WO1995025170A1 - NOVEL MUTEINS OF IFN-$g(b) - Google Patents
NOVEL MUTEINS OF IFN-$g(b) Download PDFInfo
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- WO1995025170A1 WO1995025170A1 PCT/US1995/003206 US9503206W WO9525170A1 WO 1995025170 A1 WO1995025170 A1 WO 1995025170A1 US 9503206 W US9503206 W US 9503206W WO 9525170 A1 WO9525170 A1 WO 9525170A1
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- leu
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- glu
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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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to muteins of interferon-beta ("IFN- ⁇ ") in which val (V) at position 101, when numbered in accordance with wild type IFN- ⁇ , is substituted with phe (F), trp (W), tyr (Y) or his (H).
- IFN- ⁇ interferon-beta
- Interferons are single chain polypeptides secreted by most animal cells in response to a variety of inducers, including viruses, mitogens and
- Interferons participate in regulation of cell function, and have antiviral, antiproliferative and immunomodulating properties.
- interferons are classified into three major types:
- ⁇ -IFN leukocyte
- IFN- ⁇ fibroblast
- IFN- ⁇ Native IFN- ⁇ is produced primarily by diploid fibroblast cells and in lesser amounts by lymphoblastoid cells.
- IFN- ⁇ is a glycoprotein. Its nucleic acid and amino acid sequences have been determined.
- IFN- ⁇ exhibits various biological and
- IFN- ⁇ immunological activities.
- One of IFN- ⁇ 's biological activities is its antiviral activity. This antiviral activity can be neutralized by antibodies to IFN- ⁇ . See EP-B1-41313. Preparation and purification of antibodies to IFN- ⁇ is described in EP-B1-41313 and the references cited therein. IFN- ⁇ is also able to bind to cells that express interferon receptors, such as Daudi cells or A549 cells.
- IFN- ⁇ has potential applications in immunotherapy, antitumor and anticancer therapies, and antiviral therapies.
- IFN- ⁇ antitumor and anticancer properties of both wild type and recombinant IFN- ⁇ . These include treatment of several malignant diseases such as osteosarcoma, basal cell carcinoma, cervical dysplasia, glioma, acute myeloid leukemia, multiple myeloma and Hodgkin's disease.
- IFN- ⁇ has been shown to cause local tumor regression when injected into subcutaneous tumoral nodules in melanoma and breast carcinoma-affected patients.
- IFN- ⁇ wild-type and recombinant
- viral infections including papilloma viruses, such as genital warts and condylomata of the uterine cervix; viral hepatitis, including acute/chronic hepatitis B and non-A, non-B hepatitis (hepatitis C); herpes genitalis; herpes zoster; herpetic keratitis; herpes simplex; viral encephalitis; cytomegalovirus pneumonia; and in the prophylaxis of rhinovirus.
- papilloma viruses such as genital warts and condylomata of the uterine cervix
- viral hepatitis including acute/chronic hepatitis B and non-A, non-B hepatitis (hepatitis C)
- herpes genitalis herpes zoster
- herpetic keratitis herpes simplex
- IFN- ⁇ in the treatment of multiple sclerosis have also been conducted and IFN- ⁇ is approved for sale in the United States for the treatment of multiple sclerosis.
- This invention provides muteins of IFN- ⁇ wherein the val (V) at position 101, when numbered in accordance with wild type IFN- ⁇ , is substituted with phe (F), tyr (Y), trp (W), or his (H).
- This invention also provides DNA sequences encoding these IFN- ⁇ muteins, recombinant DNA molecules containing those sequences operatively linked to expression control sequences and capable of inducing, in an appropriate host, the expression of the IFN- ⁇ muteins, hosts transformed with those recombinant DNA molecules and pharmaceutical compositions containing the IFN- ⁇ . These compositions are useful in
- Figure 1 depicts the amino acid sequence of the preferred mutein of this invention IFN- ⁇ (phe 101 ) (SEQ ID NO: 1).
- Figure 2 depicts the preferred degenerate DNA sequence encoding IFN- ⁇ (phe 101 ) and the signal sequence of native IFN- ⁇ (SEQ ID NO: 2).
- Figure 3 shows an analysis of IFN- ⁇ (phe 101 ) binding to interferon receptor bearing cells.
- Panels A and B show receptor binding data for 125 I-IFN-ß(phe 101 ) and wild type 125 I-IFN-ß, respectively, to Daudi cells.
- Panels C and D show receptor binding data for 125 I-IFN-ß(phe 101 ) and wild type 125 I-IFN-ß, respectively, to A549 cells.
- Figure 4 shows an analysis of IFN- ⁇ (phe l01 ) and wild type IFN- ⁇ by peptide mapping by
- Figure 5 shows that antibodies to wild type IFN-ß neutralize the activity of IFN- ⁇ (phe 101 ) and wild type IFN- ⁇ .
- wild type IFN- ⁇ means an IFN- ⁇ , whether native or recombinant, having the normally occurring amino acid sequence of native human IFN- ⁇ , as shown, e.g., in EP-B1-41313, Figure 4.
- IFN- ⁇ mutein means a polypeptide wherein the val (V) at position 101, when numbered in accordance with wild type IFN- ⁇ , is
- IFN- ⁇ muteins have an amino acid sequence identical to wild type IFN- ⁇ at the other residues.
- the IFN- ⁇ muteins of this invention may also be characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites in or at the other residues of the native IFN- ⁇ polypeptide chain. In accordance with this invention any such insertions, deletions, substitutions and modifications result in an IFN- ⁇ mutein that retains an antiviral activity that can be at least partially neutralized by antibodies to wild type IFN- ⁇ .
- IFN- ⁇ is known to have three cys residues, at wild-type positions 17, 31 and 141.
- 4,588,585 refers to an IFN- ⁇ mutein in which the cys (C) at position 17 has been substituted with ser (S). This substitution can also be utilized in this
- this invention contemplates an IFN- ⁇ mutein having ser (S) substituted for cys (C) at position 17 and val (V) at position 101 substituted with phe (F), trp (W), tyr (Y), or his (H), preferably phe (F), when numbered in accordance with wild type IFN- ⁇ .
- numbered in accordance with wild type IFN- ⁇ we mean identifying a chosen amino acid with reference to the position at which that amino acid normally occurs in wild type IFN- ⁇ .
- insertions or deletions are made to the IFN- ⁇ mutein, one of skill in the art will appreciate that the val (V) normally occuring at position 101, when numbered in accordance with wild type IFN- ⁇ , may be shifted in position in the mutein.
- the location of the shifted val (V) can be readily determined by inspection and correlation of the flanking amino acids with those flanking val 1Q1 in wild type IFN- ⁇ .
- the IFN- ⁇ muteins of the present invention can be produced by any suitable method known in the art.
- Such methods include constructing a DNA sequence encoding the IFN- ⁇ muteins of this invention and expressing those sequences in a suitable transformed host. This method will produce recombinant muteins of this invention. However, the muteins of this invention may also be produced, albeit less preferably, by chemical synthesis or a combination of chemical
- a DNA sequence is constructed by isolating or synthesizing a DNA sequence encoding the wild type IFN- ⁇ and then changing the codon for val 101 to a codon for phe (F), trp (W), tyr (Y) or his (H), preferably phe (F), by site-specific mutagenesis.
- This technique is well known. See, e.g., Mark et al., "Site-specific Mutagenesis of The Human Fibroblast Interferon Gene", Proc. Natl. Acad. Sci.
- Another method of constructing a DNA sequence encoding the IFN- ⁇ muteins of this invention would be chemical synthesis.
- a gene which encodes the desired IFN- ⁇ mutein may be synthesized by chemical means using an oligonucleotide synthesizer.
- Such oligonucleotides are designed based on the amino acid sequence of the desired IFN- ⁇ mutein, and preferably selecting those codons that are favored in the host cell in which the recombinant mutein will be produced.
- the genetic code is degenerate ⁇ that an amino acid may be coded for by more than one codon.
- Trp (F) is coded for by two codons, TTC or TTT
- tyr (Y) is coded for by TAC or TAT
- his (H) is coded for by CAC or CAT
- Trp (W) is coded for by a single codon, TGG.
- the DNA sequence encoding the IFN- ⁇ mutein of this invention may or may not also include DNA sequences that encode a signal
- Such signal sequence should be one recognized by the cell chosen for expression of the IFN- ⁇ mutein. It may be prokaryotic, eukaryotic or a combination of the two. It may also be the signal sequence of native IFN- ⁇ . The inclusion of a signal sequence depends on whether it is desired to secrete the IFN- ⁇ mutein from the recombinant cells in which it is made. If the chosen cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be encoded and most preferably that the wild-type IFN- ⁇ signal sequence be used.
- Standard methods may be applied to synthesize a gene encoding an IFN- ⁇ mutein according to this invention.
- the complete amino acid sequence may be used to construct a back-translated gene.
- a DNA oligomer containing a nucleotide sequence coding for IFN- ⁇ mutein may be synthesized.
- several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and then ligated.
- the individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
- the DNA sequences encoding an IFN- ⁇ mutein of this invention will be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the IFN- ⁇ mutein in the desired transformed host. Proper assembly may be confirmed by nucleotide sequencing, restriction mapping, and
- expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host/vector combinations may be employed. Useful expression vectors for
- eukaryotic hosts include, for example, vectors
- Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E.coli, including col E1, pCR1, pBR322, pMB9 and their
- plasmids such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages.
- Useful expression vectors for yeast cells include the 2 ⁇ plasmid and derivatives thereof.
- Useful vectors for insect cells include pVL 941. We prefer 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
- expression control sequences may be used in these vectors.
- useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Examples of useful expression control
- sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage lambda, for example PL, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
- Any suitable host may be used to produce the IFN- ⁇ muteins of this invention, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. More particularly, these hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli.
- CHO Chinese hamster ovary
- NS/0 African green monkey cells
- COS 1, COS 7, BSC 1, BSC 40, and BMT 10 African green monkey cells
- human cells as well as plant cells in tissue culture.
- CHO cells and COS 7 cells in cultures and particularly the CHO-DDUKY- ⁇ 1 cell line (infra, pp. 18-19).
- the host must be considered because the vector must replicate in it.
- the vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
- preferred vectors for use in this invention include those that allow the DNA
- amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g., Kaufman, United States Patent 4,470,461, Kaufman and Sharp, "Construction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient
- an expression control sequence a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the IFN- ⁇ mutein of this invention, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this invention, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.
- the IFN- ⁇ muteins obtained according to the present invention may be glycosylated or unglycosylated depending on the host organism used to produce the mutein. If bacteria are chosen as the host then the IFN- ⁇ mutein produced will be unglycosylated.
- Eukaryotic cells will glycosylate the IFN- ⁇ muteins, although perhaps not in the same way as native IFN- ⁇ is glycosylated.
- the IFN- ⁇ mutein produced by the transformed host can be purified according to any suitable method.
- Various methods are known for purifying IFN- ⁇ . See, e.g., United States patents 4,289,689, 4,359,389,
- the biological activity of the IFN- ⁇ muteins of this invention can be assayed by any suitable method known in the art.
- Such assays include antibody
- Such assays also include immunomodulatory assays (see, e.g., United States patent 4,753,795), growth inhibition assays, and measurement of binding to cells that express interferon receptors.
- the IFN- ⁇ mutein of this invention will be administered at a dose approximately paralleling that employed in therapy with wild type native or
- an effective amount of the IFN- ⁇ mutein is preferably administered.
- An "effective amount” means an amount capable of preventing or lessening the severity or spread of the condition or indication being treated. It will be apparent to those of skill in the art that the effective amount of IFN- ⁇ mutein will depend, inter alia, upon the disease, the dose, the administration schedule of the IFN- ⁇ mutein, whether the IFN- ⁇ mutein is administered alone or in conjunction with other therapeutic agents, the serum half-life of the composition, and the general health of the patient.
- the IFN- ⁇ mutein is preferably administered in a composition including a pharmaceutically
- “Pharmaceutically acceptable carrier” means a carrier that does not cause any untoward effect in patients to whom it is administered. Such pharmaceutically acceptable carriers are well known in the art. We prefer human serum albumin.
- the IFN- ⁇ muteins of the present invention can be formulated into pharmaceutical compositions by well known methods. See, e.g., Remington's
- the pharmaceutical composition of the IFN- ⁇ mutein may be formulated in a variety of forms, including liquid, gel, lyophilized, or any other suitable form. The preferred form will depend upon the particular indication being treated and will be
- the IFN- ⁇ mutein pharmaceutical composition may be administered orally, intravenously,
- the preferred mode of administration will depend upon the particular indication being treated and will be
- the pharmaceutical composition of the IFN- ⁇ mutein may be administered in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the IFN- ⁇ mutein, either concurrently or in accordance with any other acceptable treatment schedule. In addition, the IFN- ⁇ mutein pharmaceutical composition may be used as an adjunct to other therapies.
- this invention provides compositions and methods for treating viral infections, cancers or tumors, abnormal cell growth, or for
- immunomodulation in any suitable animal, preferably a mammal, most preferably human.
- Gene therapy applications contemplated include treatment of those diseases in which IFN-ß is expected to provide an effective therapy due to its antiviral activity, e.g., viral diseases, including hepatitis, and particularly HBV, or other infectious diseases that are responsive to IFN- ⁇ or infectious agents sensitive to IFN- ⁇ .
- viral diseases including hepatitis, and particularly HBV
- infectious diseases that are responsive to IFN- ⁇ or infectious agents sensitive to IFN- ⁇ .
- this invention contemplates gene therapy applications for
- Local delivery of IFN-ß using gene therapy may provide the therapeutic agent to the target area while avoiding potential toxicity problems associated with non-specific administration.
- Retrovirus-mediated DNA transfer See, e.g., Kay et al., "In Vivo Gene Therapy Of Hemophilia B:
- DNA viruses include adenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses (preferably herpes simplex virus based vectors), and parvoviruses
- retroviral vectors have been extensively studied and used in a number of gene therapy applications, these vectors are generally unsuited for infecting non-dividing cells. In addition, retroviruses have the potential for oncogenicity.
- Adenoviruses have the advantage that they have a broad host range, can infect quiescent or terminally differentiated cells, such as neurons or hepatocytes, and appear essentially non-oncogenic.
- Adenoviruses do not appear to integrate into the host genome. Because they exist extrachromosomally, the risk of insertional mutagenesis is greatly reduced. Ali et al., supra, p. 373. Adeno-associated viruses exhibit similar advantages as adenoviral-based vectors. However, AAVs exhibit site-specific integration on human chromosome 19. Ali et al., supra, p. 377.
- the IFN-ß mutein-encoding DNA of this invention is used in gene therapy for vascular smooth muscle cell proliferation after arterial injury.
- Injury of the arterial wall results in the migration of smooth muscle cells into the intimal layer of the arterial wall, where they proliferate and synthesize extracellular matrix
- gene therapy with DNA encodng the IFN-ß muteins of this invention is provided to a patient in need thereof, concurrent with, or immediately after coronary balloon angioplasty.
- any suitable gene therapy vector containing IFN-ß mutein DNA may be used in accordance with this embodiment.
- the techniques for constructing such a vector are known. See, e.g., Ohno et al., supra. p. 784; Chang et al., supra, p. 522.
- the coronary balloon angioplasty procedure is well known.
- Introduction of the IFN-ß mutein DNA-containing vector to the target artery site may be accomplished using known techniques, e.g., as described in Ohno et al., supra, p. 784.
- pBG311 plasmid pBG311 as the expression vector.
- a full description of pBG311 is given in 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
- the vector uses the SV40 early promoter, splice signal, and polyadenylation signal and was constructed using pAT153 as backbone.
- a DNA fragment containing the DNA sequence shown in Figure 2 (SEQ ID NO: 2) was cloned into pBG311 and operatively linked to the SV40 early promoter through a DNA sequence encoding the signal sequence for native IFN- ⁇ according to standard protocols.
- the resulting expression vector was designated pBeta-phe.
- the IFN- ⁇ mutein DNA sequence (SEQ ID NO: 2) encodes an IFN- ⁇ mutein having an amino acid sequence identical to wild type IFN- ⁇ except that the val (V) at position 101, numbered in accordance with wild type IFN- ⁇ , is substituted with phe (F).
- the mutein encoded by this sequence is designated IFN- ⁇ (phe 101 ).
- Competent Escherichia coli (SURETM, Stratagene) were transformed with the pBeta-phe plasmid according to standard procedures. Colonies containing the pBeta-phe plasmid (i.e., containing a DNA sequence encoding IFN- ⁇ (phe 101 ) were identified by hybridization to a oligonucleotide probe specific for IFN- ⁇ (phe 101 ) using a standard protocol (Grunstein and Hogness, 1975). Amplification Vector
- the plasmid expresses murine dihydrofolate reductase (DHFR) under the transcriptional control of the Adenovirus 2 (Ad2) major late promoter (MLP).
- a 5' splice site derived from an immunoglobulin variable region gene, is located between the Ad2 MLP and the DHFR coding sequences.
- the SV40 polyadenylation site is present downstream of the DHFR gene.
- the plasmid contains the prokaryotic origin of replication (ori) and tetracycline resistance gene from pBR322. Transformation of a Cell Line
- the CHO-DUKX-B1 DHFR- cell line was cotransformed with the pBeta-phe plasmid and plasmid pAdD26SV(a)-3.
- This cell line was derived from the wild type CHO-K1 cell line by ethyl methanesulfonate and UV irradiation induced mutagenesis. See Chasin and Urlaub, "Isolation Of Chinese Hamster Cell Mutants Deficient In Dihydrofolate Reductase Activity", Proc. Natl. Acad. Sci. USA, 77, pp. 4216-20 (1980).
- Dihydrofolate reductase catalyzes the conversion of folate to tetrahydrofolate.
- Cells without functional DHFR require exogenous ribonucleosides and
- deoxyribonucleosides for growth Inhibition of growth can be induced by methotrexate, a folate analogue, which binds to and inhibits DHFR. Titration of methotrexate, a folate analogue, which binds to and inhibits DHFR.
- methotrexate can lead to methotrexate resistance by amplification of the DHFR gene. (Kaufman & Sharp, 1982, supra). Amplification and increased expression of genes near DHFR often occurs when DHFR is amplified. Therefore, cells resistant to high levels of
- methotrexate often demonstrate increased specific productivity of nearby genes.
- the pBeta-phe plasmid (restricted with Xmn1) and plasmid pAdD26SV(a)-3 (restricted with Stul) were mixed in a ratio of 10:1, respectively.
- the DNA was transformed into CHO-DUKX-B1 DHFR- cells by
- deoxyribonucleosides 10% fetal bovine serum [FBS], 4 mM glutamine
- the medium was then exchanged for ⁇ - medium ( ⁇ MEM base without ribonucleosides and deoxyribonucleosides, 10% FBS, 4 mM glutamine) + 50 nM methotrexate (MTX).
- MTX methotrexate
- the cells were removed by trypsinization and plated at ca. 8 ⁇ 10 5 cells/10 cm tissue culture plate. After 14 days, clones were picked and grown in 96 well tissue culture plates. One clone was expanded into a 12 well tissue culture plate and then 7 days later put into a 6 well tissue culture plate in the presence of 250 nM MTX.
- This clone was expanded into a T75 flask (grown in ⁇ - medium + 250 nM MTX) and then amplified in 750 nM MTX.
- a subclone was picked into a 96 well tissue culture plate, expanded into a 48 well tissue culture plate, then a 6 well tissue culture plate and then a T75 tissue culture flask.
- IFN- ⁇ (phe 101 ) produced by culturing the above subclone (or others similar to it) and then secreted into the culture medium, can be purified by
- CNBr-Sepharose 4B resin (2 g, 7 ml) is prepared by suspending in 1 mM HCl. The gel is washed with 1 mM HCl for 15 min on a scintered glass filter. Anti-IFN- ⁇ mabs (such as B02, Yamasa, Japan) are coupled to CNBr-Sepharose 4B resin by incubating in coupling buffer (100 mM NaHCO 3 , pH 8.3, 500 mM NaCl) for 2 hours at room temperature on a rocker platform. Typically, 1-2 mg IFN- ⁇ mab per ml of resin is coupled, but this amount can be varied.
- coupling buffer 100 mM NaHCO 3 , pH 8.3, 500 mM NaCl
- the unreacted CNBr is blocked with 100 mM Tris-HCl, pH 8, 500 mM NaCl, overnight at 4°C. Alternately, the unreacted CNBr is blocked with 100 mM ethanolamine under substantially the same conditions.
- the coupled resin is washed with three cycles of alternative pH. Each cycle consists of a wash with acetate buffer (100 mM, pH 4) containing 500 mM NaCl followed by a wash with Tris buffer (100 mM, pH 8) containing 500 mM NaCl.
- a 1 cm ⁇ 3 cm column (2.3 ml bed volume) is prepared with the coupled resin.
- the column is
- IFN- ⁇ (phe 101 )-containing samples are diluted 1:3 in equilibration buffer, pH 6.8 and loaded.
- the load is chased with PBS, washed with 20 mM K 2 HPO 4 , 1 M NaCl, pH 6.8, and eluted with 200 mM Na citrate, pH 2.
- the pH of the eluate was adjusted to 6 by diluting the sample with 500 mM Mes, pH 6. Characterization By Peptide Mapping
- IFN- ⁇ (phe 101 ) An IFN- ⁇ (phe 101 ), mutein that had been produced and purified in a different and less preferred manner than described above was characterized by peptide mapping.
- FIG. 4 Panel B shows the corresponding region of a peptide map for wild type IFN- ⁇ with the arrowhead indicating the peptide TVLEEK (SEQ ID NO: 4) .
- the identity of the TFLEEK and TVLEEK were verified by protein sequence analysis. We estimate that the ⁇ -Phe 101 and wild type ⁇ -IFN were greater than 98% pure.
- IFN- ⁇ (phe 101 ) The preparation of IFN- ⁇ (phe 101 ) that was characterized by peptide mapping was analyzed in a Cytopathic Effect (CPE) assay for antiviral activity.
- CPE Cytopathic Effect
- a wild type recombinant IFN- ⁇ standard was prepared in Dulbecco's Modified Eagle Medium (DMEM), 10% FBS, 4mM glutamine at a concentration of 10,000 units/mL and stored in aliquots at -70°C.
- DMEM Dulbecco's Modified Eagle Medium
- FBS FBS
- 4mM glutamine a concentration of 10,000 units/mL
- standard, control and IFN- ⁇ phe samples were diluted in DMEM, 10% FBS, 4mM glutamine in three dilution series:
- A549 cells were added to each well at 10 5 cells/ml, 50 uL per well, in DMEM, 10% FBS, 4mM glutamine and the cells are incubated at 37°C, 5% CO 2 for 15 to 20 hours.
- the plate contents were shaken into a bleach bucket and 100 uL encephalomyocarditis virus (EMC virus) at appropriate dilution in media was added to each well.
- EMC virus encephalomyocarditis virus
- the virus and cells were incubated at 37°C, 5% CO 2 for 30 hours.
- the plate contents were then shaken into a bleach bucket and 0.75% crystal violet dye added to the plates. After 5 to 10 minutes, the plates were washed with distilled water and allowed to dry before being read visually.
- IFN- ⁇ (phe l01 ) was tested in 14 assays in duplicate. Wild type recombinant IFN- ⁇ was used as a standard. Based on these experiments, IFN- ⁇ (phe 101 ) had a specific activity of 4.8 ⁇ 10 8 U/mg with a 95% confidence interval of 3.5-6.7 ⁇ 10 Wild type IFN- ⁇ had a specific activity of approximately 2.0 ⁇ 10 units/mg with a confidence interval of 1.6-2.5 ⁇ 10 . The data in Figure 5 show a similar result.
- the IFN- ⁇ (phe 101 ) used in the CPE assay above was also analyzed for ability to bind to cells that express interferon receptors. For these studies we examined the binding of either wild type 125 I-IFN- ⁇ or 125 I-IFN- ⁇ (phe 101 ) to Daudi cells or A549 cells
- iodinated IFN- ⁇ was determined by the CPE assay, assuming a specific activity of 2 ⁇ 10 units/mg. Normally 5 ng (1 ⁇ L, 300,000 cpm) of iodinated IFN- ⁇ (either alone or in the presence of a 50 fold excess of non-iodinated
- interferon was added to 1.7 mL eppendorf tubes in a total volume of less than 10 ⁇ L.
- the labelled ligand was allowed to bind alone (-) or was competed with unlabeled IFN- ⁇ (phe 101 ), ⁇ 2-IFN ( ⁇ 2), ⁇ -IFN ( ⁇ ) or wild type recombinant IFN- ⁇ (WT).
- Both Daudi cells and A549 cells were used.
- the cells were suspended in DMEM/5% FBS at 2 ⁇ 10 6 cells/mL.
- To the samples of the IFN- ⁇ 0.5 mL of the cell suspension was added.
- the tubes were mixed by inversion and incubated at ambient temperature for 45 minutes.
- the cells were then pelleted at 1000 ⁇ g for 2 min and washed two times with 0.5 mL DMEM/10% FBS. Each wash was followed by a centrifugation step at 1000 ⁇ g for 2 min.
- the cells were resuspended in 0.1 mL, transferred into tubes for counting and binding quantified in a Beckman gamma 407 counter.
- IFN- ⁇ (phe 101 ) is very similar to that of wild type IFN- ⁇ on both cell types. Comparable amounts of wild type 125 I-IFN- ⁇ and 125 I-IFN- ⁇ (phe 101 ) were bound and competed similarly by noniodinated ⁇ -IFN, wild type IFN- ⁇ and IFN- ⁇ (phe 101 ). The binding was not affected by the addition of recombinant human ⁇ -IFN.
- A549 cells (ATCC CCL185) were seeded into 96 well plates at 3 ⁇ 10 4 cells/100 microliter media/well.
- the media used was Dulbecco's Modified Eagle Medium (DMEM), 10% FBS, 4 mM glutamine.
- DMEM Dulbecco's Modified Eagle Medium
- FBS FBS
- 4 mM glutamine The cell containing plates were then incubated at 37°C, 5% CO 2 for
- Sample wells contained either purified recombinant mutein IFN-ß(phe 101 ) or wild type
- Antibodies to wild type IFN-ß(val 101 ) were produced in rabbits immunized with recombinant wild type IFN-ß(val 101 ). Rabbit anti-IFN-ß polyclonal sera was collected from the immunized rabbits at appropriate intervals, pooled and stored until use (Rabbit IFN-ß serum pool 6/25/93; 5 ml/vial, 0.02% azide; ref.
- the media was then aspirated from the cell-containing plates prepared and replaced with aliquots (100 ⁇ l/well) of control, IFN-ß sample or IFN-ß/Ab sample, as appropriate, from the Master plates prepared.
- the cell-containing plates were incubated at 37°C, 5% CO 2 for 16-24 hours.
- the next step was the viral challenge.
- the cell-containing plate contents were then aspirated and 100 ⁇ l of a solution of encephalomyocarditis virus (EMCV) at appropriate dilution was added to each well.
- the virus and cells were incubated at 37°C, 5% CO 2 for 41-45 hours.
- the cell-containing plates were developed using the XTT/PMS colorimetric method.
- a 1 mg/ml XTT (3 ,3-[1-(phenylamino)carbonyl]-3,4-tetrazolium]-bis-(4-methoxy-6-nitro)-benzenesulfonic acid; Sigma) solution was prepared in phosphate buffered saline solution.
- a 1 mg/ml PMS (phenazine methosulfate) solution was prepared in water.
- a PMS/XTT solution was prepared at 1:50.
- the development solution was
- the cell-containing plates were aspirated and washed with 150 ⁇ l/well of phosphate buffered saline solution. Each well then received 150 ⁇ l of
- the plates were incubated at 37°C, 5% CO 2 for 30-60 minutes.
- Figure 5 shows that in the absence of rabbit anti-IFN- ⁇ polyclonal sera, samples of mutein
- IFN- ⁇ (phe 101 ) (closed square; ⁇ ) and of wild type
- IFN- ⁇ (val 101 ) (closed diamond; ⁇ ) protected the A549 cells from the EMCV. This is indicated by the increase in absorbance (indicating more living cells) with increasing mutein IFN- ⁇ or wild type IFN- ⁇
- Figure 5 also shows that in the presence of rabbit anti-IFN- ⁇ polyclonal sera, samples of mutein IFN- ⁇ (phe 101 ) (open square; ⁇ ) and of wild type
- IFN- ⁇ (val 101 ) failed to protect the A549 cells from the EMCV. This is shown by the baseline absorbance value for any mutein IFN-ß or wild type IFN-ß concentration, indicating that almost all A549 cells were dead.
- Figure 5 shows that the antiviral activity of mutein IFN-ß(phe 101 ) was neutralized by antibodies to wild-type IFN-ß (i.e., rabbit anti-IFN-ß antibodies) .
- Both balloons are inflated, and the segment is irrigated with heparinized saline.
- the adenoviral inoculum is instilled for 20 min in the central space of the catheter. The catheter is removed and antegrade blood flow as restored.
- the injured arteries of all pigs are infected with 10 10 plaque-forming units (PFU) per milliliter of an ADV- ⁇ E1 vector containing an insert encoding porcine IFN- ⁇ or with an ADV- ⁇ E1 vector lacking the insert.
- PFU plaque-forming units
- the vessel segments in these pigs are excised 21 or 42 days later. Each artery is processed in an identical manner. The region of instillation between the two double balloons is cut into five cross-sections of identical size. Sections 1 and 4 are fixed in methyl Carnoy and sections 3 and 5 are fixed in
- adenoviral-based Ad- ⁇ El vector direct gene transfer may also be used.
- One suitable construct is a plasmid derived from the RSV backbone, with the RSV-LTR promoter driving expression of IFN- ⁇ , with the SV-40 poly A' signal 3' to the IFN- ⁇ DNA sequence. See, e.g., Gorman et al., Science, 221, pp. 551-53 (1983).
- SEQ ID NO: 2 DNA sequence encoding IFN- ⁇ (phe 101 ),
- SEQ ID NO: 3 Amino acid sequence of peptide TFLEEK.
- SEQ ID NO: 4 Amino acid sequence of peptide TVLEEK.
- E.coli K-12 containing plasmid pBeta-phe (which contains a DNA sequence encoding IFN- ⁇ (phe 101 ) and the native IFN- ⁇ signal sequence) has been
- the deposit was made in accordance with the Budapest Treaty and was deposited at the American Type Culture Collection, Rockville, Maryland, U.S.A. on March 11, 1994. The deposit received the accession number 69584.
Abstract
Description
Claims
Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP95914045A EP0750668B1 (en) | 1994-03-15 | 1995-03-13 | Novel muteins of ifn-beta |
JP52414595A JP3822903B2 (en) | 1994-03-15 | 1995-03-13 | Novel mutant protein of IFN-β |
DE69535883T DE69535883D1 (en) | 1994-03-15 | 1995-03-13 | MUTINS OF IFN-BETA |
DK95914045T DK0750668T3 (en) | 1994-03-15 | 1995-03-13 | New muteins of IFN-beta |
NZ283217A NZ283217A (en) | 1994-03-15 | 1995-03-13 | Ifn-beta muteins and medicaments |
AU21202/95A AU695208B2 (en) | 1994-03-15 | 1995-03-13 | Novel muteins of IFN-beta |
CA002185352A CA2185352C (en) | 1994-03-15 | 1995-03-13 | Novel muteins of ifn-.beta. |
NO19963837A NO318989B1 (en) | 1994-03-15 | 1996-09-13 | New muteins of IFN-beta |
FI963630A FI120356B (en) | 1994-03-15 | 1996-09-13 | New muteins in IFN-beta |
MXPA/A/1996/004073A MXPA96004073A (en) | 1994-03-15 | 1996-09-13 | Novel muteins of ifn-'beta' |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/213,448 US5545723A (en) | 1994-03-15 | 1994-03-15 | Muteins of IFN-β |
US08/213,448 | 1994-03-15 |
Publications (2)
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WO1995025170A1 true WO1995025170A1 (en) | 1995-09-21 |
WO1995025170B1 WO1995025170B1 (en) | 1995-10-05 |
Family
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PCT/US1995/003206 WO1995025170A1 (en) | 1994-03-15 | 1995-03-13 | NOVEL MUTEINS OF IFN-$g(b) |
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US (2) | US5545723A (en) |
EP (1) | EP0750668B1 (en) |
JP (2) | JP3822903B2 (en) |
AT (1) | ATE414152T1 (en) |
AU (1) | AU695208B2 (en) |
CA (1) | CA2185352C (en) |
DE (1) | DE69535883D1 (en) |
DK (1) | DK0750668T3 (en) |
FI (1) | FI120356B (en) |
NO (1) | NO318989B1 (en) |
NZ (2) | NZ283217A (en) |
WO (1) | WO1995025170A1 (en) |
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- 1995-03-13 AU AU21202/95A patent/AU695208B2/en not_active Expired
- 1995-03-13 DK DK95914045T patent/DK0750668T3/en active
- 1995-03-13 AT AT95914045T patent/ATE414152T1/en not_active IP Right Cessation
- 1995-03-13 NZ NZ283217A patent/NZ283217A/en not_active IP Right Cessation
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- 1995-03-13 DE DE69535883T patent/DE69535883D1/en not_active Expired - Lifetime
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Cited By (7)
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US6531122B1 (en) | 1999-08-27 | 2003-03-11 | Maxygen Aps | Interferon-β variants and conjugates |
US7144574B2 (en) | 1999-08-27 | 2006-12-05 | Maxygen Aps | Interferon β variants and conjugates |
US7238344B2 (en) | 1999-08-27 | 2007-07-03 | Maxygen, Inc. | Interferon-β variants and conjugates |
US7338788B2 (en) | 1999-08-27 | 2008-03-04 | Maxygen Aps | Interferon-β variants and conjugates |
US7431921B2 (en) | 2000-04-14 | 2008-10-07 | Maxygen Aps | Interferon beta-like molecules |
EP2080771A2 (en) | 2001-02-27 | 2009-07-22 | Maxygen Aps | New interferon beta-like molecules |
US8114630B2 (en) | 2007-05-02 | 2012-02-14 | Ambrx, Inc. | Modified interferon beta polypeptides and their uses |
Also Published As
Publication number | Publication date |
---|---|
DK0750668T3 (en) | 2009-03-02 |
ATE414152T1 (en) | 2008-11-15 |
DE69535883D1 (en) | 2008-12-24 |
CA2185352C (en) | 2005-02-22 |
NZ283217A (en) | 1998-05-27 |
US6127332A (en) | 2000-10-03 |
NO318989B1 (en) | 2005-05-30 |
NO963837L (en) | 1996-11-14 |
FI963630A0 (en) | 1996-09-13 |
JPH10500563A (en) | 1998-01-20 |
MX9604073A (en) | 1997-12-31 |
FI120356B (en) | 2009-09-30 |
NZ329970A (en) | 2000-01-28 |
EP0750668A1 (en) | 1997-01-02 |
JP2006199711A (en) | 2006-08-03 |
US5545723A (en) | 1996-08-13 |
EP0750668B1 (en) | 2008-11-12 |
AU2120295A (en) | 1995-10-03 |
AU695208B2 (en) | 1998-08-06 |
NO963837D0 (en) | 1996-09-13 |
CA2185352A1 (en) | 1995-09-21 |
FI963630A (en) | 1996-09-13 |
JP3822903B2 (en) | 2006-09-20 |
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