded amino acid, or any functional substituent of a natural or non-natural amino acid, or any substituent or functional group added to a natural or non-natural amino acid.
  • the molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more.
  • the molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da.
  • the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1 ,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. [499]
  • the present invention provides substantially homogenous preparations of polymerrprotein conjugates. "Substantially homogenous" as used herein means that polymer :protein conjugate molecules are observed to be greater than half of the total protein.
  • the polymer:protein conjugate has biological activity and the present "substantially homogenous" PEGylated IFN beta polypeptide preparations provided herein are those which are homogenous enough to display the advantages of a homogenous preparation, e.g., ease in clinical application in predictability of lot to lot pharmacokinetics.
  • the polymer selected may be water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer may be branched or unbranched.
  • the polymer will be pharmaceutically acceptable.
  • polymers include but are not limited to polyalkyl ethers and alkoxy- capped analogs thereof (e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogs thereof, especially polyoxyethylene glycol, the latter is also known as polyethyleneglycol or PEG); polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide and derivatives thereof); polyhydroxyalkyl acrylates; polysialic acids and analogs thereof; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives, e.g., carboxymethyldextran, dextran sulfates,
  • the proportion of polyethylene glycol molecules to protein molecules will vary, as will their concentrations in the reaction mixture.
  • the optimum ratio in terms of efficiency of reaction in that there is minimal excess unreacted protein or polymer
  • the molecular weight of the polyethylene glycol selected and on the number of available reactive groups available As relates to molecular weight, typically the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein. Similarly, branching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polyme ⁇ protein ratio.
  • the term "therapeutically effective amount” refers to an amount which gives the desired benefit to a patient. The amount will vary from one individual to another and will depend upon a number of factors, including the overall physical condition of the patient and the underlying cause of the condition to be treated. The amount of IFN beta polypeptide used for therapy gives an acceptable rate of change and maintains desired response at a beneficial level. A therapeutically effective amount of the present compositions may be readily ascertained by one of ordinary skill in the art using publicly available materials and procedures.
  • the water soluble polymer may be any structural form including but not limited to linear, forked or branched.
  • the water soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water soluble polymers can also be employed.
  • PEG poly(ethylene glycol)
  • PEG is used to describe certain embodiments of this invention.
  • PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods known to those of ordinary skill in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161).
  • PEG polyethylene glycol molecule
  • n 2 to 10,000 and X is H or a terminal modification, including but not limited to, a C 1-4 alkyl, a protecting group, or a terminal functional group.
  • a PEG used in the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH 3 ("methoxy PEG").
  • the PEG can terminate with a reactive group, thereby forming a bifunctional polymer.
  • Typical reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the 20 common amino acids (including but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups present in non-naturally encoded amino acids (including but not limited to, azide groups, alkyne groups).
  • Y may be an amide, carbamate or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the N- terminus) of the polypeptide.
  • Y may be a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine).
  • Y may be a linkage to a residue not commonly accessible via the 20 common amino acids.
  • an azide group on the PEG can be reacted with an alkyne group on the IFN beta polypeptide to form a Huisgen [3+2] cycloaddition product.
  • an alkyne group on the PEG can be reacted with an azide group present in a non-naturally encoded amino acid to form a similar product.
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JP2010506615A JP2010525821A (ja) 2007-05-02 2008-04-30 修飾ＩＦＮβポリペプチドおよびこれらの使用
BRPI0810622-3A BRPI0810622A2 (pt) 2007-05-02 2008-04-30 polipeptídeos de interferon beta modificados e seus usos
EP08747233.8A EP2076533B1 (en) 2007-05-02 2008-04-30 Modified interferon beta polypeptides and their uses
NZ580686A NZ580686A (en) 2007-05-02 2008-04-30 Modified interferon beta polypeptides and their uses
MX2009011870A MX2009011870A (es) 2007-05-02 2008-04-30 Polipeptidos de interferon beta modificados y usos de los mismos.
AU2008247815A AU2008247815B2 (en) 2007-05-02 2008-04-30 Modified interferon beta polypeptides and their uses
CA002685596A CA2685596A1 (en) 2007-05-02 2008-04-30 Modified interferon beta polypeptides and their uses
CN200880014461.7A CN101675071B (zh) 2007-05-02 2008-04-30 经修饰干扰素β多肽和其用途
IL201791A IL201791A (en) 2007-05-02 2009-10-28 Interferon-adapted polypeptides in the cell
HK09111900.7A HK1134822A1 (en) 2007-05-02 2009-12-17 Modified interferon beta polypeptides and their uses

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010131015A1 (en) * 2009-05-15 2010-11-18 Almac Sciences (Scotland) Limited Method of labelling interferons with peg
JP2012511326A (ja) * 2008-12-10 2012-05-24 ザ スクリプス リサーチ インスティチュート 化学的に反応性の非天然アミノ酸を使用するキャリア−ペプチド接合体の製造
JPWO2011024887A1 (ja) * 2009-08-26 2013-01-31 国立大学法人 岡山大学 環状ペプチドを含む結合体およびその作製方法
US9238878B2 (en) 2009-02-17 2016-01-19 Redwood Bioscience, Inc. Aldehyde-tagged protein-based drug carriers and methods of use
US9452222B2 (en) 2010-08-17 2016-09-27 Ambrx, Inc. Nucleic acids encoding modified relaxin polypeptides
US9540438B2 (en) 2011-01-14 2017-01-10 Redwood Bioscience, Inc. Aldehyde-tagged immunoglobulin polypeptides and methods of use thereof
US9567386B2 (en) 2010-08-17 2017-02-14 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
US9732349B2 (en) 2007-12-11 2017-08-15 The Scripps Research Institute In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris
US10266578B2 (en) 2017-02-08 2019-04-23 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof
US11208632B2 (en) 2016-04-26 2021-12-28 R.P. Scherer Technologies, Llc Antibody conjugates and methods of making and using the same

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010525821A (ja) * 2007-05-02 2010-07-29 アンブルックス，インコーポレイテッド 修飾ＩＦＮβポリペプチドおよびこれらの使用
AR080993A1 (es) * 2010-04-02 2012-05-30 Hanmi Holdings Co Ltd Formulacion de accion prolongada de interferon beta donde se usa un fragmento de inmunoglobulina
CN101942026B (zh) * 2010-10-14 2014-08-27 成都正能生物技术有限责任公司 长效干扰素融合蛋白及其用途
KR101838919B1 (ko) * 2015-03-03 2018-03-15 에이비온 주식회사 인간 인터페론-베타 변이체가 접합된 면역사이토카인 및 이의 제조방법
EP3875477A1 (en) 2015-04-17 2021-09-08 Alpine Immune Sciences, Inc. Immunomodulatory proteins with tunable affinities
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EP3454887B1 (en) * 2016-05-13 2021-01-20 Orionis Biosciences BV Targeted mutant interferon-beta and uses thereof
US11129906B1 (en) 2016-12-07 2021-09-28 David Gordon Bermudes Chimeric protein toxins for expression by therapeutic bacteria
WO2019073315A1 (en) 2017-10-09 2019-04-18 Mansour Poorebrahim ANALOGUE PEPTIDE OF INTERFERON-BETA
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WO2023288226A2 (en) * 2021-07-12 2023-01-19 Nektar Therapeutics Polymer engineered forms of interferon-gamma and methods of use

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4462940A (en) 1982-09-23 1984-07-31 Cetus Corporation Process for the recovery of human β-interferon-like polypeptides
US4518584A (en) 1983-04-15 1985-05-21 Cetus Corporation Human recombinant interleukin-2 muteins
US4894330A (en) 1986-12-23 1990-01-16 Cetus Corporation Purification of recombinant beta-interferon incorporating RP-HPLC
US4966843A (en) 1982-11-01 1990-10-30 Cetus Corporation Expression of interferon genes in Chinese hamster ovary cells
US5702699A (en) 1982-09-23 1997-12-30 Cetus Corporation Process for the recovery of lipophilic proteins
US5814485A (en) 1995-06-06 1998-09-29 Chiron Corporation Production of interferon-β (IFN-β) in E. coli
US20020137895A1 (en) 2000-10-27 2002-09-26 Chiron Corporation Methods of protein purification and recovery
US6514729B1 (en) 1999-05-12 2003-02-04 Xencor, Inc. Recombinant interferon-beta muteins
US6531111B1 (en) 1999-05-21 2003-03-11 Micro Therapeutics, Inc. High viscosity embolizing compositions
US6800735B2 (en) 1998-10-16 2004-10-05 Biogen, Inc. Interferon-beta fusion proteins and uses
US20050054053A1 (en) 2002-10-01 2005-03-10 Xencor, Inc. Interferon variants with improved properties
US6887462B2 (en) 2001-04-09 2005-05-03 Chiron Corporation HSA-free formulations of interferon-beta
US6962978B2 (en) 1998-10-16 2005-11-08 Biogen, Inc. Polymer conjugates of interferon beta-1a and uses
US7144574B2 (en) 1999-08-27 2006-12-05 Maxygen Aps Interferon β variants and conjugates

Family Cites Families (303)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) * 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4289872A (en) * 1979-04-06 1981-09-15 Allied Corporation Macromolecular highly branched homogeneous compound based on lysine units
US6410697B1 (en) 1979-04-20 2002-06-25 Schering Corporation Process for purifying human leukocyte interferon
US5554513A (en) * 1979-11-21 1996-09-10 Yeda Research & Development Co. Ltd. Production of recombinant human interferon-beta2
IE57069B1 (en) 1980-04-03 1992-04-22 Biogen Inc Dna sequences,recombinant dna molecules and processes for producing human fibroblast interferon
US6610830B1 (en) 1980-07-01 2003-08-26 Hoffman-La Roche Inc. Microbial production of mature human leukocyte interferons
US4414150A (en) * 1980-11-10 1983-11-08 Genentech, Inc. Hybrid human leukocyte interferons
US4456748A (en) * 1981-02-23 1984-06-26 Genentech, Inc. Hybrid human leukocyte interferons
FR2504010B1 (fr) * 1981-04-15 1985-10-25 Sanofi Sa Medicaments anticancereux contenant la chaine a de la ricine associee a un anticorps antimelanome et procede pour leur preparation
US4551433A (en) * 1981-05-18 1985-11-05 Genentech, Inc. Microbial hybrid promoters
JPS57206622A (en) * 1981-06-10 1982-12-18 Ajinomoto Co Inc Blood substitute
US4485045A (en) * 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4614651A (en) * 1981-07-12 1986-09-30 Damon Biotech, Inc. Interferon epsilon
NZ201705A (en) 1981-08-31 1986-03-14 Genentech Inc Recombinant dna method for production of hepatitis b surface antigen in yeast
US4678751A (en) * 1981-09-25 1987-07-07 Genentech, Inc. Hybrid human leukocyte interferons
US5096705A (en) * 1981-10-19 1992-03-17 Genentech, Inc. Human immune interferon
AU561343B2 (en) * 1981-10-19 1987-05-07 Genentech Inc. Human immune interferon by recombinant dna
US4672108A (en) * 1981-12-07 1987-06-09 Hoffmann-La Roche Inc. Crystalline human leukocyte interferon
JPS58110600A (ja) 1981-12-25 1983-07-01 Kyowa Hakko Kogyo Co Ltd ヒトβ型インタ−フエロン遺伝子を含む組みかえ体プラスミド
US4973479A (en) * 1982-01-15 1990-11-27 Cetus Corporation Interferon-α61
US5098703A (en) * 1982-01-15 1992-03-24 Cetus Corporation Interferon-alpha 76
US4975276A (en) * 1982-01-15 1990-12-04 Cetus Corporation Interferon-alpha 54
EP0098876A1 (en) * 1982-01-19 1984-01-25 Cetus Corporation Multiclass hybrid interferons
EP0088046B1 (de) * 1982-02-17 1987-12-09 Ciba-Geigy Ag Lipide in wässriger Phase
US4671958A (en) * 1982-03-09 1987-06-09 Cytogen Corporation Antibody conjugates for the delivery of compounds to target sites
US6936694B1 (en) * 1982-05-06 2005-08-30 Intermune, Inc. Manufacture and expression of large structural genes
DE3218121A1 (de) 1982-05-14 1983-11-17 Leskovar, Peter, Dr.-Ing., 8000 München Arzneimittel zur tumorbehandlung
US4588585A (en) * 1982-10-19 1986-05-13 Cetus Corporation Human recombinant cysteine depleted interferon-β muteins
US4511502A (en) * 1982-12-22 1985-04-16 Genentech, Inc. Purification and activity assurance of precipitated heterologous proteins
US4512922A (en) * 1982-12-22 1985-04-23 Genentech, Inc. Purification and activity assurance of precipitated heterologous proteins
US4511503A (en) * 1982-12-22 1985-04-16 Genentech, Inc. Purification and activity assurance of precipitated heterologous proteins
US4820352A (en) * 1983-01-10 1989-04-11 Bausch & Lomb Incorporated Cleaning and conditioning solutions for contact lenses and methods of use
US5089398A (en) * 1983-02-22 1992-02-18 Chiron Corporation Enhanced yeast transcription employing hybrid GAPDH promoter region constructs
US4876197A (en) * 1983-02-22 1989-10-24 Chiron Corporation Eukaryotic regulatable transcription
US4755465A (en) * 1983-04-25 1988-07-05 Genentech, Inc. Secretion of correctly processed human growth hormone in E. coli and Pseudomonas
US4859600A (en) * 1983-04-25 1989-08-22 Genentech, Inc. Recombinant procaryotic cell containing correctly processed human growth hormone
US4544545A (en) * 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
GB8317880D0 (en) * 1983-07-01 1983-08-03 Searle & Co Structure and synthesis of interferons
US4689406A (en) * 1983-08-10 1987-08-25 Amgen Enhancement of microbial expression of polypeptides
US4681930A (en) 1983-09-20 1987-07-21 Hoffmann-La Roche Inc. Immune interferon and a method for its extraction and purification
EP0142641B1 (de) * 1983-09-26 1991-01-16 Udo Dr. Ehrenfeld Mittel und Erzeugnis für die Diagnose und Therapie von Tumoren sowie zur Behandlung von Schwächen der zelligen und humoralen Immunabwehr
US4855238A (en) 1983-12-16 1989-08-08 Genentech, Inc. Recombinant gamma interferons having enhanced stability and methods therefor
US4880734A (en) * 1984-05-11 1989-11-14 Chiron Corporation Eukaryotic regulatable transcription
GB8412564D0 (en) * 1984-05-17 1984-06-20 Searle & Co Structure and properties
CA1302320C (en) * 1984-06-11 1992-06-02 Hideo Niwa Expression of human cromosomal interferon-_ in animal cells
JPS6156199A (ja) * 1984-08-27 1986-03-20 Shionogi & Co Ltd 新規ヒトインタ−フエロンα類
US5120832A (en) * 1984-08-27 1992-06-09 Genentech, Inc. Distinct family of human leukocyte interferons
US4542225A (en) * 1984-08-29 1985-09-17 Dana-Farber Cancer Institute, Inc. Acid-cleavable compound
US4675483A (en) * 1984-09-10 1987-06-23 Siemens Aktiengesellschaft Contact arrangement for vacuum switches
US4738921A (en) * 1984-09-27 1988-04-19 Eli Lilly And Company Derivative of the tryptophan operon for expression of fused gene products
US4659839A (en) * 1984-10-10 1987-04-21 Mallinckrodt, Inc. Coupling agents for radiolabeled antibody fragments
US4837148A (en) * 1984-10-30 1989-06-06 Phillips Petroleum Company Autonomous replication sequences for yeast strains of the genus pichia
US4959314A (en) * 1984-11-09 1990-09-25 Cetus Corporation Cysteine-depleted muteins of biologically active proteins
US4885166A (en) * 1985-06-11 1989-12-05 Ciba-Geigy Corporation Hybrid interferons
DE3675588D1 (de) * 1985-06-19 1990-12-20 Ajinomoto Kk Haemoglobin, das an ein poly(alkenylenoxid) gebunden ist.
US4917888A (en) * 1985-06-26 1990-04-17 Cetus Corporation Solubilization of immunotoxins for pharmaceutical compositions using polymer conjugation
US4766106A (en) * 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US4810643A (en) 1985-08-23 1989-03-07 Kirin- Amgen Inc. Production of pluripotent granulocyte colony-stimulating factor
US6004548A (en) 1985-08-23 1999-12-21 Amgen, Inc. Analogs of pluripotent granulocyte colony-stimulating factor
US4680338A (en) * 1985-10-17 1987-07-14 Immunomedics, Inc. Bifunctional linker
AU6543286A (en) 1985-10-25 1987-05-19 Zymogenetics Inc. Method of using bar1 for secreting foreign proteins
US4699784A (en) * 1986-02-25 1987-10-13 Center For Molecular Medicine & Immunology Tumoricidal methotrexate-antibody conjugate
EP0260350B1 (en) 1986-09-05 1992-02-12 Cetus Oncology Corporation Oxidation-resistant interferon-beta muteins and their production; formulations containing such muteins
US5186933A (en) 1986-12-30 1993-02-16 Baylor College Of Medicine Synthesis and immunogenicity of rotavirus genes using a baculovirus expression system
US4908432A (en) * 1987-01-09 1990-03-13 New York University Novel polypeptide having gamma-interferon activity
JPH02502876A (ja) 1987-03-16 1990-09-13 アメリカン　バイオジェネティック　サイエンシズ　インク ワクチンと生物学的殺虫剤における組換えバクロウイルス閉塞体
DE3712564A1 (de) 1987-04-14 1988-11-24 Bioferon Biochem Substanz Verfahren zur konstruktion einer animalen zellinie fuer die herstellung von humanem interferon-beta
US5229490A (en) * 1987-05-06 1993-07-20 The Rockefeller University Multiple antigen peptide system
AU2136788A (en) 1987-07-24 1989-03-01 Cetus Corporation Production of ricin toxins in a baculovirus-insect cell expression system
AU2251488A (en) 1987-07-24 1989-03-01 Cetus Corporation Production of biologically active forms of csf using a baculovirus (acnpv)-insect cell expression system
US5080891A (en) * 1987-08-03 1992-01-14 Ddi Pharmaceuticals, Inc. Conjugates of superoxide dismutase coupled to high molecular weight polyalkylene glycols
US4929555A (en) * 1987-10-19 1990-05-29 Phillips Petroleum Company Pichia transformation
GB8725529D0 (en) 1987-10-30 1987-12-02 Delta Biotechnology Ltd Polypeptides
US4904584A (en) * 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
CA1340772C (en) 1987-12-30 1999-09-28 Patricia Tekamp-Olson Expression and secretion of heterologous protiens in yeast employing truncated alpha-factor leader sequences
US5674706A (en) 1988-05-06 1997-10-07 Chiron Corporation High level expression of proteins in yeast
CA1324969C (en) 1988-05-06 1993-12-07 Jeffrey R. Shuster High level expression of proteins in yeast
FR2631974B1 (fr) * 1988-05-31 1992-12-11 Agronomique Inst Nat Rech Baculovirus modifie, son procede de preparation et son application en tant que vecteur d'expression de genes
AU4197389A (en) 1988-08-05 1990-03-05 Mount Sinai School Of Medicine Of The City University Of New York, The In vivo infection of live insects with a recombinant baculovirus
GB8819453D0 (en) 1988-08-16 1988-09-21 Roy P Production of bluetongue virus non-structural proteins using baculovirus expression vector
NZ230425A (en) 1988-09-02 1992-07-28 Molecular Eng Ass Production of paramyxovirus fusion (f) protein using recombinant baculovirus expression vector
US5218092A (en) * 1988-09-29 1993-06-08 Kyowa Hakko Kogyo Co., Ltd. Modified granulocyte-colony stimulating factor polypeptide with added carbohydrate chains
US5997856A (en) 1988-10-05 1999-12-07 Chiron Corporation Method and compositions for solubilization and stabilization of polypeptides, especially proteins
US6780613B1 (en) 1988-10-28 2004-08-24 Genentech, Inc. Growth hormone variants
WO1990004788A1 (en) 1988-10-28 1990-05-03 Genentech, Inc. Method for identifying active domains and amino acid residues in polypeptides and hormone variants
EP0446299A4 (en) 1988-11-18 1992-05-13 The Regents Of The University Of California Method for site-specifically incorporating unnatural amino acids into proteins
US5047335A (en) 1988-12-21 1991-09-10 The Regents Of The University Of Calif. Process for controlling intracellular glycosylation of proteins
EP0401384B1 (en) 1988-12-22 1996-03-13 Kirin-Amgen, Inc. Chemically modified granulocyte colony stimulating factor
US4902502A (en) * 1989-01-23 1990-02-20 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
AU5187190A (en) 1989-02-23 1990-09-26 University Of Ottawa Improved baculovirus expression system capable of producing foreign gene proteins at high levels
WO1990010277A1 (en) 1989-02-24 1990-09-07 Cell Analysis Systems, Inc. Method and apparatus for determining a proliferation index of a cell sample
US6372206B1 (en) 1989-03-02 2002-04-16 University Of Florida Orally-administered interferon-TAU compositions and methods
US5705363A (en) 1989-03-02 1998-01-06 The Women's Research Institute Recombinant production of human interferon τ polypeptides and nucleic acids
US5122614A (en) * 1989-04-19 1992-06-16 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
US5324844A (en) * 1989-04-19 1994-06-28 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
ATE300519T1 (de) 1989-04-19 2005-08-15 Enzon Inc Verfahren zum herstellen von modifizierten polypeptiden die eine polypeptid und ein polyalkylenoxid enthalten
ATE92107T1 (de) 1989-04-29 1993-08-15 Delta Biotechnology Ltd N-terminale fragmente von menschliches serumalbumin enthaltenden fusionsproteinen.
US5766883A (en) 1989-04-29 1998-06-16 Delta Biotechnology Limited Polypeptides
WO1990014428A1 (en) 1989-05-17 1990-11-29 University Of Georgia Research Foundation, Inc. Improved baculovirus expression vectors
FR2649120B1 (fr) 1989-06-30 1994-01-28 Cayla Nouvelle souche et ses mutants de champignons filamenteux, procede de production de proteines recombinantes a l'aide de ladite souche et souches et proteines obtenues selon ce procede
IL91562A0 (en) 1989-09-07 1990-04-29 Yeda Res & Dev Interferon-gamma receptor fragment and its production
US5889151A (en) 1989-10-20 1999-03-30 Societe Leb-Tech Purified human alpha interferon receptor
US5312808A (en) 1989-11-22 1994-05-17 Enzon, Inc. Fractionation of polyalkylene oxide-conjugated hemoglobin solutions
US5162601A (en) * 1989-11-22 1992-11-10 The Upjohn Company Plant potyvirus expression vector with a gene for protease
US5219564A (en) * 1990-07-06 1993-06-15 Enzon, Inc. Poly(alkylene oxide) amino acid copolymers and drug carriers and charged copolymers based thereon
FR2664905B1 (fr) * 1990-07-18 1994-08-12 Agronomique Inst Nat Rech Baculovirus modifie, son procede d'obtention, et vecteurs d'expression obtenus a partir dudit baculovirus.
WO1992001800A1 (en) 1990-07-20 1992-02-06 Chiron Corporation Method for integrative transformation of yeast using dispersed repetitive elements
EP0541721A4 (en) 1990-08-02 1993-12-29 Chiron Corporation Expression of human cmv glycoprotein-h using the baculovirus-insect cell expression system
WO1992004363A1 (en) * 1990-09-04 1992-03-19 The Salk Institute Biotechnology/Industrial Associates, Inc. Production of insulin-like growth factor-1 in methylotrophic yeast cells
EP0513332A4 (en) 1990-11-14 1993-03-17 Cargill, Incorporated Conjugates of poly(vinylsaccharide) with proteins for the stabilization of proteins
US5252714A (en) * 1990-11-28 1993-10-12 The University Of Alabama In Huntsville Preparation and use of polyethylene glycol propionaldehyde
ATE164395T1 (de) 1990-12-03 1998-04-15 Genentech Inc Verfahren zur anreicherung von proteinvarianten mit geänderten bindungseigenschaften
US5231178A (en) * 1991-01-16 1993-07-27 The Salk Institute Biotechnology/Industrial Associates, Inc. Method for the purification of intact, correctly-folded insulin-like growth factor-1
EP0576589A4 (en) 1991-03-18 1994-07-27 Enzon Inc Hydrazine containing conjugates of polypeptides and glycopolypeptides with polymers
AU1651992A (en) 1991-03-19 1992-10-21 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Expression of influenza nucleoprotein antigens in baculovirus
US5595732A (en) * 1991-03-25 1997-01-21 Hoffmann-La Roche Inc. Polyethylene-protein conjugates
US6013433A (en) 1991-04-26 2000-01-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Baculovirus expression vectors and recombinant antigens for detecting type-specific antibodies to herpes simplex virus
US5281698A (en) * 1991-07-23 1994-01-25 Cetus Oncology Corporation Preparation of an activated polymer ester for protein conjugation
US5290686A (en) * 1991-07-31 1994-03-01 The United States Of America As Represented By The Department Of Health And Human Services Expression of influenza a M2 protein in baculovirus
DE4128319A1 (de) 1991-08-27 1993-03-04 Bioferon Biochem Substanz Neues rekombinantes human-ifn-beta, verfahren zu dessen herstellung und dieses enthaltende pharmazeutische zubereitungen
US5540923A (en) * 1991-12-06 1996-07-30 Landsforeningen Til Kraeftens Bekaemplse Interferon proteins
IT1260468B (it) 1992-01-29 1996-04-09 Metodo per mantenere l'attivita' di enzimi proteolitici modificati con polietilenglicole
FR2686899B1 (fr) 1992-01-31 1995-09-01 Rhone Poulenc Rorer Sa Nouveaux polypeptides biologiquement actifs, leur preparation et compositions pharmaceutiques les contenant.
JPH07505915A (ja) 1992-04-14 1995-06-29 コーネル　リサーチ　ファウンデーション、インコーポレーテッド 樹枝状巨大分子およびその製造法
US5516657A (en) * 1992-05-11 1996-05-14 Cambridge Biotech Corporation Baculovirus vectors for expression of secretory and membrane-bound proteins
ZA933926B (en) * 1992-06-17 1994-01-03 Amgen Inc Polyoxymethylene-oxyethylene copolymers in conjuction with blomolecules
WO1994004193A1 (en) 1992-08-21 1994-03-03 Enzon, Inc. Novel attachment of polyalkylene oxides to bio-effecting substances
US5382657A (en) 1992-08-26 1995-01-17 Hoffmann-La Roche Inc. Peg-interferon conjugates
US5298643A (en) 1992-12-22 1994-03-29 Enzon, Inc. Aryl imidate activated polyalkylene oxides
AU6029594A (en) 1993-01-15 1994-08-15 Enzon, Inc. Factor viii - polymeric conjugates
US5349001A (en) 1993-01-19 1994-09-20 Enzon, Inc. Cyclic imide thione activated polyalkylene oxides
US5321095A (en) 1993-02-02 1994-06-14 Enzon, Inc. Azlactone activated polyalkylene oxides
US5532142A (en) * 1993-02-12 1996-07-02 Board Of Regents, The University Of Texas System Method of isolation and purification of fusion polypeptides
IL104734A0 (en) * 1993-02-15 1993-06-10 Univ Bar Ilan Bioactive conjugates of cellulose with amino compounds
US5441734A (en) 1993-02-25 1995-08-15 Schering Corporation Metal-interferon-alpha crystals
EP0626448A3 (de) 1993-05-26 1998-01-14 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Verfahren zur Herstellung und Reinigung von alpha-Interferon
WO1994028024A1 (en) 1993-06-01 1994-12-08 Enzon, Inc. Carbohydrate-modified polymer conjugates with erythropoietic activity
WO1994029344A1 (en) 1993-06-11 1994-12-22 Pestka Biomedical Laboratories, Inc. Super proteins including interferons and interleukins
WO1995000162A1 (en) 1993-06-21 1995-01-05 Enzon, Inc. Site specific synthesis of conjugated peptides
GB9317618D0 (en) 1993-08-24 1993-10-06 Royal Free Hosp School Med Polymer modifications
US5762939A (en) 1993-09-13 1998-06-09 Mg-Pmc, Llc Method for producing influenza hemagglutinin multivalent vaccines using baculovirus
US5919455A (en) 1993-10-27 1999-07-06 Enzon, Inc. Non-antigenic branched polymer conjugates
US5643575A (en) 1993-10-27 1997-07-01 Enzon, Inc. Non-antigenic branched polymer conjugates
US5491076A (en) 1993-11-01 1996-02-13 The Texas A&M University System Expression of foreign genes using a replicating polyprotein producing virus vector
US5605792A (en) 1993-11-04 1997-02-25 The Ohio State University Research Foundation Infectious bursal disease virus VP2 fusion protein expressed by baculovirus, use as diagnostic
US5951974A (en) 1993-11-10 1999-09-14 Enzon, Inc. Interferon polymer conjugates
EP0730470B1 (en) 1993-11-10 2002-03-27 Enzon, Inc. Improved interferon polymer conjugates
US5446090A (en) 1993-11-12 1995-08-29 Shearwater Polymers, Inc. Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules
FR2715664B1 (fr) 1994-01-31 1996-04-12 Proteine Performance Sa Baculovirus recombinant et son utilisation pour la production d'anticorps monoclonaux.
JP3090586B2 (ja) 1994-03-15 2000-09-25 片倉工業株式会社 システインプロテアーゼ遺伝子欠損バキュロウイルスおよびその製造法並びにこれを利用する有用タンパク質の製造法
US5545723A (en) 1994-03-15 1996-08-13 Biogen Inc. Muteins of IFN-β
US5473034A (en) * 1994-03-18 1995-12-05 Hyogo Prefectural Government Method for producing protein-synthetic polymer conjugate and said conjugate produced thereby
US5629384A (en) 1994-05-17 1997-05-13 Consiglio Nazionale Delle Ricerche Polymers of N-acryloylmorpholine activated at one end and conjugates with bioactive materials and surfaces
US5686110A (en) 1994-06-02 1997-11-11 Enzon, Inc. Water soluble complex of an alkyl or olefinic end capped polyalkylene oxide and a water insoluble substance
US5730990A (en) 1994-06-24 1998-03-24 Enzon, Inc. Non-antigenic amine derived polymers and polymer conjugates
US6403375B1 (en) 1994-08-24 2002-06-11 Boyce Thompson Institute For Plant Research, Inc. Establishment of Trichoplusia ni cell lines in serum-free medium for recombinant protein and baculovirus production
US5650234A (en) 1994-09-09 1997-07-22 Surface Engineering Technologies, Division Of Innerdyne, Inc. Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces
US5871986A (en) 1994-09-23 1999-02-16 The General Hospital Corporation Use of a baculovirus to express and exogenous gene in a mammalian cell
US5766582A (en) 1994-10-11 1998-06-16 Schering Corporation Stable, aqueous alfa interferon solution formulations
US5824784A (en) 1994-10-12 1998-10-20 Amgen Inc. N-terminally chemically modified protein compositions and methods
CA2204726A1 (en) 1994-11-09 1996-12-27 Robin E. Offord Functionalized polymers for site-specific attachment
US5738846A (en) 1994-11-10 1998-04-14 Enzon, Inc. Interferon polymer conjugates and process for preparing the same
IL116085A (en) 1994-12-16 1999-12-31 Ortho Pharma Corp Spray dried erythropoietin
US5932462A (en) 1995-01-10 1999-08-03 Shearwater Polymers, Inc. Multiarmed, monofunctional, polymer for coupling to molecules and surfaces
WO1996025496A1 (en) 1995-02-17 1996-08-22 Instituut Voor Dierhouderij En Diergezondheid (Id-Dlo) PRODUCTION OF BIOLOGICALLY ACTIVE RECOMBINANT BOVINE FOLLICLE STIMULATING HORMONE (REC bFSH) IN THE BACULOVIRUS EXPRESSION SYSTEM
FR2732035B1 (fr) 1995-03-23 1997-05-30 Agronomique Inst Nat Rech Procede de regulation de l'expression d'un gene dans un baculovirus, par un site de fixation d'un recepteur de l'acide retinoique, et vecteur pour la mise en oeuvre du dit procede
US6184344B1 (en) 1995-05-04 2001-02-06 The Scripps Research Institute Synthesis of proteins by native chemical ligation
NZ308772A (en) 1995-05-17 1999-04-29 Du Pont Recombinant baculovirus insecticides
AU5893696A (en) 1995-06-07 1996-12-30 Novo Nordisk A/S Modification of polypeptides
US5672662A (en) 1995-07-07 1997-09-30 Shearwater Polymers, Inc. Poly(ethylene glycol) and related polymers monosubstituted with propionic or butanoic acids and functional derivatives thereof for biotechnical applications
US5840565A (en) 1995-08-22 1998-11-24 The Regents Of The University Of California Methods for enhancing the production of viral vaccines in PKR-deficient cell culture
US5908621A (en) 1995-11-02 1999-06-01 Schering Corporation Polyethylene glycol modified interferon therapy
WO1997026332A1 (en) 1996-01-17 1997-07-24 Schering Corporation Baculovirus expression system for human interleukin 5 receptor and method of screening for interleukin 5 antagonists
US5861279A (en) 1996-01-17 1999-01-19 Schering Corporation Baculovirus expression system for human interleukin 5 receptor and method of screening for interleukin 5 antagonists
US5747639A (en) 1996-03-06 1998-05-05 Amgen Boulder Inc. Use of hydrophobic interaction chromatography to purify polyethylene glycols
KR20000029673A (ko) 1996-08-02 2000-05-25 오르토-맥네일 파마슈티칼, 인코퍼레이티드 단일의공유결합된ｎ-말단수용성폴리머를갖는폴리펩티드
US5980948A (en) 1996-08-16 1999-11-09 Osteotech, Inc. Polyetherester copolymers as drug delivery matrices
US6214966B1 (en) 1996-09-26 2001-04-10 Shearwater Corporation Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
AU5794598A (en) 1996-12-13 1998-07-03 Patricia Tekamp-Olson Method for expression of heterologous proteins in yeast
BR9606270A (pt) 1996-12-18 1998-09-22 Univ Minas Gerais Processo para a produção da proteína do interferon beta-cis humano recombinante e proteína de interferon beta-cis humano recombinante
ATE200030T1 (de) 1997-01-29 2001-04-15 Polymasc Pharmaceuticals Plc Pegylationsverfahren
GB9703406D0 (en) 1997-02-19 1997-04-09 Chiron Spa Expression of heterologous proteins
WO1998047089A1 (en) 1997-04-11 1998-10-22 California Institute Of Technology Apparatus and method for automated protein design
DE19717864C2 (de) 1997-04-23 2001-05-17 Fraunhofer Ges Forschung Humanes rekombinantes Interferon-beta, Verfahren zu seiner Herstellung und seine Verwendung
WO1998048837A1 (en) 1997-04-30 1998-11-05 Enzon, Inc. Polyalkylene oxide-modified single chain polypeptides
US5990237A (en) 1997-05-21 1999-11-23 Shearwater Polymers, Inc. Poly(ethylene glycol) aldehyde hydrates and related polymers and applications in modifying amines
US5965393A (en) 1997-07-01 1999-10-12 National Institute Of Immunology Method for enhancing foreign gene expression in baculovirus expression vector system
US7270809B2 (en) * 1997-07-14 2007-09-18 Bolder Biotechnology, Inc. Cysteine variants of alpha interferon-2
AU751898B2 (en) 1997-07-14 2002-08-29 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins
GB9715660D0 (en) 1997-07-25 1997-10-01 Zeneca Ltd Proteins
AU8688798A (en) 1997-08-05 1999-03-01 Chiron Corporation Novel (pichia pastoris) gene sequences and methods for their use
DE19735593C2 (de) 1997-08-15 1999-08-26 Hepavec Ag Fuer Gentherapie Hüllprotein-modifizierter Baculovirus-Vektor für die Gentherapie
US6090584A (en) 1997-08-21 2000-07-18 University Technologies International Inc. Baculovirus artificial chromosomes and methods of use
US5989868A (en) 1997-09-12 1999-11-23 The Board Of Regents Of The University Of Oklahoma Fusion protein systems designed to increase soluble cytoplasmic expression of heterologous proteins in esherichia coli
PT1015576E (pt) 1997-09-16 2005-09-30 Egea Biosciences Llc Metodo para a sintese quimica completa e montagem de genes e de genomas
WO1999016318A1 (en) 1997-09-26 1999-04-08 Uab Research Foundation Reduced antigenic cells and uses therefor
US6004573A (en) 1997-10-03 1999-12-21 Macromed, Inc. Biodegradable low molecular weight triblock poly(lactide-co-glycolide) polyethylene glycol copolymers having reverse thermal gelation properties
US6201072B1 (en) 1997-10-03 2001-03-13 Macromed, Inc. Biodegradable low molecular weight triblock poly(lactide-co- glycolide) polyethylene glycol copolymers having reverse thermal gelation properties
DE19748489A1 (de) 1997-11-03 1999-05-06 Roche Diagnostics Gmbh Polyethylenglykol-derivatisierte Biomoleküle und deren Verwendung in heterogenen Nachweisverfahren
US6448369B1 (en) 1997-11-06 2002-09-10 Shearwater Corporation Heterobifunctional poly(ethylene glycol) derivatives and methods for their preparation
JP2001526033A (ja) 1997-12-08 2001-12-18 ジェネンテク・インコーポレイテッド ヒトインターフェロン−イプシロンというｉ型インターフェロン
ZA9811070B (en) 1997-12-08 2000-07-03 Genentech Inc Type I interferons.
EP0924298A1 (en) 1997-12-18 1999-06-23 Stichting Instituut voor Dierhouderij en Diergezondheid (ID-DLO) Protein expression in baculovirus vector expression systems
US5985263A (en) 1997-12-19 1999-11-16 Enzon, Inc. Substantially pure histidine-linked protein polymer conjugates
US5981709A (en) 1997-12-19 1999-11-09 Enzon, Inc. α-interferon-polymer-conjugates having enhanced biological activity and methods of preparing the same
CN1292033A (zh) 1998-03-04 2001-04-18 昂尼克斯药物公司 用于遗传物质高通量表达的杆状病毒表达系统和方法
WO1999045026A1 (en) 1998-03-05 1999-09-10 Chiron Corporation Method for increasing the serum half-life of a biologically active molecule
ATE268609T1 (de) 1998-03-12 2004-06-15 Nektar Therapeutics Al Corp Polyethylenglycolderivate mit benachbarten reaktiven gruppen
US6180096B1 (en) 1998-03-26 2001-01-30 Schering Corporation Formulations for protection of peg-interferon alpha conjugates
KR100264953B1 (ko) 1998-04-03 2001-03-02 박현우 재조합 베큘로바이러스, 이의 제조방법 및 이를 포함하는 미생물 살충제
CN100335503C (zh) * 1998-04-28 2007-09-05 应用研究系统Ars股份公司 多元醇干扰素β偶联物
CZ299460B6 (cs) 1998-05-07 2008-08-06 Shire Human Genetic Therapies, Inc. DNA konstrukt, izolovaná molekula nukleové kyseliny, homologne rekombinantní bunka a její použití azpusob produkce G-CSF
US6451986B1 (en) 1998-06-22 2002-09-17 Immunex Corporation Site specific protein modification
US6168932B1 (en) 1998-07-13 2001-01-02 Parker Hughes Institute Recombinant DTctGMCSF fusion toxin in a baculovirus expression vector system
US6472512B1 (en) 1998-07-21 2002-10-29 Human Genome Sciences, Inc. Keratinocyte derived interferon
US6245528B1 (en) 1998-07-28 2001-06-12 Academia Sinica Latent baculovirus expression system
US6368825B1 (en) 1998-08-10 2002-04-09 Academia Sinica Baculovirus containing minimal CMV promoter
ATE258946T1 (de) 1998-08-28 2004-02-15 Gryphon Sciences Verfahren zur herstellung von polyamidketten von genauer länge und deren konjugate mit proteinen
WO2000020032A1 (en) 1998-10-06 2000-04-13 Trustees Of Dartmouth College RECOMBINANT CAT ALLERGEN, Fel dI, EXPRESSED IN BACULOVIRUS FOR DIAGNOSIS AND TREATMENT OF CAT ALLERGY
US6420339B1 (en) 1998-10-14 2002-07-16 Amgen Inc. Site-directed dual pegylation of proteins for improved bioactivity and biocompatibility
US6403312B1 (en) 1998-10-16 2002-06-11 Xencor Protein design automatic for protein libraries
CA2348822A1 (en) 1998-10-30 2000-05-11 Novozymes A/S Glycosylated proteins having reduced allergenicity
US6451346B1 (en) 1998-12-23 2002-09-17 Amgen Inc Biodegradable pH/thermosensitive hydrogels for sustained delivery of biologically active agents
US6433144B1 (en) 1999-01-12 2002-08-13 Viragen, Inc. Compositions of highly-purified natural mixtures of type I Interferon derived from leukocytes and methods
US6350589B1 (en) 1998-12-31 2002-02-26 Viragen, Inc. Compositions of highly-purified natural mixtures of type I interferon derived from leukocytes and methods
US6703225B1 (en) 1999-01-12 2004-03-09 Sumitomo Pharmaceuticals Company, Limited Interferon-α
US6281211B1 (en) 1999-02-04 2001-08-28 Euro-Celtique S.A. Substituted semicarbazides and the use thereof
US6994986B2 (en) 1999-03-17 2006-02-07 The Board Of Trustees Of The Leland Stanford University In vitro synthesis of polypeptides by optimizing amino acid metabolism
JP4707237B2 (ja) 1999-03-17 2011-06-22 ザ ボード オブ トラスティーズ オブ ザ リーランド スタンフォード ジュニア ユニバーシティ 外因性アミノ酸および新規ａｔｐ再生システムを用いたインビトロ高分子生合成の方法
US6342216B1 (en) 1999-03-17 2002-01-29 The Board Of Regents, The University Of Texas System Therapy of cancer by insect cells containing recombinant baculovirus encoding genes
US6337191B1 (en) 1999-03-22 2002-01-08 The Board Of Trustees Of The Leland Stanford Junior University Vitro protein synthesis using glycolytic intermediates as an energy source
CA2370330C (en) 1999-04-16 2009-01-20 Wm. Marsh Rice University Biodegradable poly(propylene fumarate) networks cross linked with poly(propylene fumarate)-diacrylate macromers
JP2003525592A (ja) 1999-06-22 2003-09-02 ユニバシティ オブ メリーランド カレッジ パーク インターフェロンタウ変異体及びその製法
US6261805B1 (en) 1999-07-15 2001-07-17 Boyce Thompson Institute For Plant Research, Inc. Sialyiation of N-linked glycoproteins in the baculovirus expression vector system
US6531122B1 (en) 1999-08-27 2003-03-11 Maxygen Aps Interferon-β variants and conjugates
US6485937B1 (en) 1999-10-15 2002-11-26 The Rockefeller University System for rapid generation of recombinant baculovirus-based expression vectors for silkworm larvae
US6348558B1 (en) 1999-12-10 2002-02-19 Shearwater Corporation Hydrolytically degradable polymers and hydrogels made therefrom
ES2321800T5 (es) 1999-12-22 2017-02-17 Nektar Therapeutics Procedimiento de preparación de ésteres de 1-benzotriazolil carbonato de polímeros solubles en agua
WO2001046291A1 (en) 1999-12-22 2001-06-28 Shearwater Corporation Sterically hindered derivatives of water soluble polymers
US6413507B1 (en) * 1999-12-23 2002-07-02 Shearwater Corporation Hydrolytically degradable carbamate derivatives of poly (ethylene glycol)
US6646110B2 (en) 2000-01-10 2003-11-11 Maxygen Holdings Ltd. G-CSF polypeptides and conjugates
US6602498B2 (en) 2000-02-22 2003-08-05 Shearwater Corporation N-maleimidyl polymer derivatives
AU780270B2 (en) 2000-02-23 2005-03-10 Association Francaise Contre Les Myopathies Treatment of immune diseases
AU2001257577A1 (en) 2000-02-28 2001-09-03 Shearwater Corporation Water-soluble polymer conjugates of artelinic acid
GB0012997D0 (en) 2000-05-26 2000-07-19 Eurogene Limited Gene delivery
US6586207B2 (en) 2000-05-26 2003-07-01 California Institute Of Technology Overexpression of aminoacyl-tRNA synthetases for efficient production of engineered proteins containing amino acid analogues
JP2002030098A (ja) 2000-07-17 2002-01-29 Institute Of Immunology Co Ltd バキュロウィルスの発芽ウイルスからウイルスエンベロープを回収する方法
KR100396983B1 (ko) 2000-07-29 2003-09-02 이강춘 고반응성의 가지 달린 고분자 유도체 및 고분자와 단백질또는 펩타이드의 접합체
JP2004508338A (ja) 2000-09-08 2004-03-18 グリフォン　セラピューティクス，インコーポレーテッド ポリマー修飾合成タンパク質
US7118737B2 (en) * 2000-09-08 2006-10-10 Amylin Pharmaceuticals, Inc. Polymer-modified synthetic proteins
PT1343518E (pt) 2000-11-07 2009-08-03 Novartis Vaccines & Diagnostic Composições de interferão estabilizadas
US6436386B1 (en) 2000-11-14 2002-08-20 Shearwater Corporation Hydroxyapatite-targeting poly (ethylene glycol) and related polymers
TW593427B (en) 2000-12-18 2004-06-21 Nektar Therapeutics Al Corp Synthesis of high molecular weight non-peptidic polymer derivatives
TWI246524B (en) 2001-01-19 2006-01-01 Shearwater Corp Multi-arm block copolymers as drug delivery vehicles
JP2004529633A (ja) 2001-03-15 2004-09-30 メルク　パテント　ゲゼルシャフト　ミット　ベシュレンクテル　ハフトング 低減された免疫原性を有する修飾されたヒトインターフェロンベータ
WO2002079415A2 (en) 2001-03-30 2002-10-10 Lexigen Pharmaceuticals Corp. Reducing the immunogenicity of fusion proteins
WO2002080959A1 (en) 2001-04-03 2002-10-17 Universitair Medisch Centrum Utrecht Method of treatment of hypoxia/ischaemia blutflusswiderstand
MXPA03009566A (es) 2001-04-19 2004-12-06 Scripps Research Inst Metodos y composicion para la produccion de pares trna-aminoaciltrna sintetasa ortogonales.
US6566132B1 (en) 2001-04-26 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Interferon gamma receptor 1 expression
GB0113657D0 (en) 2001-06-05 2001-07-25 Geneprot Inc Improved native chemical ligation with three or more components
US20040138412A1 (en) 2001-09-07 2004-07-15 Paolo Botti Extended native chemical ligation
US6908963B2 (en) 2001-10-09 2005-06-21 Nektar Therapeutics Al, Corporation Thioester polymer derivatives and method of modifying the N-terminus of a polypeptide therewith
JP4758608B2 (ja) 2001-11-07 2011-08-31 ネクター セラピューティックス 分枝ポリマーおよびそれらの結合体
US7884182B2 (en) 2001-11-14 2011-02-08 Amylin Pharmaceuticals, Inc. Chemical peptide ligation with three or more components
JP5424521B2 (ja) * 2001-12-21 2014-02-26 ヒューマン ジノーム サイエンシーズ， インコーポレイテッド アルブミン融合タンパク質
US6716821B2 (en) 2001-12-21 2004-04-06 Immunogen Inc. Cytotoxic agents bearing a reactive polyethylene glycol moiety, cytotoxic conjugates comprising polyethylene glycol linking groups, and methods of making and using the same
ATE437946T1 (de) 2002-02-15 2009-08-15 Univ New York State Res Found Ribozyme mit breiter trna-aminoacylierungswirkung
KR20040104504A (ko) 2002-03-12 2004-12-10 맥시겐 에이피에스 스트로크의 치료를 위한 인터페론 베타-유사 분자
DE60332725D1 (de) 2002-05-30 2010-07-08 Scripps Research Inst Kupferkatalysierte ligierung von aziden und acetylenen
WO2004002500A1 (en) 2002-06-28 2004-01-08 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method of treating autoimmune diseases with interferon-beta and il-2r antagonist
WO2004017921A2 (en) 2002-08-23 2004-03-04 Zymogenetics, Inc. Method for treating inflammatory bowel disease
EP1581631A4 (en) * 2002-10-01 2007-09-05 Xencor Inc INTERFERON VARIANTS HAVING IMPROVED PROPERTIES
KR20050072441A (ko) 2002-10-16 2005-07-11 더 스크립스 리서치 인스티튜트 케토 아미노산의 단백질로의 부위 특이적 도입
ATE445709T1 (de) 2002-10-16 2009-10-15 Scripps Research Inst Glycoproteinsynthese
US7314613B2 (en) * 2002-11-18 2008-01-01 Maxygen, Inc. Interferon-alpha polypeptides and conjugates
EP1583816A4 (en) 2002-12-22 2007-06-13 Scripps Research Inst PROTEIN NETWORKS
UA90654C2 (ru) * 2002-12-26 2010-05-25 Маунтин Вью Фармасьютикалз, Инк. Полимерные конъюгаты интерферона-бета с повышенной биологической активностью
MXPA05006944A (es) 2002-12-26 2005-12-14 Mountain View Pharmaceuticals Conjugados polimericos de interferon-beta cion potencia biologica aumentada.
JP2006518725A (ja) 2003-02-25 2006-08-17 アプライド リサーチ システムズ エーアールエス ホールディング ナームロゼ フェンノートシャップ 脱髄疾患におけるリバビリンとインターフェロンベータの併用
WO2004094593A2 (en) 2003-04-17 2004-11-04 The Scripps Research Institute Expanding the eukaryotic genetic code
WO2005007870A2 (en) 2003-07-07 2005-01-27 The Scripps Research Institute COMPOSITIONS OF ORTHOGONAL LEUCYL-tRNA AND AMINOACYL-tRNA SYNTHETASE PAIRS AND USES THEREOF
WO2005019415A2 (en) 2003-07-07 2005-03-03 The Scripps Research Institute Compositions of orthogonal lysyl-trna and aminoacyl-trna synthetase pairs and uses thereof
US7527943B2 (en) 2003-07-07 2009-05-05 The Scripps Research Institute Compositions of orthogonal glutamyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof
CN100408090C (zh) 2003-07-11 2008-08-06 舍林股份公司 改良的重组人干扰素-β-1b多肽
ES2831379T3 (es) * 2003-10-09 2021-06-08 Ambrx Inc Derivados poliméricos para la modificación selectiva de proteínas
RS52218B (en) 2003-12-11 2012-10-31 Ares Trading S.A. STABILIZED FLUID INTERFERON FORMULATIONS
EP1718664A4 (en) 2004-02-02 2008-08-13 Ambrx Inc MODIFIED HUMAN FOUR FIXED PROPELLANT (4HB) BEAM POLYPEPTIDES, AND USE THEREOF
WO2005091944A2 (en) 2004-03-17 2005-10-06 Eli Lilly And Company Glycol linked fgf-21 compounds
ZA200609412B (en) 2004-05-17 2008-07-30 Ares Trading Sa Hydrogel interferon formulations
EP1765382A1 (en) 2004-06-07 2007-03-28 Nastech Pharmaceutical Company Inc. Intranasal formulations of interferon beta free of stabilizers that are proteins or polypeptides
US7670595B2 (en) 2004-06-28 2010-03-02 Merck Patent Gmbh Fc-interferon-beta fusion proteins
EP1786917A4 (en) * 2004-07-26 2008-05-28 Enzon Pharmaceuticals Inc OPTIMIZED INTERFERON BETA-GEN
KR100781666B1 (ko) 2004-11-02 2007-12-03 신영기 인간 인터페론-베타 변이체
KR101330626B1 (ko) 2004-11-10 2013-11-18 노바티스 백신즈 앤드 다이아그노스틱스 인코포레이티드 탈아미드화된 인터페론-베타
TW200633718A (en) 2004-12-16 2006-10-01 Applied Research Systems Treatment of hepatitis c in the asian population
CA2927595C (en) 2004-12-22 2023-01-31 Ambrx, Inc. Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides
ATE541934T1 (de) 2004-12-22 2012-02-15 Ambrx Inc Zusammensetzungen von aminoacyl-trna-synthetase und verwendungen davon
CN101247821B (zh) 2005-06-03 2013-01-23 Ambrx公司 经改良人类干扰素分子和其用途
DK1937824T3 (da) 2005-08-18 2013-04-02 Ambrx Inc Sammensætninger af tRNA og anvendelser deraf
SG155183A1 (en) 2005-08-26 2009-09-30 Ares Trading Sa Process for the preparation of glycosylated interferon beta
JP2009507003A (ja) 2005-09-01 2009-02-19 アレス トレーディング ソシエテ アノニム 視神経炎の治療
FI20051003A0 (fi) 2005-10-07 2005-10-07 Faron Pharmaceuticals Oy Menetelmä iskemiareperfuusiovaurion tai monielinhäiriön hoitamiseksi tai estämiseksi
JP2010525821A (ja) * 2007-05-02 2010-07-29 アンブルックス，インコーポレイテッド 修飾ＩＦＮβポリペプチドおよびこれらの使用

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4462940A (en) 1982-09-23 1984-07-31 Cetus Corporation Process for the recovery of human β-interferon-like polypeptides
US5702699A (en) 1982-09-23 1997-12-30 Cetus Corporation Process for the recovery of lipophilic proteins
US4966843A (en) 1982-11-01 1990-10-30 Cetus Corporation Expression of interferon genes in Chinese hamster ovary cells
US5376567A (en) 1982-11-01 1994-12-27 Berlex Laboratories, Inc. Expression of interferon genes in Chinese hamster ovary cells
US5795779A (en) 1982-11-01 1998-08-18 Berlex Laboratories, Inc. Human interferon-β (IFN-β) produced in Chinese hamster ovary (CHO) cells
US4518584A (en) 1983-04-15 1985-05-21 Cetus Corporation Human recombinant interleukin-2 muteins
US4894330A (en) 1986-12-23 1990-01-16 Cetus Corporation Purification of recombinant beta-interferon incorporating RP-HPLC
US5814485A (en) 1995-06-06 1998-09-29 Chiron Corporation Production of interferon-β (IFN-β) in E. coli
US6800735B2 (en) 1998-10-16 2004-10-05 Biogen, Inc. Interferon-beta fusion proteins and uses
US6962978B2 (en) 1998-10-16 2005-11-08 Biogen, Inc. Polymer conjugates of interferon beta-1a and uses
US6514729B1 (en) 1999-05-12 2003-02-04 Xencor, Inc. Recombinant interferon-beta muteins
US6531111B1 (en) 1999-05-21 2003-03-11 Micro Therapeutics, Inc. High viscosity embolizing compositions
US7144574B2 (en) 1999-08-27 2006-12-05 Maxygen Aps Interferon β variants and conjugates
US20040115169A1 (en) 2000-10-27 2004-06-17 Chiron Corporation Methods of protein purification and recovery
US20020137895A1 (en) 2000-10-27 2002-09-26 Chiron Corporation Methods of protein purification and recovery
US6887462B2 (en) 2001-04-09 2005-05-03 Chiron Corporation HSA-free formulations of interferon-beta
US20050054053A1 (en) 2002-10-01 2005-03-10 Xencor, Inc. Interferon variants with improved properties

Non-Patent Citations (31)

* Cited by examiner, † Cited by third party
Title
"ACS Symposium Series", vol. 469, 1991, AMERICAN CHEMICAL SOCIETY, article "POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS"
"Nektar Molecular Engineering Catalog", 2003, article "Polyethylene Glycol and Derivatives for Advanced PEGylation", pages: 1 - 17
"Polyethylene Glycol and Derivatives for Biomedical Applications", SHEARWATER CORPORATION'S CATALOG, 2001
"Remington's Pharmaceutical Sciences", 1985
A. FORSTER ET AL., PROC. NATL ACAD. SCI. (USA, vol. 100, 2003, pages 6353
ARAKAWA ET AL., PHARM. RES., vol. 8, no. 3, 1991, pages 285 - 291
BASU ET AL., BIOCONJUGATE CHEM, vol. 17, 2006, pages 618 - 630
CANNELLA ET AL., PNAS, vol. 95, no. 10100, 1998, pages 5
DELGADO ET AL., CRITICAL REVIEWS IN THERAPEUTIC DRUG CARRIER SYSTEMS, vol. 9, 1992, pages 249 - 304
G. T. HENNANSON ET AL.: "IMMOBILIZED AFFINITY LIGAND TECHNIQUES", 1993, ACADEMIC PRESS
GAERTNER ET AL., BIOCONJUG. CHEM., vol. 3, 1992, pages 262 - 268
GAERTNER ET AL., J. BIOL. CHEM., vol. 269, 1994, pages 7224 - 7230
GCNAIN; HAUSER J., MOL. MED., vol. 75, no. 187, 1997, pages 97
GEOGHEGAN, K.; STROH, J., BIOCONJUG. CHERN., vol. 3, 1992, pages 138 - 146
GOEDDEL ET AL., NUCLEIC ACIDS RES., vol. 8, 1980, pages 4057
HARRIS, MACROMOL. CHEM. PHYS., vol. C25, 1985, pages 325 - 373
HASSOUNA ET AL., J. UROLOGY, vol. 130, no. 806, 1983, pages 10
J. CHIN ET AL., SCIENCE, vol. 301, 2003, pages 964 - 7
L. WANG ET AL., SCIENCE, vol. 292, 2001, pages 498 - 500
MURRAY ET AL., J. NEUROSCI., vol. 18, no. 7306, 1998, pages 14
PIKAL, M., BIOPHARM., vol. 3, no. 9, 1990, pages 26 - 30
R. F. TAYLOR: "PROTEIN IMMOBILISATION. FUNDAMENTAL AND APPLICATIONS", 1991, MARCEL DEKKER
ROSTOVTSEV ET AL., ANGEW. CHEM. INT. ED., vol. 41, 2002, pages 2596 - 2599
S. S. WONG: "CHEMISTRY OF PROTEIN CONJUGATION AND CROSSLINKING", 1992, CRC PRESS
SCOUTEN, METHODS IN ENZYMOLOGY, vol. 135, 1987, pages 30 - 65
See also references of EP2076533A4
SPENCER ET AL., J. BIOL. CHEM., vol. 263, 1988, pages 7862 - 7867
TOMOE ET AL., J. ORG. CHEM., vol. 67, 2002, pages 3057 - 3064
WONG ET AL., ENZYME MICROB. TECHNOL., vol. 14, 1992, pages 866 - 874
ZALIPSKY, BIOCONJUGATE CHEM., vol. 6, 1995, pages 150 - 165
ZAPRIANOVA ET AL., MORFOLOGIIA, vol. 112, no. 25, 1997, pages 8

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9732349B2 (en) 2007-12-11 2017-08-15 The Scripps Research Institute In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris
JP2012511326A (ja) * 2008-12-10 2012-05-24 ザ スクリプス リサーチ インスティチュート 化学的に反応性の非天然アミノ酸を使用するキャリア−ペプチド接合体の製造
US9238878B2 (en) 2009-02-17 2016-01-19 Redwood Bioscience, Inc. Aldehyde-tagged protein-based drug carriers and methods of use
US9879249B2 (en) 2009-02-17 2018-01-30 Redwood Bioscience, Inc. Aldehyde-tagged protein-based drug carriers and methods of use
CN102421447A (zh) * 2009-05-15 2012-04-18 阿尔麦克科学（苏格兰）有限公司 用peg标记干扰素的方法
WO2010131015A1 (en) * 2009-05-15 2010-11-18 Almac Sciences (Scotland) Limited Method of labelling interferons with peg
JPWO2011024887A1 (ja) * 2009-08-26 2013-01-31 国立大学法人 岡山大学 環状ペプチドを含む結合体およびその作製方法
US9962450B2 (en) 2010-08-17 2018-05-08 Ambrx, Inc. Method of treating heart failure with modified relaxin polypeptides
US11311605B2 (en) 2010-08-17 2022-04-26 Ambrx, Inc. Methods of treating heart failure and fibrotic disorders using modified relaxin polypeptides
US11786578B2 (en) 2010-08-17 2023-10-17 Ambrx, Inc. Modified relaxin polypeptides and their uses
US9452222B2 (en) 2010-08-17 2016-09-27 Ambrx, Inc. Nucleic acids encoding modified relaxin polypeptides
US9567386B2 (en) 2010-08-17 2017-02-14 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
US10253083B2 (en) 2010-08-17 2019-04-09 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
US11439710B2 (en) 2010-08-17 2022-09-13 Ambrx, Inc. Nucleic acids encoding modified relaxin polypeptides
US10702588B2 (en) 2010-08-17 2020-07-07 Ambrx, Inc. Modified relaxin polypeptides comprising a non-naturally encoded amino acid in the A chain
US10751391B2 (en) 2010-08-17 2020-08-25 Ambrx, Inc. Methods of treatment using modified relaxin polypeptides comprising a non-naturally encoded amino acid
US10183998B2 (en) 2011-01-14 2019-01-22 Redwood Bioscience, Inc. Aldehyde-tagged immunoglobulin polypeptides and methods of use thereof
US9540438B2 (en) 2011-01-14 2017-01-10 Redwood Bioscience, Inc. Aldehyde-tagged immunoglobulin polypeptides and methods of use thereof
US11208632B2 (en) 2016-04-26 2021-12-28 R.P. Scherer Technologies, Llc Antibody conjugates and methods of making and using the same
US11788066B2 (en) 2016-04-26 2023-10-17 R.P. Scherer Technologies, Llc Antibody conjugates and methods of making and using the same
US11185570B2 (en) 2017-02-08 2021-11-30 Bristol-Myers Squibb Company Method of treating cardiovascular disease and heart failure with modified relaxin polypeptides
US11364281B2 (en) 2017-02-08 2022-06-21 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and pharmaceutical compositions thereof
US10266578B2 (en) 2017-02-08 2019-04-23 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof

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