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Patent US6545124 - Peptide linkers for covalent linking polypeptide cell modulators - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsDescribed is a new class of polypeptide cell modulators characterized by being composed of two covalently linked cell modulators in a linear polypeptide sequence. Such dual function polypeptides have new and particularly useful activities when the component polypeptide cell modulators are interferons,...http://www.google.com/patents/US6545124?utm_source=gb-gplus-sharePatent US6545124 - Peptide linkers for covalent linking polypeptide cell modulatorsAdvanced Patent SearchPublication numberUS6545124 B1Publication typeGrantApplication numberUS 09/382,738Publication dateApr 8, 2003Filing dateAug 25, 1999Priority dateDec 2, 1985Fee statusLapsedAlso published asCA1304024C, DE3686397D1, DE3686397T2, EP0225579A2, EP0225579A3, EP0225579B1, US4935233, US5114711Publication number09382738, 382738, US 6545124 B1, US 6545124B1, US-B1-6545124, US6545124 B1, US6545124B1InventorsLeslie David Bell, Keith Graham McCullagh, Alan George PorterOriginal AssigneePharmacia CorporationExport CitationBiBTeX, EndNote, RefManPatent Citations (13), Non-Patent Citations (11), Referenced by (1), Classifications (46), Legal Events (3) External Links: USPTO, USPTO Assignment, EspacenetPeptide linkers for covalent linking polypeptide cell modulators
US 6545124 B1Abstract
Described is a new class of polypeptide cell modulators characterized by being composed of two covalently linked cell modulators in a linear polypeptide sequence. Such dual function polypeptides have new and particularly useful activities when the component polypeptide cell modulators are interferons, lymphokines or cytotoxins which act through different and specific cell receptors to initiate complementary biological activities. Also described is a peptide linker segment comprising a mouse IgG gamma 2 b �hinge� in which the four cysteines of the �hinge� have been converted to serines.
What is claimed is: 1. A peptide linker segment comprising the amino acid sequence;
Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro. Description
This is a continuation of pending U.S. Ser. No. 07/848,887 which was filed on Mar. 10, 1999 abandoned, which is a divisional of U.S. Ser. No. 07/379,509 filed on Jul. 13, 1989, now U.S. Pat. Ser. No. 5,114,711 issued May 19, 1992, which is a divisional of U.S. Ser. No. 06/803,748 filed Dec. 2, 1985, which issued as U.S. Pat. No. 4,935,233 on Jun. 19, 1990, entitled Covalently Linked Polypeptide Cell Modulators.
In mixtures, much lower concentrations of type I and type II interferons can achieve a particular level of response. Several authors have also described IFN-gamma/hLT and IFN-alpha/hLT synergy or related synergies (Lee, S. H. et al. J. Immunol. 133, 1083, 1984; Stone-wolff, D. S. et al. J. Exp. Med. 159, 828, 1984; Williams, T. W. Lymphokine Res. 3, 113, 1984), European Patent Application (EPO 107 498), (EPO 128009).
Additional patent publications have described the primary amino acid sequences of human IFN-gamma (GB 2 107 718 A), the IFN-gamma (IFN X918) described herein (PCT 83/04053), IFN-alphas (U.S. Pat. No. 4,414,150- Aug. 11, 1983) and IFN-beta (e.g. GB 0689 70B; GB 2098996A). A modified IFN-beta (IFN X430) described herein is identical to human fibroblast IFN-beta except that amino acids 36 to 48 inclusive are replaced with amino acids 34 to 46 inclusive from human IFN-alpha 1 (European Patent Application 85105914.7 and (Taniguchi, T. at al. Nature 285, 547, 1980).
R1�L�R2 where R1 is a polypeptide cell modulator with one activity, R2 is a polypeptide cell modulator with a different but complementary activity. By complementary activity is meant activity which enhances or changes the response to another cell modulator. The polypeptide cell modulators are either directly bonded to one another or are each bound to a polypeptide linker segment. Thus L represents a chemical bond or a polypeptide linker segment to which both R1 and R2 are bound, most commonly L is a linear peptide to which R1 and R2 are bound by amide bonds linking the carboxy terminus of R1 to the amino terminus of L and the carboxy terminus of L to the amino terminus of R2. The linking group is generally a polypeptide of between 1 and 500 amino acids in length.
Compounds of this invention are preferably made by genetic engineering techniques. Thus genetic material (DNA) coding for one polypeptide cell regulator, peptide linker segment and the other polypeptide cell regulator is inserted into a suitable vector which is used to transform bacteria, yeast or mammalian cells. The transformed organism is grown and the protein isolated by standard techniques. The resulting product is therefore a new protein which has two complementary cell regulatory regions joined by a peptide linker segment as shown in the formula R1�L�R2, wherein R1 and R2 represent polypeptide cell regulator regions and L represents the peptide linker segment.
FIG. 1 shows the enhanced antiproliferative activity of IFN X601 and a mixture of IFN X198 and IFN X430 against HEp-2 carcinoma cells.
FIG. 3 (Chart 1A) shows the path to construction of the plasmid vector PGC269, which expresses IFN X601.
FIGS. 4 & 5 (Charts 1Aa & 1Ab) show preparation of starting plasmid pAP8.
FIG. 6 (Chart1B) shows the path to construction of the plasmid vector pZZ102, which expresses IFN X603.
FIG. 7 (Chart 2A) shows the ligated DNA duplex coding for the spacer amino acids and used to prepare an intermediate plasmid (pGC262) in the construction of pGC269 and Chart 2B shows the DNA duplex coding for (Ala-Gly-Ser)7, an alternative spacer for linking IFN X918 to IFN X430.
FIGS. 8 & 9 (Chart 3) show the complete nucleotide and amino acid sequence of the IFN X601 gene and IFN X601, respectively.
FIGS. 10 & 11 (Chart 4) show the complete nucleotide and amino acid sequences of the IFN X602 gene and IFN X602, respectively.
FIGS. 12 & 13 (Chart 5) show the complete nucleotide and amino acid sequences of the IFN X603 gene and IFN X603, respectively.
FIGS. 14 & 15 (Chart 6) show the complete nucleotide and amino acid sequences of the IFN X604 gene and IFN X604, respectively.
FIGS. 16 & 17 (Chart 7) show SDS-PAGE analysis of immunoprecipitates of 35S-labeled E. coli extracts made with anti IFN-β anti IFN-α monoclonal antibodies.
FIGS. 18 & 19 (Chart 8) show Western blotting confirmation of co-identity of IFN-β immunoreactivity with IFN X601 36 kd protein.
Specifically included within this cytokine class are the following polypeptides: interleukins 1, 2 and 3, alpha interferons (all types), beta interferon, gamma interferon, lymphotoxin, tumour necrosis factor, epidermal growth factor or urogastrone, B-cell growth factor, insulin like growth factors I & II, bone-derived growth factor, chondrocyte growth factor, T-cell growth factors, endothelial-derived growth factors, nerve growth factor, macrophage-derived growth factor, platelet-derived growth factor, neurotrophic growth factors, transforming growth factor (Type I or II), transforming growth factors, T-cell replacing factor, cartilage-derived growth factor, growth hormone, colony-stimulating factors, insulin, endothelial-cell growth factors, placental lactogen, erthropoietin, plasminogen activators, eye-derived growth factor, prolactin, fibroblast-derived growth factor, relaxin, fibroblast growth factors, thrombin, glial growth factor, transferrin, osteosarcoma-derived growth factor, vasopressin, thymosin, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, calcitonin, adrenal corticotropin, melanocyte stimulating hormone, parathyroid hormone, oxytocin, glucagon, secretin, cholecystokinin, gastrin, angiotensin, angiogenin and the polypeptide releasing factors from the hypothalamus.
R1�L�R2 which is a peptide in which R1 and R2 are regions which have sequences which have the above described polypeptide cell modulator activity and L is a peptide linker segment. Large numbers of variations will produce equivalent results. The invention also encompasses glycosylated proteins which for example are produced as a result of expression in yeast or mammalian cells. Also encompassed are variations in the composition of oligasaccharide chains attached to the protein through specific amino acid glycosylation sites. Such variations can be introduced by expression in cells or organisms of varying type or by modification of amino acid glycosylation sites by genetic enginering techniques.
Another preferred embodiment of the present invention is IFN X601, which is composed of sequentially from the N-terminus 1. IFN gamma in which the N-terminal cys-tyr-cys has been replaced by met (designated IFN X918; Chart 3); 2) a 22 amino acid peptide linker segment coded by synthetic DNA (chart 2A), related to the mouse IgG 2b �hinge� region (Chart 3, amino acids 145 to 167; and Nature 283, 786, 1980), except that the four cysteines are replaced by serines (Chart 3; serine residues 156, 159, 162 and 165); 3) IFN X430, which is identical to human IFN-beta, except that amino acid residues 36 to 48 inclusive are replaced by the equivalent residues from human IFN-alpha 1 (chart 3, residues 202 to 214).
Any suitable peptide linker segment may be used which correctly aligns and separates the two polypeptides comprising the polypeptide cell modulator, for example, the mouse IgG gamma 2b �hinge� region (Nature 283, 786, 1980) with the four cysteines converted to serines (e.g., Chart 3; residues 145 to 167); or a seven times repeated unit coding for alanine-glycine-serine (Chart 2B; and Chart 4; residues 145 to 165) which separates IFN X918 and IFN X430, giving rise to IFN X602 (Chart 4).
Alternatively, any suitable peptide linker segment may be used which results in significant potentiation of biological activity, but preferably the mouse IgG gamma 2b �hinge� with the four cysteines converted to serines. This modified hinge region may be inserted between IFN X918 and hLT (Chart 6).
Expression of polypeptide cell modulators, as in example 2, may be in E.coli K12 HB 101, or other E.coli strain; from any strong promoter and ribosome binding site combination of prokaryotic or eukaryotic origin, but preferably the E.coli strain; from any strong promoter and ribosome binding site combination of prokaryotic or eukaryotic origin, but preferably the E.coli trp promoter minus attenuator (Patent applications EP 130 564 and EP 130 564 A) linked to the following ribosome binding site sequence:
A A G G G T A T C G A T C G A A T G
S.D. I.C.
where S.D. is the Shine Dalgarno region and I.C. is the Initiation codon of IFNsX601, or X602, or X603 or X604.
The novel, polypeptide cell modulators of the present invention can be formulated by methods well known for pharmaceutical compositions, wherein the active chimaeron is combined in admixture with a pharmaceutically acceptable carrier substance, the nature of which depends on the particular mode of administration being used. Remington's Pharmaceutical Sciences by E. W. Martin, hereby incorporated by reference, describes compositions and formulations suitable for delivery of the compounds of the present invention. For instance, parenteral formulations are usually injectable fluids that use physiologically acceptable fluids such as saline, balanced salt solutions, or the like as a vehicle.
The novel, polypeptide cell modulators of the invention may be administered to humans or other animals on whose cells they are effective in various ways such as orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally or subcutaneously. Administration of the polypeptide cell modulators is indicated for patients with malignancies or neoplasms, whether or not immunosuppressed, or in patients requiring immunomodulation, or antiviral treatment. Dosage and dose rates may parallel those employed in conventional therapy with naturally occurring interferons�approximately 105 to 108 antiviral units daily. Dosages significantly above or below these levels may be indicated in long term administration or during acute short term treatment. A novel, polypeptide cell modulators may be combined with other treatments or used in association with other chemotherapeutic or chemopreventive agents for providing therapy against the above mentioned diseases and conditions, or other conditions against which it is effective.
Oligodeoxyribonucleotides were synthesized by the phosphoramidite method (M. H. Caruthers, in �Chemical and Enzymatic Synthesis of Gene Fragments�, ed. H. G. Gasen and A. Lang, Verlag chemie, 1982, p.71) on controlled pore glass (H. Koster et al., Tetrahedron, 1984, 40, 103). fully protected 2′-deoxyribonucleotide 3′-phosphoramidites were synthesized from the protected deoxyribonucleotide and chloro-N, N-(diisopropylamino) methoxyphosphine (L. J. McBride and M. H. Caruthers, Tetrahedron Lett., 1983, 24, 245 and S. A. Adams et al., J. Amer. Chem. Soc., 1983, 105, 661). Controlled pore glass supports were synthesized as described (F. Chow et al., Nuc. Acids Res., 1981, 9, 2807) giving 30-50 umol deoxynucleoside per gram.
After completion of the synthesis, the protecting groups were removed and the oligomer cleaved from the support by sequential treatment with 3% (v/v) dichloroacetic acid/dichloromethane (120s), thiophenol/triethylamine/dioxane 1/1/2v/v) (1 hour) and concentrated ammonia at 70� C. (4hour). The deprotected oligonucleotides were purified either by HPLC on a PartisilR 10 SAX column using a gradient from 1M to 4M triethylammonium acetate pH4.9 at 50� C. or by electrophoresis on a denaturing 15% polyacrylamide gel (pH8.3).
500 pmole aliquots of the oligonucleotides were phosphorylated with 1 unit of T4 induced polynucleotide kinase in 20 ul of a solution containing 1000 pmole [32p] gamma-ATP (2.5 Ci/mMole), 100 uM spermidine, 20 mM DTT, 10 mM MgCl2,50 mM Tris-HCl (pH9.0) and 0.1 mM EDTA for 60 minutes at 37� C. The mixtures were then lyophilized and each oligonucleotide purified in a denaturing 15% polyacrylamide gel (pH8.3). After elution from the gel, the recovery was determined by counting the radioactivity.
Blocks (length 30-50 bases were assembled by combining 25 pmole of each phosphorylated component with equimolar amounts of the unphosphorylated oligomers from the complementary strand. The mixtures were lyophilized and then taken up in 15 ul water and 2 ul 10�ligase buffer (500 mM Tris-HCl pH7.6, 100 mM mgCl2). The blocks were annealed at 90� C. for 2 minutes, then slowly cooled to room temperature (20� C). 2 ul 200 mM DTT and 0.5 ul 10 mM ATP were added to give final concentrations of 20 mM DTT and 250uM ATP in 10 ul. 1.25 units of T4 DNA ligase were also added. After 18 hours at 20� C., the products were purified in a 15% polyacrylamide gel under denaturing conditions.
The final duplexes were then constructed from the single-stranded pieces. 1.5 pmole of each piece was taken and the mixtures lyophilized. Annealing was carried out in 15 ul water and 2 ul 10�ligase buffer at 100� C. for 2 minutes, then slowly cooled to 10� C. 2 ul 200 mM DTT, 0.5ul 10 mM ATP and 1.25 units T4 DNA ligase were added. The reaction was left at 10� C. for 18 hours. The final products were then purified in a 10% native polyacrylamide gel.
A 171 bp chemically synthesized duplex (Chart 2A) coding for the C-terminal 13 amino acids of IFN X918, 22 amino acids of the mouse immunoglobulin gamma 2b �hinge� region (cys- ser) and 20 N-terminal amino acids of IFN X430, was ligated to the BglII to SalI large vector fragment of pJB9 (Chart 1A). The resultant plasmid, pGC 262 (table 1) contains a HindIII site for insertion of the remainder of the IFN X430 gene.
Plasmid pJB9 (Chart 1B) was cut with BGlII and SalI and a 106 bp chemically synthesized duplex, coding for the C-terminal 13 amino acids of IFN X918 (as in Chart 2A); and a single methionine followed by the 21 N-terminal amino acids of human lymphotoxin (Chart 5; residues 132 to 166) was ligated to the BglII to SalI large vector fragment of pJB9 (Chart 1B). The resultant plasmid, pZZ101, contains an NsiI site at hLT codons 20 and 21 (Gray, P. W. et al. Nature 312, 721, 1984) for insertion of the remainder of the hLT gene, i.e.
NsiI SalI
...A T G. C A T. T A G A A G T C G A C...
Plasmid pLT101 was cleaved with NsiI and Sall and the resultant 725bp small fragment was ligated to the NsiI and SalI large vector fragment of ppZZ101 (Chart 1B) to give plasmid pZZ102, which codes for the IFN X918-lymphotoxin polypeptide cell modulator, designated IFN X603 (Chart 5).
Overnight cultures (10 ml.) of transformed bacteria were grown in M9/casamino acids medium (EP 131 816A) supplemented with tryptophan (40 ug/ml) and ampicillin (100 ug/ml). Inocula (0.5 ml.) were added to 50 ml. M9/casamino acids medium containing 100 ug/ml. ampicillin. Growth was continued at 37� C. until the A 670 nm had reached 0.5, at which time the cultures were made 20 ug/ml. with respect to beta�indole acrylic acid in order to induce the synthesis of polypeptide cell modulators. Growth was at 37� C. with vigorous shaking, and samples for biological assay (as described in example 3 below) and electrophoretic analysis were removed at 4 hours after induction.
b) SDS-Polyacrylamide Gel Electrophoresis of Total E.coli Proteins for Estimation of Expressed Protein Content
The volume of cells equivalent to 0.5 optical density units at 670 nm was removed from the culture immediately and at 4 hours after adding IAA, and the bacteria recovered by centrifugation. The cells were immediately resuspended in 50 ul of 60 mM tris-HCl pH6.8, 0.05% bromophenol blue, 5% glycerol, 1% sodium dodecylsulphate, 0.5% 2-mercaptoethanol, heated at 100� C. for 3 min. and quick frozen on dry ice. The boiling-freezing cycles were repeated 2-3 times to reduce the viscosity of the sample before a final boiling 5 minutes prior to loading 7.5 μl on a 15% SDS-polyacrylamide gel (Molecular Cloning, A Laboratory Manual, ibid.). The gel was stained with coomassie brilliant blue and dried. The dried gel was scanned with a Joyce-Loebl �chromascan 3� gel scanner, which computes the percentage of total protein for each polypeptide band.
This polypeptide is absent from cultures of E.coli K12 HB 101 harbouring plasmid pJB9 expressing IFN X918 (�17K) or pIL201 expression IFN X430 (�19K).
Alternatively, lysis without sonication was used as follows. 10 ml. culture was centrifugated and the bacterial pellet resuspended in 2 ml. 30 mM NaCl, 50 mM tris-HClpH 7.5, 0.05 to 1 mg/ml lysozyme. Following incubation at 25� C. for 10 min. and 0� C. for 15-30 min. three freeze-thaw cycles were performed (−70� C.). The supernatant from a 15,000 rpm, 15 min. centrifugation was divided for gel analysis, protein estimation and assay.
The cellular extract prepared as in Example 2 (together with 1 log dilutions to 10−6) was assayed for antiviral activity by monitoring the protection conferred on Vero (African Green Monkey) cells against the cytopathic effect of encephalomyocarditis (EMC) virus infection in an in vitro microplate assay system; for example, Dahl, H. and Degre, M. Acta.Path.Microbiol.Scan., 1380, 863, 1972.
A comparison is made in Table 3 of the antiviral (AV) activity in crude bacterial extracts of IFN X601 and the parental IFNs, derived from equivalent numbers of bacterial cells. IFN X601 consistently exhibited 2.5-3.0 fold higher AV activity than IFN X430 and a 4-6 fold higher AV activity than IFN X918, despite a �2-fold lower level of protein expression (Table 2).
A 1:1 mixture of the separately expressed IFNs X918 and X430 also exhibited a significantly enhanced AV activity, which was 4 fold higher than the value expected if the AV activities of the individual IFNs X918 and X430 were additive (Table 3). This is a reflection of the known synergy between Type I and Type II IFNs (Czarniecki, C. W. et al. J.Virol. 49, 490, 1985; and EP 0107 498).
Antiproliferative (AP) activity was assessed by the ability of the polvpeptide cell modulator to inhibit the replication of Daudi (lymphoblastoid) cells (Horoszewicz et al. Science 206, 1091, 1979). Daudi cells in log phase were cultured for 6 days in 96 well plates in the presence of various dilutions of chimaeron or IFN. The phenol red in the medium changes from red to yellow (more acid) with progressive cell growth. Liquid paraffin was added to prevent pH change on exposure to the atmosphere, and the pH change in the medium measured calorimetrically on a Dynatech plate reader. Inhibition of cell growth is reflected by a corresponding reduction in the colour change.
A comparison is made in Table 4A of the Daudi lymphoblastoid cell antiproliferative activity in crude bacterial extracts of IFN X601 and the parental IFNs derived from equivalent numbers of bacterial cells. Daudi cells are known to be unresponsive to IFN-gamma and in a similar fashion did not respond to the antiproliferative action of IFN X918, being more than 100X less sensitive to IFN X918 than to IFN X430 (Table 4A). By contrast, IFN X601 exhibited similar activity to that of IFN X430. Mixtures of IFN X918 and IFN X430 gave a lower titre than with IFN X430 alone i.e., synergy was not evident. These results are expected as the Daudi cell line is capable of responding to the antiproliferative effect of only the IFN X430 portion of the polypeptide cell rodulator. These results also indicate that the IFN X430 portion of the polypeptide cell modulator is functionally active, contributing to its biological activity (Tables 3 and 4B).
Potentiation of AP activity by mixtures of IFN X918 and IFN X430 is a reflection of the synergy which can be demonstrated between IFN-gamma (equivalent to IFN X918) and IFN X430 and is illustrated by the results presented in Table 5. Where the FIC index (as defined in Table 5) is less than: 0.5, synergy is evident. Maximum synergy was observed at equivalent numbers of antiviral units of IFN-gamma and IFN X430 (10 U/ml). Since the specific activities of IFN-gamma and IFN X430 differ only by a factor of approximately two, similar amounts of IFN protein are also present.
Fibrobiasts are grown to confluence in DMEM/10%FCS (Dulbecco's Modified Eagles Medium) in 96-well tissue culture plates. IFN-gamma or modified IFN is serially diluted in DMEM/0.1% BSA and dilutions are added to the medium on the fibroblasts. The fibroblasts are incubated at 37� C. for a further 3 days and then the medium is removed and the cells are washed once with PBS. Admixtures in Herpes-buffered DMEM of a monoclonal antibody directed against HLA-DR and peroxidase conjugated antibody against mouse IgG, is added to the cells and incubated at room temperature for 2 hours. The cells are washed five times with PBS and then the amount of anti-DR antibody bound to the cells is measured by assaying for bound peroxidase using tetramethyl benzidine (TMB) as a chromogen. The colour generated is measured with a Dynatech� microelisa reader.
96 well microplates (Nunc Immunoplate 1) are coated with a goat anti human beta interferon antibody (Rega Institute). To each well of a microplate, is added 100 microlitre of a 5 microgram/ml solution of immunoglobulin (obtained by a 40% ammonium sulphate precipitation of the interferon antibody) in 0.05 M sodium carbonate buffer, pH 9.8, and incubated for two hours at room temperature. After removal of the well contents, unoccupied binding situes are blocked by incubation with 100 microlitres of phosphate buffered saline containing 0.5% casein (PBS/C), for 30 minutes at room temperature. The plates are then washed six times with phosphate buffered saline containing 0.05% Tween 20 (PBS/T), and stored at +4� C. in a covered moist box until required.
100 microlitres of peroxidase conjugated goat anti-mouse immunoglobin (Sigma a7282, diluted 1/2000 in PBS/T), are added to each well and incubated for thirty minutes at room temperature. The well contents are removed and the plates are washed six times with PBS/T. 100 microlitres of TMB (Tetramethyl benzidine, Sigma, 50 mcg/ml in 0.1N acetate/citrate buffer pH 6.0, containing 0.0022% hydrogen peroxide) are added and incubated for one hour at room temperature. 25 microlitres of 2.5 M sulphuric acid is added to stop the reaction and the optical density read at 450 nm in an automatic plate reader (Titertek Multiscan MC). Data is fed into a computer and the 50% end points determined by linear regression analysis of the logic log transformed data. Corrections are then made to the internal standard included on each plate.
Intererons were labelled by including 35S-methionine in bacterial growth medium and extracts were prepared by treatment by lysozyme and sonication. 35S-labelled E. coli extracts were immunoprecipitated with either monoclonal antibodies directed against IFN-β or IFN-γ and the immunoprecipitates were analyzed by SDS-PAGE.
Chart 8 shows that anti-IFNβ monoclonal antibody detects IFN X430 in lanes A, does not recognize IFN X918 in lanes B and recognizes a �36 kd band in the IFN X601 extract in lanes C. This again demonstrates that a band in the IFN X601 extract which is recognized by anti-IFN-β monoclonal antibody has the predicted MW for the chimaeric protein IFN X601.
Table 10 shows that IFN X603 retains both lymphotoxin and interferon-like activities. Antiproliferative activity against mouse L cells is characteristic of LT activity, while AV, HLA DR and ELISA give characteristic IFN-gammal activities. (HEp-2 antiproliferative activity could be due to IFN-gamma or lymphotoxin/IFN-gamma combination but not to lymphotoxin alone.)
Charts 1Aa and 1Ab illustrate the path to constructing a high level expression vector for IFN-β[β(36-48)→α1(34-46)][cys17→ser17], also referred to as IFNX416, in the host E.coli HB101 (European Patent No. 85105914.7). The starting vector was p1/24C (�4,440bp) which was identical to plasmid p1/24 U.K. Patent 8,102,051, except for the underlined sequences which follows:
Nucleotide Sequence of Trp Promoter Region of IFN-β Expression Plasmid p1/24/C
The technique of �site-directed mutagenesis� (Zoller and Smith, Nucl. Acids Res., 10, 6487, 1982) was employed to introduce two base changes, one each in the IFN-β codons 74 and 75 so as not to change the encoded amino acid sequence. Supercoiled DNA resulting from transcription/ligation was separated from non-ligated DNA in a 1% agarose gel and used to transform E.coli JM101. Total plasmid DNA was prepared.
Mutant DNA bearing a unique XhoI site was separated from non-mutant DNA by XhoI restriction and electrophoresis in 1% agarose. The linear DNA was electroeluted from the agarose (Molecular cloning, A Laboratory Manual, eds. Maniatis et al., p.168, Cold Spring Harbor Laboratories). Following self-ligation of the linear DNA and transformation of E.coli JM101, M13 clones were obtained all of which had a unique XhoI site, one of which was designated mAP3.
The complete IFN-β gene with an XhoI site spanning codons 74-76 was recloned back in pAT153. This generated a vector (pAP4) similar to p1/24� C., except for the changed codons 74 and 75 and the deletion of the �546 base pair BglII-BamHI fragment, originally lying 3′ to the IFN-β coding sequence. The new sequence of the Serine codons 74 and 75 is given in Chart 1Aa.
The �230 bp synthetic DNA fragment, assembled as described above, was cloned in the ClaI-XhoI sites of plasmid pAP4 to give pAP8 (Chart 1Ab), a plasmid expressing IFNX416 in the host E.coli HB101.
Table of Plasmids
Expression vector coding for
EP 85105914.7
IFN X416 gene
U.K. Patent 8,102,051,
Chart 1Aa and 1Ab
Amino acids 19/20
IFN X416 gene plus HindIII
coded by AAG.CTT
instead of AAG.CTC
(pAp8)
pGC262
Intermediate vector in
construction of pCC269 − codes
for IFN-gamma + 22 amino acid
mouse gamma 2b IgG �hinge�
pCC203
Chart 1A and
synthetic human IFN-gamma
PCT 83/04053
synthetic IFN-gamma gene
with DNA coding for N-terminal
Cys-Tyr-Cys deleted and replaced
by Met. (IFN X918)
LT3/1
Charts 1A, 3
synthetic human lymphotoxin
Nature 312, 721,
pGC279
construction of pZZ102; codes
for IFN X918 plus 22 N-terminal
amino acids of lymphotoxin
pZZ102
Charts 1B, 5
IFN X603 gene (IFN X918-met-
lymphotoxin polypeptide
cell modulator).
pGC269
IFN X601 gene.
Molecular Weight and Expression in E. coli of IFN X601
(from polyacrylamide
(% of total bacterial
X918*
13.6 − 15.6 (N = 14.6) X430+ 19,000
12.3 − 17.0 (N = 14.65)
5.4 − 10.0 (N = 7.7) *IFN-gamma with N-terminal cys�tyr�cys deleted and replaced by met (Chart 3) +IFN-beta with amino acids 36 to 48 inclusive replaced by amino acids 34 to 46 inclusive from IFN-alpha 1. N mean. TABLE 3
Antiviral Activity of IFN X601
I.U/ml at 10 A670
IFN X430
IFN X918
X9181 0.59
X918 + X4302 3.47
*IU/ml10 A670 � 10−6. Mean of 3 determinations in 2 separate experiments: 1IFN-gamma with N-terminal Cys-Tyr-Cys replaced by Met (chart 3). 2Approximately 1:1 mixture of each IFN (protein). TABLE 4
A. Daudi lymphoblastoid cells
X918 plus X4301 1.9
B. HEp-2 carcinoma cells
X918 plus X4301 4.9
* Units/ml � 10−4 = dilution of IFN at 50% cell growth inhibition. Mean of 2 determinations. 1Mixture 1:1 w/w TABLE 5
IFN X430/IFN-gamma synergy on HEp-2 carcinoma cells
A. IFN X430
Antiviral Units/ml
(�A� + �B�)
*FIC. Fractional Inhibitory Concentration-Ratio: antiviral units at 50% cell growth inhibition of a given IFN (e.g. �A�) in combination with another IFN 9(e.g. �B�) to antiviral units of IFN-�A� alone. Concentration of IFN alone or in combination required to produce 50% inhibition of HEp-2 growth. Synergy is present when FIC index is equal to or less than 0.5 TABLE 6
ACTIVITY (LOG UNITS/ML)
IFN X430 (= beta)
Interferon X601
Mixture of A and B (1:1)
Notes 1. E represents the 50% end points 2. F represents the corrected activities 3. ND is not detectable activity TABLE 7
COMPETITION BY IFN X601 FOR THE BINDING
OF 125 I-IFN alpha 2 TO DAUDI CELL RECEPTORS
Activity Log U/ml.*
* IFN α2 antiviral unit equivalents. The activity in each sample was calculated by interpolation from a standard dose curve of the competition by IFN α2 for the binding of 125 I-IFNα2. TABLE 8
MONOCLONAL ANTIBODY AFFINITY PURIFICATION
OF CRUDE LYSATES OF IFN X601
IFN Activity*
Hep-2**
Anti IFN-Beta Column
Anti IFN Gamma Column
*Log units/ml = dilution of IFN at 50% assay end point. **Enhanced antiproliferative activity seen. TABLE 9
BIOLOGICAL ACTIVITY OF IFN X602 COMPARED WITH IFN X601
Lung Fi-
Antiviral plus Beta and Gamma ELISA activities expressed as Log IU/ml/10 A670. Antiproliferative, HLA DR and Mixed ELISA activities expressed as Log dilution/ml/10 A670 at 50% end point. 1. Assayed in presence of anti IFN beta monoclonal antibody to overcome inhibitory activity of the X430 domain. *Enhanced growth inhibitory activity typical of IFN gamma/IFN X430 mixtures. TABLE 10
BIOLOGICAL ACTIVITY OF IFN X603
HLA DR Induction
EMC/Vero
Antiviral and Gamma ELISA activities expressed as Log IU/ml/10 A670. Antiproliferative and HLA DR activities expressed as Log dilution/ml/10 A670 at 50% end point. Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS4468382Jul 15, 1982Aug 28, 1984New England Medical Center, Inc.Polypeptide-toxin hybrid proteinUS4691009Dec 26, 1984Sep 1, 1987Repligen CorporationHybrid proteins produced by an ultrahigh prokaryotic expression systemUS4734491Aug 31, 1984Mar 29, 1988University Patents, Inc.DNA sequences encoding hybrid lymphoblastoid-leukocyte human interferonsUS4758428Jul 15, 1985Jul 19, 1988Cetus CorporationMulticlass hybrid interferonsUS4816567 *Apr 8, 1983Mar 28, 1989Genentech, Inc.Recombinant immunoglobin preparationsUS4921699 *Oct 14, 1988May 1, 1990Hoffman-La Roche Inc.Polypeptides having interferon activityUS5292668 *Dec 5, 1990Mar 8, 1994Boston Biomedical Research Institute, Inc.Bispecific antibody determinantsUSRE32637Jul 3, 1986Apr 5, 1988Asahi Kasei Kogyo Kabushiki KaishaPhysiologically active peptideEP0141484A2Jun 5, 1984May 15, 1985Biogen, Inc.Methods of producing hybrid DNA sequences and hybrid polypeptides and DNA sequences produced by themEP0158198A1Mar 23, 1985Oct 16, 1985Takeda Chemical Industries, Ltd.DNA and use thereofEP0237019A2Mar 10, 1987Sep 16, 1987Toray Industries, Inc.Interferon conjugate and production thereof using recombinant geneWO1983002461A1Jan 18, 1983Jul 21, 1983Cetus CorpMulticlass hybrid interferonsWO1985002198A1Oct 30, 1984May 23, 1985AmgenMicrobial expression of type i transforming growth factor, polypeptide analogs thereof and hybrid egf/tgf polypeptides* Cited by examinerNon-Patent CitationsReference1 *Adlersberg et al. Proc. Natl. Acad. Sci. USA 1975;73(2):723-727.*2 *de Preval et al. Nature 1970;228:930-932.*3Hiramatsu et al. Recombinant Plasmid Containing Human Urokinase etc., Chem. Abstract; pp. 159 1987, Abstract #106:62196j.4 *Ito et al. Biochem. 1985; 24:6467-6474.*5 *Ngo et al. in "The Protein Folding Problem and Tertiary Structure Prediction" K. Merz, Jr and S. Le Grand, ed; Birkhauser Boston 1994; see pp. 14-491-495.*6 *Oi et al. Nature Jan. 1984; 307:136-140.*7Rudinger, "Characteristics of the Amino Acids as etc." in Peptide Hormones ed J.A. Pargone, University Park Press 1976, pp. 1-7.8Shepard et al. "A Single Amino Acid Change in IFN-BETA1 etc." Nature vol. 294 pp. 563-565 1981.9 *Takayasu et al. Biochem. Biophys. Res. Com. 1982;105(3):1066-71.*10 *Wolfenstein-Todel et al. Biochem. 1972;11(21):3971-5.*11 *Yamawaki-Kataoka at el. Nature 283:786-789 1980.** Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS7790870 *Nov 24, 2000Sep 7, 2010Heinrich-Pette-InstitutMembrane-anchored GP41 peptides that inhibit subsequent HIV viral entry* Cited by examinerClassifications U.S. Classification530/326, 530/300, 530/387.1, 530/387.3International ClassificationC12N15/09, C07K14/54, C07H21/04, C12N15/00, C12N1/21, C07K19/00, C12P21/00, A61K38/22, A61P37/00, A61P35/00, C12R1/19, C07K14/525, C07K14/00, A61K38/21, C07K14/555, A61K38/00, A61P31/12, C12P21/02, C12N15/70, C07K14/565, C07K14/57, C07K14/475, C12N15/62, C07K14/52Cooperative ClassificationY10S930/142, Y10S930/143, C12N15/62, C07K2319/55, C07K14/57, C07K14/565, C07K14/52, C12N15/70, C07K2319/75, C07K2319/00, C07K14/475, A61K38/00European ClassificationC07K14/57, C07K14/565, C12N15/62, C12N15/70, C07K14/52, C07K14/475Legal EventsDateCodeEventDescriptionJun 5, 2007FPExpired due to failure to pay maintenance feeEffective date: 20070408Apr 8, 2007LAPSLapse for failure to pay maintenance feesOct 25, 2006REMIMaintenance fee reminder mailedRotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services