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Patent US4935233 - Covalently linked 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/US4935233?utm_source=gb-gplus-sharePatent US4935233 - Covalently linked polypeptide cell modulatorsAdvanced Patent SearchPublication numberUS4935233 APublication typeGrantApplication numberUS 06/803,748Publication dateJun 19, 1990Filing dateDec 2, 1985Priority dateDec 2, 1985Fee statusPaidAlso published asCA1304024C, DE3686397D1, DE3686397T2, EP0225579A2, EP0225579A3, EP0225579B1, US5114711, US6545124Publication number06803748, 803748, US 4935233 A, US 4935233A, US-A-4935233, US4935233 A, US4935233AInventorsLeslie D. Bell, Keith G. McCullagh, Alan G. PorterOriginal AssigneeG. D. Searle And CompanyExport CitationBiBTeX, EndNote, RefManPatent Citations (4), Non-Patent Citations (2), Referenced by (220), Classifications (47), Legal Events (7) External Links: USPTO, USPTO Assignment, EspacenetCovalently linked polypeptide cell modulatorsUS 4935233 AAbstract 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.
What is claimed is: 1. A composition represented by the formula R1 --L--R2 wherein R1 is gamma interferon or a biologically active modified gamma interferon; R2 is beta interferon or a biologically active modified beta interferon; and L is a peptide linker segment of 1 to 500 amino acid residues. 2. A composition according to claim 1 wherein R1 is gamma interferon and R2 is a modified beta interferon wherein amino acids 36-48 of natural beta interferon have been replaced by amino acids 34-46 of alpha interferon.
3. A composition according to claim 1 wherein R1 is gamma interferon and R2 is a modified beta interferon wherein amino acids of 36-48 of natural beta interferon have been replaced by amino acids 34-46 of alpha interferon and the cysteine at position 17 of natural beta interferon is replaced by serine.
4. The protein identified in FIG. 8 and FIG. 9 as IFNX 601 and having the amino acid sequence shown in FIG. 8 and FIG. 9.
5. The protein identified in FIG. 10 and FIG. 11 as IFNX 602 and having the amino acid sequence shown in FIG. 10 and FIG. 11.
6. A pharmaceutical composition for use in the treatment of viral infections, regulating cell growth or regulating the immune system in an animal comprising a therapeutically effective amount of the composition of claim 1 admixed with a pharmaceutically acceptable carrier.
7. A pharmaceutical composition for use in the treatment of viral infections, regulating cell growth or regulating the immune system in an animal comprising a therapeutically effective amount of the protein of claim 4 admixed with a pharmaceutically acceptable carrier.
8. A pharmaceutical composition for use in the treatment of viral infections, regulating cell growth or regulating the immune system in an animal comprising a therapeutically effective amount of the protein of claim 5 admixed with a pharmaceutically acceptable carrier.
9. A method of treating viral infections in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the protein of claim 4.
10. A method of regulating cell growth in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the protein of claim 4.
11. A method of regulating the immune system in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the protein of claim 4.
12. A method of treating viral infections in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the protein of claim 5.
13. A method of regulating cell growth in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the protein of claim 5.
14. A method of regulating the immune system in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the protein of claim 5.
This invention relates to covalently linked polypeptide cell modulators, each of which acts through a different and specific cell receptor to initiate complementary biological activities. Polypeptide cell modulators include lymphokines, monokines, interferons, polypeptide hormones or cytotoxins as well as modifications and active segments of such peptides. Also described are DNA sequences, plasmids and hosts capable of expressing the linked polypeptide cell modulators.
One class of polypeptide cell modulators can be defined whose members exert an antiproliferative effect almost specifically on tumour cells and possess immunomodulatory activity, but lack antiviral activity. Among the members of this class are human lymphotoxin and tumour necrosis factor (Gray, P. W. et al. Nature 312, 721, 1984; Pennica D. et al. Nature 312, 724, 1984).
Human lymphotoxin (hLT) is a cytotoxin induced in lymphocytes by a specific antigen or by bacteria or parasites and has a cytotoxic or cytostatic action on a variety of tumour cells in vivo or in vitro. hLT has been implicated to play a role in cell-mediated immunity and its potent anti-tumour effect suggests it may be of value therapeutically (Ruddle, N. H. et al. Lymphokine Res. 2, 23, 1983).
Another class of lymphokine can be defined whose members induce an antiviral state in responsive cells, and also have antiproliferative and immunomodulating activity. Among the members of this class are leukocyte interferon (IFN-alpha), fibroblast interferon (IFN-beta) and immune interferon (IFN-gamma).
It has been reported that mixtures of type I interferons (IFN-beta or IFN-alpha) and type II interferons (IFN-gamma) are highly synergistic in exerting an antiviral or antiproliferative effect. (Fleishmann, W. R. et al. Infect. Immun. 26, 248, 1979; Czarniecki, C. W. et al. J. Virol. 49, 490, 1984).
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).
However, in these instances, there was no disclosure of covalent linkage of the two classes of molecules that were synergistic.
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. 4 414 150-08.11.83) 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. et al. Nature 285, 547, 1980).
BRIEF DESCRIPTION OF THE INVENTION This invention encompasses mixed function proteins formed from covalently linked polypeptide cell modulators, each of which acts through a different and specific cell receptor to initiate complementary biological activities. Novel compounds of this invention are represented by the formula
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.
The term polypeptide cell modulator encompasses a large variety of peptides which elicit a biological response by binding to a specific binding site on a cell. It is known that mixtures of polypeptide cell modulators such as beta and gamma interferon exhibit a synergistic effect. In this invention the polypeptide cell modulators are bound together to produce the same synergistic effect as a mixture of the polypeptide cell modulators or a further enhanced effect or a different effect with the advantage of a single dosage form.
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.
BRIEF DESCRIPTION OF THE CHARTS, TABLES, AND FIGURES Table 1 shows the origin and identification of the plasmids used in the construction of polypeptide cell modulators.
Table 2 shows expression and molecular weight data for IFN X601.
Table 3 shows a comparison of the antiviral activity of IFN X601 with that of the parental IFNs.
Table 4 shows a comparison of the antiproliferative activity of IFN X601 on Daudi lymphoblastoid cells and HEp-2 carcinoma cells with that of the parental IFNs.
Table 5 demonstrates synergy between human IFN-gamma and IFN X430.
Table 6 shows the antigenic properties of IFN X601 as judged by enzyme-linked immunoadsorbent assay (ELISA).
Table 7 shows a comparison of the binding to Daudi cell IFN alpha 2 receptors of IFN X601 with that of the parental interferons, IFN X918 and IFN X430.
Table 8 shows the antiviral, antiproliferative and HLA DR inducing activity of IFN X601 eluted from monoclonal antibody affinity columns.
Table 9 shows the antiviral, antiproliferative, HLA DR inducing and ELISA activity of IFN X602 compared with IFN X601.
Table 10 shows the antiviral, antiproliferative, HLA DR inducing and ELISA activity of IFN X603.
FIG. 3 depicting Chart 1A shows the path to construction of the plasmid vector pGC269, which expresses IFN X601. FIG. 4 and FIG. 5 depicting Charts 1Aa and 1Ab show preparation of starting plasmid pAP8.
FIG. 6 depicting Chart 1B shows the path to construction of the plasmid vector pZZ102, which expresses IFN X603.
FIG. 7 depicts Chart 2; 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.
Chart 2B shows the DNA duplex coding for (Ala-Gly-Ser)7, an alternative spacer for linking IFN X918 to IFN X430.
FIG. 8 and FIG. 9 depicting Chart 3 shows the complete nucleotide and amino acid sequences of the IFN X601 gene and IFN X601, respectively.
FIG. 10 and FIG. 11 depicting Chart 4 shows the complete nucleotide and amino acid sequences of the IFN X602 gene and IFN X602, respectively.
FIG. 12 and FIG. 13 depicting Chart 5 shows the complete nucleotide and amino acid sequences of the IFN X603 gene and IFN X603, respectively.
FIG. 14 and FIG. 15 depicting Chart 6 shows the complete nucleotide and amino acid sequences of the IFN X604 gene and IFN X604, respectively.
FIG. 16 and FIG. 17 depicting Chart 7 shows SDS-PAGE analysis of immunoprecipitates of 35 S-labelled E. coli extracts made with anti IFN-β anti IFN-γ monoclonal antibodies.
FIG. 18 and FIG. 19 depicting Chart 8 shows Western blotting confirmation of co-identity of IFN-β immunoreactivity with IFN X601 36 kd protein.
FIG. 1 shows the enhanced antiproliferative activity of IFN X601 and a mixture of IFN X918 and IFN X430 against HEp-2 carcinoma cells.
FIG. 2 shows the activity of IFN X601 in inducing HLA DR expression on human fibroblasts in comparison with the parental IFNs used either individually or as a mixture.
DETAILED DESCRIPTION OF THE INVENTION Polypeptide cell modulators include soluble protein modulators released by differentiated cells which have their principle effect on other cell types and include lymphokines, monokines, peptide hormones or peptide growth factors.
Among the polypeptide cell modulators are cytokines, that is, all soluble protein modulators released by a differentiated cell that have their principle effect on other cell types. Included within this cytokine class are lymphokines, monokines, products of the endocrine, paracrine or autocrine hormone systems and polypeptide growth factors.
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 factors, 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, melanoctye stimulating hormone, parathyroid hormone, oxytocin, glucagon, secretin, cholecystokinin, gastrin, angiotensin, angiogenin and the polypeptide releasing factors from the hypothalmus.
Those skilled in the biochemical arts will recognize that modification of the polypeptide cell modulators such as changing amino acid sequences and derived or synthetic portions or regions of active cell modulators are equally useful as polypeptide cell modulators and are included as polypeptide cell modulators.
These polypeptide cell modulators are either linked directly or through a peptide linker segment. The peptide linker segment is generally a polypeptide derived from 1 to 500 amino acids. Other peptide linker segments such as dicarboxylic acids, diaminoalkyls and the like are useful for chemically linking polypeptide cell modulators. Peptide linker segments from the hinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptide cell modulators. Especially useful are those hinge region sections where the cysteines are replaced by serines.
Since the preferred methods for preparing these linked polypeptide cell modulators are through genetic engineering, it is understood that variations in the genetic code can produce polypeptide cell modulators which have the general structure of
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 of by modification of amino acid glycosylation sites by genetic engineering techniques.
DESCRIPTION OF PREFERRED EMBODIMENTS Plasmids used in the construction of, or expression of linked polypeptide cell modulator genes are listed in Table 1. One preferred embodiment of the present invention is plasmid pGC269 which codes for IFN X601 (Chart 3) and was derived from plasmids pGC262 (Chart 1A) and pJA39 (Chart 1A). Plasmid pGC262 was derived from plasmid pCC203 (deposited at ATCC no. 39,494) via plasmid pJB9 (Chart 1A); pJA39, which codes for the IFN X430 gene, was derived from plasmid pAP8.
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 147; and Nature 283, 786, 1980), except that the four cysteines are replaces by serines (Chart 3; serine residues 156, 159, 162 and 166); (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).
The plasmid pGC269 of example 1 below (Chart 1A; Table 1) was used in the expression of a polypeptide cell modulator (IFN X601) of example 2 having the antiviral, antiproliferative and immunomodulatory properties described in example 3.
IFN X918 is just one version of IFN-gamma which may be used (i.e., the N-terminal cys-tyr-cys may be present). IFN X430 is just one example of a type I IFN which may be linked to IFN-gamma, or a modified IFN-gamma, such as IFN X918. Other type I IFNs which may be used include IFN-beta or any IFN-alpha (e.g., IFN-alpha 2; Streuli, M. et al. Science 209, 1343, 1980).
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, l786, 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).
A further embodiment is expression plasmid pZZ102 of example 1 which codes for IFN X603 (Chart 5), which was derived from plasmids pZZ101 and pLT101 (Chart 1B and Table 1). Plasmid pZZ101 was derived from plasmid pJB9 by insertion of a 106 bp peptide linker segment coding for the C-terminus of IFN X918 and the amino-terminal 21 amino acids of hLT (Chart 5; resdidues 132 to 166); plasmid pLT 101 contains a synthetic human lymphotoxin gene (i.e., amino acid residues 146 to 316; Chart 5) cloned between the ClaI and BamHI sites of plasmid pAT153 (Twigg, A. J. Nature 283, 216, 1980). IFN X603 is composed of sequentially from the N-terminus; 1) IFN X918; a single methionine; and (2) human lymphotoxin (Chart 5).
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).
It must be appreciated that the DNA sequences coding for IFN X601, IFN X602, IFN X603 and IFN X604 disclosed in charts 3 to 6, are examples of many possible combinations given that alternative triplet codons exist for all amino acids except methionine and tryptophan. Other DNA sequences can code for the amino acid sequences defined in the charts (e.g., Gln-2 in IFN X601 in Chart 3 may be coded by CAG or CAA, etc.).
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: ##STR1## 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 phsiologically 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.
EXAMPLE 1 Chemical Synthesis of Oligonucleotide Fragments; and Plasmid Constructions (a) Chemical Synthesis of Oligonucleotides 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, Tetrahydron 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 1/1/2 v/v) (1 hour) and concentrated ammonia at 70� C. (4 hour). The deprotected oligonucleotides were purified either by HPLC on a Partisil� 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).
(b) Ligation of Oligonucleotide Blocks 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 [32 p]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 radiactivity.
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 250 uM 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, 0.5 ul and 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.
(c) Plasmid Constructions (i) Plasmid pGC269 (Table 1) STEP 1
DNA corresponding to the amino-terminal cys-tyr-cys of human IFN-gamma in the plasmid pCC203 (ATCC No. 39, 494) was deleted by ClaI/BamH double restriction enzyme digestion as in Chart 1A (Methods in Molecular Cloning, a Laboratory manual, eds. Maniatis et al., Cold Spring Harbor Laboratory, 1982). The resultant expression plasmid, pJ89, codes for IFN X918 which has the cys-tyr-cys replaced by methionine (PCT No. 83/04053).
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.
To create an IFN X416 gene (European Patent application No. 85105914.7) with a unique HindIII site, plasmid pAP8 was cut with ClaI and XhoI (chart IA), and the 230 bp fragment replaced by an identical chemically synthesized fragment except that codons 19 and 20 are AAGCTT (HindIII) instead of AAGCTC. The resultant plasmid was designated pJA39 (Table 1).
Since IFN X416 and IFN X430 are identical except at amino acid position 17, the HindIII to SalI 719 bp fragment from pJA39 (equivalent to amino acids 19 to 166 of IFN X430 or IFN X416) was ligated to the large HindIII/SalI vector fragment of pGC262 to give plasmid pGC269, which codes for the IFN X918-IFN X430 polypeptide cell modulator, designated IFN X601 (Chart 3).
(ii) Plasmid pZZ102 (Table 1)
A similar strategy was used to construct pZZ102.
Plasmid pJ89 (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. ##STR2##
Plasmid pZZ101 was cleaved with NsiI and SalI and the large vector fragment isolated in preparation for insertion of the remainder of the hLT gene, which was isolated from pLT101 (Table 1; chart 1B).
pLT101 contains a complete synthetic hLT gene modified from Gray, P. W. et al. Nature 312, 721, 1984 (equivalent to amino acid residues 145 to 316 in Chart 5). The hLT gene in pLT 101 was cloned on a ClaI to BamHI fragment in the ClaI/BamHI sites of plasmid pAT153. The nucleotide sequences of the ClaI and BamHI junctions are, respectively: A T C G A T A A G C T A T G. and T A G A G G A T C C (ATG=initiation codon, TAG=termination codon).
Plasmid pLT101 was cleaved with NsiI and SalI and the resultant 725 bp 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).
EXAMPLE 2 Expression and Isolation of Polypeptide Cell Modulators (a) Expression of plasmids coding for IFN X601, X602, X603 and X604 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 μg/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 pH 6.8, 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.
Table 2 shows that for IFN X601, a polypeptide of approximately the size expected for an IFN X918/hinge/IFN X430 fusion is expressed in the range 5.4 to 10% of total bacterial protein.
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).
(c) Preparation of bacterial extracts for biological assay 10 to 20 ml. of bacterial culture was removed at the optical density (670 nm) of 1.5-2.0 (middle to late log phase of growth) and centrifuged to recover the cells. After suspension in 25 mM tris-HClpH 7.5, 50 mM NaCl (1 ml.) and 1 mM EDTA (1.4 ml.) at 0� C., 28 ul lysozyme was added to a final concentration of 50 ug/ml and the suspension incubated at 0� C. for 30 min. The suspension was sonicated for 24 sec., the cell debris removed by centrifugation and the supernatants assayed for biological activity as described in Example 3 or gel analysis as described in Example 2.
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-HCl pH 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.
EXAMPLE 3 Biological Activity of Polypeptide Cell Modulators in Crude Bacterial Extracts (a) Antiviral 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 XFN 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).
In conclusion, the polypeptide cell modulator IFN X601 displayed a significant enhancement of AV activity compared with the parental IFNs, which was similar to that of equimolar mixtures of IFN X918 and IFN X430.
(b) Antiproliferative assays (i) Daudi (lymphoblastoid) cells
Antiproliferative (AP) activity was assessed by the ability of the polypeptide 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 colorimetrically 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 modulator. 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).
Consistent with these findings is the observation that there is a similar level of binding of IFN X430 and IFN X601 to Daudi receptors (Table 7), while the lack of AP activity of IFN X918 correlates with very low receptor binding.
(ii) HEp-2 (human laryngeal carcinoma) cells
Antiproliferative activity was also assessed in HEp-2 cells Growth inhibition was measured by methylene blue staining of the cell monolayer by a modification of the method of Ito. (Ito, M. J. Interferon Res. 4, 603, 1984). Inhibitory concentration (IC50) end point is the log dilution giving 50% reduction of methylene blue staining.
A comparison is made in Table 4B of the HEp-2 antiproliferative activity in crude bacterial extracts of IFN X601 and the parental IFNs, derived from equivalent numbers of bacterial cells. IFN X601 consistently displayed a 3 fold higher AP activity than IFN X430 and a 15 fold higher AP activity than IFN X918, despite a �2-fold lower level of protein expression (Table 2). Furthermore, when equivalent antiviral units of these interferons were compared it was seen that IFN X601 had an enhanced antiproliferative effect as shown in FIG. 1. For the individual IFNs X430 and X918 there is a maximum achievable level of growth inhibition which cannot be increased despite adding a hundredfold excess of interferon. This is not seen with IFN X601 where a markedly increased level of growth inhibition is seen.
These properties of IFN X601 are reminiscent of the antiproliferative effect of mixtures of IFN X430 and IFN X918. For example, Table 4B shows that equivalent concentrations of these two IFNs mixed together gave 1.8-8.6 fold higher AP activity than either alone. In this case, AP activity was almost 3 fold higher than the value expected if the AP activities of the individual IFNs X918 and X430 were additive (Table 4B). Further, like IFN X601, equimolar mixtures of IFN X918 and IFN X430 have enhanced antiproliferative activity against HEp-2 cells (FIG. 1).
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.
Taken together, these results indicate that (i) a covalent combination of IFN X918 and IFN X430 via a peptide linker segment potentiates cytotoxicity in a manner analogous to simple mixtures; (ii) a covalent combination of IFN X918 and IFN X430 is a suitable ratio to potentiate biological activity; (iii) the IC50 end point on HEp-2 cells for IFN X601 was significantly higher than the values for the parental IFNs. Potentiation was similar to that observed with synergistic mixtures of IFN X918 and IFN X430.
(c) HLA-DR Antigen presentation on human fibroblasts IFN-gamma, but not IFN-beta or IFN X430, induces the expression on the surface of normally DR-negative human foetal lung fibroblasts (17/1 strain). This is detected and measured by the binding of monoclonal antibody against HLA-DR.
Fibroblasts 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.
IFN X601 and IFN X918 clearly caused expression of HLA-DR antigens on the surface of 17/1 fibroblasts while IFN X430 did not (table 9). The level of HLA DR induction by IFN X601 was markedly lower than that induced by equivalent antiviral units of IFN X918. This may be due to suppression by the IFN X430 domain because the HLA DR induction by IFN X918 was seen to be reduced in a 1:1 mixture with IFN X430. The HLA DR induction by IFN X601 can be increased more than ten fold by blocking the activity of the IFN X430 domain with anti IFN-β monoclonal antibody. These results demonstrate that IFN-gamma biological activity is present in the polypeptide cell modulator IFN X601.
(d) Analysis of IFN X601 with Antibodies Against beta and gamma-IFNs (i) Enzyme linked immunoadsorbent assay (ELISA) for interferon
The ELISA for both beta and gamma interferons utilizes an indirect two site sandwich technique. Dilutions of the interferon samples (or standards) are allowed to bind to interferon antibodies attached to the wells of a 96 well microplate. A second antibody to interferon, but raised in a different species from that attached to the plate, is included in the incubation mixture, which then binds to a second epitope on the interferon molecule. After washing away the unbound molecules, an enzyme labelled antispecies antibody is added which binds to the second interferon antibody. The presence of bound enzyme is detected by adding a substrate which changes color in the presence of enzyme. The amount of color produced is proportional to the amount of interferon, since the other reagents are present in excess.
For the beta and gamma interferon ELISA's, two antibodies against the corresponding interferon are used, while for a hybrid ELISA, an antibody directed against beta interferon is bound to the plate, while the second antibody used is one directed against gamma interferon.
The general scheme of the assay is illustrated below:
ANTIBODY TO INTERFERON
INTERFERON SAMPLE
SECOND ANTIBODY TO INTERFERON
ANTI SPECIES ANTIBODY
(ENZYME LABELLED)
BETA INTERFERON ELISA 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 microliter of a 5 microgram/ml solution of immunoglobulin (obtained by a 40% ammonium sulphate precipitation of the interferon antibody) in 0.05M 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 microliters 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.
Serial dilutions of interferon samples are made in the plates, by dilution in PBS/C containing a mouse monoclonal antibody to beta interferon at a 1/100 dilution. Each plate also contains an internal standard which has been calibrated against the International Reference Standard. After incubation overnight at +4� C., the well contents are removed and the plates washed six times with PBS/T.
100 microliters 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 microliters 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 microliters of 2.5M 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.
GAMMA INTERFERON ELISA This assay is carried out in the same way as the beta ELISA, with the following changes: the plates are coated with a mouse monoclonal antibody to gamma interferon (Meloy Laboratories) at 1/200 in carbonate buffer. Serial dilutions of the gamma interferon samples are made in PBS/C containing a rabbit antiserum to human gamma interferon (Immunomodulator laboratories, diluted to 1/5000). A peroxidase conjugated goat anti rabbit immunoglobulin (Tago Laboratories, diluted to 1/3000) is used as the indicator molecule.
HYBRID BETA/GAMMA INTERFERON ELISA The only difference from the beta ELISA is that the interferon samples are diluted in PBS/C containing a mouse monoclonal to human gamma interferon (Meloy Laboratories, at a dilution of 1/1000). This assay will only detect interferon molecules containing both a beta and a gamma epitope.
The results of testing the polypeptide cell modulator IFN X601 and the appropriate controls in the beta, gamma and hybrid ELISA's are given in Table 6. In the beta ELISA, IFN X430 (equivalent to beta) reacts, the gamma interferon shows no sign of cross reactivity, while a 50/50 mixture of the two gives a titre reduced by 0.4 log unit/ml, close to the expected 0.3 reduction. The IFN X601 also reacts strongly, showing that the two beta interferon epitopes are still available to bind antibodies.
In the gamma ELISA, the gamma interferon reacts, the IFN X430 shows no cross reactivity, while a 50/50 mixture of the two gives a titre reduced by the expected 0.3 log units/ml. IFN X601 also reacts, though with a reduced titre compared to the other positive reactions, which might indicate that one of the gamma epitopes is slightly sterically affected by the presence of the beta hybrid interferon.
In the hybrid ELISA, the only sample to react is IFN X601, which conclusively demonstrates that the molecule contains both beta and gamma epitopes covalently bonded to each other. Quantitatively the results from this assay cannot be compared to the other two ELISA's since there is no standard available and the 50% end points are dependent on relative affinities and concentrations of the various reagents used, which differ for the three assays used. However, the results indicate that a substantial proportion of the polypeptide cell modulators is present in the covalently linked state in sample X601.
(ii) Immunoprecipitation
Interferons were labelled by including 35 S-methionine in bacterial growth medium and extracts were prepared by treatment by lysozyme and sonication. 35 S-labelled E. coli extracts were immunoprecipitated with either monoclonal antibodies directed againt IFN-β or IFN-γ and the immunoprecipitates were analyzed by SDS-PAGE.
The results in Chart 7 show that anti IFN-β monoclonal antibody precipitates IFN X430 but not IFN X918, anti IFN-γ monoclonal antibody precipitates IFN X918 but not IFN X430 while both monoclonal antibodies precipitate a �36 kd protein in the IFN X601 extract. The material precipitated from the IFN X601 extracts by both antibodies therefore has the predicted molecular weight for the chimaeric protein and has both X430 and X918 antigenic activity.
(iii) Western Blot Analysis
Bacterial extracts containing IFNs were run out on SDS-PAGE and analyzed by Western blotting with anti IFN-β monoclonal antibody.
Chart 8 shows that anti-IFN-β 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.
(iv) Monoclonal antibody affinity column purification
Bacterial extracts containing IFN X601 were loaded on to monoclonal antibody affinity columns consisting of either anti-IFN-β bound to CNBr sepharose or anti-IFN-γ bound to CNBr sepharose (Celltech MAb). The loaded columns were extensively washed, bound material was eluted and fractions were assayed for antiproliferative activity against Daudi and HEp-2 cells and for HLA DR inducing activity on human lung fibroblasts.
The results in Table 8 demonstrate that material from an E. coli lysate containing IFN X601 can be bound to and eluted from both anti-IFN-β and anti-IFN-γ affinity columns. The material eluted from the anti-IFN-β column must have IFN X430 antigenicity and has been shown to have IFN X430 biological activity (Daudi antiproliferative assay) as well as IFN X918 activity in the HLA DR induction assay. The material eluted from the anti-IFN-γ column must have IFN X918 antigenicity and has been shown to have IFN X918 biological activity (HLA DR induction activity) as well as IFN X430 activity in the Daudi antiproliferative assay. In addition, eluted material from both columns showed enhanced antiproliferative activity against HEp-2 cells which is taken to indicate that both the IFN X430 and IFN X918 domains are biologically active.
Biological Activity of IFN X602 (IFN X918 (AGS), IFN X430)
Table 9 shows X602 to have similar biological properties as X601.
Biological Activity of IFN X603 (IFN X918-LT)
Table 10 shows that IFN X602 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-gamma activities. (HEp-2 antiproliferative activity could be due to IFN-gamma or lymphotoxin/IFN-gamma combination but not to lymphotoxin alone.)
EXAMPLE 4 Construction of the Plasmid pAP8 Expressing IFNX416 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 pl/24C (�4,440 bp) which was identical to plasmid pl/24 U.K. Patent 8,102,051, except for the underlined sequences which follows: ##STR3##
Step 1 (Chart 1Aa)
The subcloning of the natural human IFN-β gene from plasmid pl/24C (Taniguchi et al., Gene, 10, 11, 1980) in phage M13mp8 (Sanger, F. et al., J. Mol. Biol., 143, 161, 1981) was performed, and the presence of the whole fragment was confirmed by restriction endonuclease mapping of M13 plasmid mAP2.
Step 2 (Chart 1Aa)
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.
Step 3 (Chart 1Aa)
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.
Step 4 (Chart 1Ab)
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 pl/24C, 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.
Step 5 (Chart 1Ab)
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.
Modifications of the above described mode for carrying out the invention such as, without limitation, use of alternative vectors, alternative expression control systems, and alternative host micro-organisms and other therapeutic or related uses of the novel polypeptide cell modulators that are obvious to those of ordinary skill in the biotechnology, pharmaceutical medical and/or related fields are intended to be within the scope of the following claims.
TABLE 1______________________________________Table of PlasmidsPlasmid  Properties         Source______________________________________pAP8   Expression vector coding for                     EP 85105914.7  IFN X416 gene      U.K. Patent 8,102,051,                     Chart 1Aa and 1Ab                     and example 4pJA39  Expression vector containing                     Amino acids 19/20  IFN X416 gene plus HindIII                     coded by AAG.CTT  site               instead of AAG.CTC                     (pAp8)pGC262 Intermediate vector in                     Chart 1A  construction of pGC269 -  codes for IFN-gamma + 22  amino acid mouse gamma 2b  IgG "hinge"pCC203 Expression vector containing                     Chart 1A and  synthetic human IFN-gamma                     PCT 83/04053  genepJB9   Expression vecror containing                     Chart 1A and  synthetic IFN-gamma gene                     PCT 83/04053  with DNA coding for N-  terminal Cys--Tyr--Cys  deleted and replaced by Met.  (IFN X918)LT3/1  Expression vector containing                     Charts 1A, 3  synthetic human lymphotoxin                     Nature 312, 721,  gene               1984pGC279 Intermediate vector in                     Chart 1B  construction of pZZ102; codes  for IFN X918 plus 22 N-  terminal amino acids of  lymphotoxinpGC282 Expression vector containing                     Charts 1B, 5  IFN X603 gene (IFN X918 -  metlymphotoxin polypeptide  cell modulator).pGC269 Expression vector containing                     Charts 1A, 3  IFN X601 gene.______________________________________
TABLE 2______________________________________Molecular Weight and Expressionin E. coli of IFN X601    Molecular weight                    Range of expression    (from polyacrylamide                    (% of total bacterialInterferon    gel)            protein)______________________________________X918*    17,000          13.6-15.6 (N = 14.6)X430+    19,000          12.3-17.0 (N = 14.65)X601     37,500           5.4-10.0 (N = 7.7)______________________________________ *IFN-gamma with Nterminal cystyr-cys deleted and replaced by met (Chart 3 + IFNbeta with amino acids 36 to 48 inclusive replaced by amino acid 34 to 46 inclusive from IFNalpha 1. N mean.
TABLE 3______________________________________Antiviral Activity of IFN X601   Antiviral activity               Increase compared   I.U/ml at 10 A670 �               with:Interferon     10 -6         IFN X430  IFN X918______________________________________.sup. X9181     0.59          (0.5X)    --X430      1.1           --        2.9XX601      2.87          2.6X      4.9XX918 + X4302     3.47          3.2X      5.9X______________________________________ *IU/m110 A670 � 10-6. Mean of 3 determinations in 2 separate experiments: 1. IFNgamma with Nterminal Cys--Tyr--Cys replaced by Met (chart 3). 2. Approximately 1:1 mixture of each IFN (protein).
TABLE 4______________________________________               Increase compared    Antiproliferative               with:Interferon Activity*    IFN X430  IFN X918______________________________________A. Daudi lymphoblastoid cellsX918       0.004        --        --X430       2.7          --        --X601       3.3          1.2X      --X918 plus X4301      1.9          (0.7X)    --B. HEp-2 carcinoma cellsX918       0.57         (0.2X)    --X430       2.8          --        4.9XX601       9.0          3.2X      15.8XX918 plus X4301      4.9          1.8X      8.6X______________________________________ *Units/ml � 10-4 = dilution of IFN at 50% cell growth inhibition. Mean of 2 determinations. Mixture 1:1 w/w
TABLE 5______________________________________IFN X430/IFN-gamma synergy on HEp-2 carcinoma cellsA. IFN X430      B. IFN-gamma+Antiviral    FIC*    Anitiviral   FIC*  FIC IndexUnits/ml "A"     units/ml     "B"   ("A" + "B")______________________________________168      1.000   0            0.000 1.00056       0.334   0.3          0.003 0.33740       0.230   1.0          0.009 0.23932       0.188   3.1          0.029 0.21710       0.059   10           0.094 0.1533.1      0.018   27           0.252 0.2702.2      0.013   32           0.298 0.3111.0      0.006   81           0.767 0.7730.8      0.004   100          0.940 0.9440        0       106          1.000 1.000______________________________________ *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 to IFN`A` alone. Concentration of IFN alone or in combination required to produce 50� inhibition of HEp2 growth. Synergy is present when FIC index is equal to or less than 0.5
TABLE 6______________________________________       ACTIVITY (LOG UNITS/ML)       Beta    Gamma     Hybrid       ELISA   ELISA     ELISA       E    F      E      F    E    F______________________________________A Gamma interferon         ND     ND     4.47 5.44 ND   NDB IFN X430 (= beta)         3.95   5.84   ND   ND   ND   NDC Interferon X601         4.13   6.02   2.98 3.95 3.73 --D Mixture of A and B         3.59   5.48   4.16 5.13 ND   ND(1:1)______________________________________ Notes 1. E represents the 50% end points 2. F represents teh corrected activities 3. ND is not detectable activity
TABLE 7______________________________________COMPETITION BY IFN X601 FOR THE BINDINGOF 125 I-IFN alpha 2 TO DAUDI CELL RECEPTORSIFN        Activity Log U/ml.*______________________________________X430       7.0X918       3.6X601       6.6______________________________________ *IFN &#945;2 antiviral unit equivalents. The activity in each sample was calculated by interpolation from a standard dose curve of the compet:tion by IFN &#945;2 for the binding of 125 IIFN&#945;2.
TABLE 8______________________________________MONOCLONAL ANTIBODY AFFINITYPURIFICATION OF CRUDE LYSATES OF IFN X601   IFN Activity*Fraction  Daudi       HEp-2**  HLA DR______________________________________Anti IFN-Beta Column3         3.00        Not done 2.34         3.25        2.89     2.35         4.25        3.79      2.476         4.20        3.85      2.657         3.82        3.25     Not doneAnti IFN Gamma Column3         3.24        2.72     2.34         3.72        4.31     2.45         3.70        4.15     2.36         3.28        3.95     2.37         3.22        3.67     Not done______________________________________ *Log units/ml = dilution of IFN at 50% assay end point. **Enhanced antiproliferative activity seen.
TABLE 9__________________________________________________________________________BIOLOGICAL ACTIVITY OF IFN X602COMPARED WITH IFN X601Antiviral    Antiproliferative             HLA DR Induction                       ELISAIFN   EMC/Vero    HEp-2         Daudi             Lung Fibroblasts                       Beta                          Gamma                               Mixed__________________________________________________________________________X601   6.49  4.74*         4.28             3.30      5.93                          4.08 3.50X602   6.46  3.89*         3.55             2.81      5.94                          3.46 2.75__________________________________________________________________________ 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 X603Antiviral Antiproliferative               HLA DR Induction                          ELISAIFN   EMC/Vero     HEp-2          L Cell               Lung Fibroblasts                          Gamma__________________________________________________________________________X603   4.47   3.19 4.02 2.80       4.31__________________________________________________________________________ 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 dateApplicantTitleUS4468382 *Jul 15, 1982Aug 28, 1984New England Medical Center, Inc.Polypeptide-toxin hybrid proteinUS4691009 *Dec 26, 1984Sep 1, 1987Repligen CorporationHybrid proteins produced by an ultrahigh prokaryotic expression systemUS4734491 *Aug 31, 1984Mar 29, 1988University Patents, Inc.DNA sequences encoding hybrid lymphoblastoid-leukocyte human interferonsUS4758428 *Jul 15, 1985Jul 19, 1988Cetus CorporationMulticlass hybrid interferons* Cited by examinerNon-Patent CitationsReference1 *Shepard et al., Nature, vol. 294, pp. 563 565, 1981.2Shepard et al., Nature, vol. 294, pp. 563-565, 1981.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS5073627 *Aug 14, 1990Dec 17, 1991Immunex CorporationAnimal growth regulatorUS5108910 *Mar 22, 1991Apr 28, 1992Immunex CorporationDNA sequences encoding fusion proteins comprising GM-CSF and IL-3US5238823 *Aug 22, 1990Aug 24, 1993Veterinary Infectious Disease OrganizationInterleukin-2-leukotoxin gene fusions and uses thereofUS5273889 *Oct 16, 1991Dec 28, 1993University Of SaskatchewanGamma-iterferon-leukotoxin gene fusions and uses thereofUS5334379 *Jul 16, 1990Aug 2, 1994American Cyanamid CompanyCytokine and hormone carriers for conjugate vaccinesUS5359035 *Oct 19, 1992Oct 25, 1994Hoechst AktiengesellschaftStable antitumor agentsUS5496924 *Dec 28, 1992Mar 5, 1996Hoechst AktiengesellschaftFusion protein comprising an interleukin-2 fragment ballast portionUS5585380 *Jan 25, 1995Dec 17, 1996Fred Hutchinson Cancer Research CenterModulation of cellular response to external stimuliUS5594107 *Dec 20, 1993Jan 14, 1997University Of SaskatchewanChimeric protein comprising an RTX-family cytotoxin and interferon-2 or interferonUS5684136 *May 5, 1995Nov 4, 1997Genentech, Inc.Fusion proteins and immunoglobulinsUS5705478 *Nov 4, 1994Jan 6, 1998Washington UniversityCovalently linked β subunits of the glycoprotein hormones as antagonistsUS5763584 *May 5, 1995Jun 9, 1998Genentech, Inc.Receptor activation with hepatocyte growth factor agonistsUS5770704 *Jan 31, 1997Jun 23, 1998Genentech, Inc.Receptor activation with inactive hepatocyte growth factor ligandsUS5883230 *Jun 5, 1996Mar 16, 1999Genetics Institute, Inc.Multidomain hematopoiesis stimulatorsUS5945397 *May 16, 1996Aug 31, 1999Immunex CorporationA soluble protein capable binding tumor necrosis factor for use in raising antibodies, in diagnosis and therapy, regulating the immune activities of cytokineUS6010873 *Jun 7, 1995Jan 4, 2000Washington UniversityModified complement system regulatorsUS6028177 *Jul 11, 1997Feb 22, 2000Washington UniversityMethods of detecting single-chain forms of the glycoprotein hormone quartetUS6083724 *Mar 5, 1996Jul 4, 2000Commonwealth Scientific And Industrial Research OrganizationRecombinant avian interferon-gamma (IFN-γ)US6096320 *Oct 20, 1997Aug 1, 2000University Of SaskatchewanVaccines with chimeric protein comprising gamma-interferon and leukotoxin derived from pasteurella haemolyticaUS6143866 *Jan 19, 1995Nov 7, 2000Amgen, Inc.Analog of a defined amino acid sequence; for treatment of septic shock and cachexiaUS6171824Nov 8, 1993Jan 9, 2001Fred Hutchinson Cancer Research CenterPolypeptide includes four alpha-helical and three linking sequences from leukemia inhibitory factor, granulocyte colony stimulating factor, interleukin-6, interleukin-11, ciliary neurotrophic factor, and oncostatin-m; therapyUS6201105Jan 20, 1999Mar 13, 2001Craig A. SmithAmino acid sequence; treatment of septic shock and cachexia; diagnosisUS6204364 *Jul 27, 1993Mar 20, 2001Fred Hutchinson Research CenterCell growth factor; for the treatment of cell proliferation and differentiation defectsUS6271346Jun 7, 1995Aug 7, 2001Amgen Inc.TNF Receptors, TNF binding proteins and DNAs coding for themUS6294352 *Feb 1, 1995Sep 25, 2001Amgen Inc.Nucleotide sequence which code polypeptide which binds tumor necrosis factor; for the reduction of side effects associated with tumor necrosis factor treatmentsUS6306820Jun 4, 1999Oct 23, 2001Amgen Inc.Combination therapy using a TNF binding protein for treating TNF-mediated diseasesUS6417158 *Jun 7, 1995Jul 9, 2002Amgen, Inc.Method of ameliorating the harmful effects of TNF using a polypeptide having the ability to bind to TNFUS6440693Jun 7, 1995Aug 27, 2002Rudolf HauptmannGiven amino acid sequence; recombinant tumor necrosis factor (tnf)-binding protein useful in pharmaceuticals for inhibiting tnfUS6521450Dec 2, 1999Feb 18, 2003Washington UniversityCells expressing a modified regulator of complement activationUS6541610Mar 20, 1995Apr 1, 2003Immunex CorporationGenetic engineering; drugsUS6541620Jun 7, 1995Apr 1, 2003Angen Inc.Antagonist for tumor necrosis factor; antiinflammatory agents; rheumatic diseasesUS6552170Jun 14, 1994Apr 22, 2003Amgen Inc.Derivatives of polyethylene glycol and related hydrophilic polymers and to polypeptides that have been covalently bonded to such active derivativesUS6572852Jan 12, 2001Jun 3, 2003Immunex CorporationGenetic engineeringUS6642032Nov 19, 1999Nov 4, 2003Commonwealth Scientific And Industrial Research OrganisationUses of avian interferon gamma (IFN-γ)US6897290 *Sep 24, 1993May 24, 2005Washington UniversityRegulators of complement activation (RCA); diagnosis/therapy of complement system for autoimmune disease treatment; open reading frames; point mutations; membrane cofactor/decay accelerating factor/factor HUS6989147Jun 15, 2001Jan 24, 2006Amgen Inc.Tumor necrosis factor receptor protein for use in the treatment of arthritisUS7033807Oct 30, 2001Apr 25, 2006Pharmacia CorporationAspergillus ochraceus 11 alpha hydroxylase and oxidoreductaseUS7057022Apr 23, 2003Jun 6, 2006Immunex CorporationAntibodies which specifically bind to TNF-RUS7083957Feb 12, 2002Aug 1, 2006Reasearch Development FoundationModified proteins, designer toxins, and methods of making thereofUS7101977Jul 17, 2002Sep 5, 2006Research Development FoundationTargeted killing of a cell utilizing a chimeric polypeptide comprising a cell-specific targeting moiety and a signal transduction pathway factorUS7122361Oct 10, 2003Oct 17, 2006WyethCompositions employing a novel human kinaseUS7186810Aug 29, 2003Mar 6, 2007Amgen Inc.Modified peptides as therapeutic agentsUS7208306Oct 24, 2003Apr 24, 2007WyethComprises nucleotide sequences coding calcineurin-like protein phosphatase 1 (CLPP1) for diagnosis, prevention and treatment of inflammation, cancer, arteriosclerosis and psoriasisUS7238507Jul 28, 2004Jul 3, 2007Pharmacia CorporationAspergillus ochraceus 11 alpha hydroxylase and oxidoreductaseUS7238776Jul 3, 2001Jul 3, 2007Amgen Inc.TNF binding proteinsUS7264944Mar 15, 2000Sep 4, 2007Amgen Inc.TNF receptors, TNF binding proteins and DNAs coding for themUS7282483Jul 3, 2001Oct 16, 2007Amgen Inc.Method of ameliorating the harmful effects of TNF using a polypeptide having the ability to bind to TNFUS7285635Apr 25, 2006Oct 23, 2007Research Development FoundationReducing the antigenicity of a proteinaceous compound while maintaining the compound's biological activityUS7297525Nov 7, 2003Nov 20, 2007WyethComposition employing a novel human kinaseUS7371723May 1, 2006May 13, 2008Research Development FoundationTherapeutic agents comprising pro-apoptotic proteinsUS7407792Aug 17, 2006Aug 5, 2008WyethCompositions, organisms and methodologies employing a novel human kinaseUS7459528Oct 28, 2005Dec 2, 2008Immunex CorporationDNA sequences encoding tumor necrosis factor receptors; recombinant expression vectorsUS7494788Jul 11, 2006Feb 24, 2009Molecular Kinetics, Inc.Entropic bristle domain sequences and their use in recombinant protein productionUS7528232Dec 15, 2005May 5, 2009The University Of Kentucky Research FoundationUtility of phylloplanins as antibiotics, selective fungicides and for enhancing microbial resistance in crop plantsUS7576183Dec 24, 2003Aug 18, 2009Los Alamos National Security, LlcStructure-based receptor MIMICS targeted against bacterial superantigen toxinsUS7611839Nov 21, 2003Nov 3, 2009WyethMethods for diagnosing RCC and other solid tumorsUS7732587May 10, 2005Jun 8, 2010Amgen Inc.for treatment of TNF-mediated diseases, particularly including chronic inflammatory diseasesUS7741278Sep 17, 2007Jun 22, 2010Research Development FoundationModified proteins, designer toxins, and methods of making thereofUS7759091Feb 29, 2008Jul 20, 2010Research Development FoundationTargeted killing of a cell utilizing a chimeric polypeptide comprising a cell-specific targeting moiety and a signal transduction pathway factor; apoptosis-inducing factorUS7763250Apr 28, 2006Jul 27, 2010Rinat Neuroscience Corp.Antibodies directed against amyloid-beta peptide and nucleic acids encoding sameUS7807165Aug 1, 2005Oct 5, 2010Rinat Neuroscience Corp.comprises an Fc region having impaired effector function; treating a disease characterized by aberrant deposition of a protein in the brain of a subject; Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathyUS7906277Aug 16, 2006Mar 15, 2011Corixa CorporationCompound and methods for diagnosis of tuberculosisUS7927594Aug 1, 2005Apr 19, 2011Rinat Neuroscience Corp.Monoclonal antibody 9TL that comprises an Fc region having impaired effector function; treating a disease characterized by aberrant deposition of a protein in the brain of a subject; Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathyUS7927609Oct 29, 2007Apr 19, 2011Corixa CorporationCompounds and methods for immunotherapy and diagnosis of tuberculosisUS7927610Oct 30, 2007Apr 19, 2011Corixa CorporationCompounds and methods for diagnosis and immunotherapy of tuberculosisUS7927611Oct 30, 2007Apr 19, 2011Corixa CorporationCompounds and methods for diagnosis and immunotherapy of tuberculosisUS7935353Jun 27, 2006May 3, 2011Corixa CorporationCompounds and methods for diagnosis and immunotherapy of tuberculosisUS7943571May 24, 2010May 17, 2011Research Development FoundationModified proteins, designer toxins, and methods of making thereofUS7973153Oct 30, 2007Jul 5, 2011Corixa CorporationAntigen, nucleic acid encodes fusion protein; polynucleotides; nucleoside sequencesUS7976844May 5, 2006Jul 12, 2011Corixa CorporationFusion proteins of Mycobacterium tuberculosisUS7982025May 30, 2007Jul 19, 2011Corixa CorporationFusion proteins of Mycobacterium tuberculosisUS7989605Oct 29, 2007Aug 2, 2011Corixa CorporationCompounds and methods for immunotherapy and diagnosis of tuberculosisUS8034771Sep 10, 2007Oct 11, 2011Amgen Inc.IL-1F6 polypeptidesUS8043831May 26, 2010Oct 25, 2011Research Development FoundationTherapeutic agents comprising pro-apoptotic proteinsUS8063182May 19, 1995Nov 22, 2011Hoffman-Laroche Inc.Human TNF receptor fusion proteinUS8067016Jun 24, 2009Nov 29, 2011Corixa CorporationFusion proteins of Mycobacterium tuberculosisUS8071747Jun 23, 2009Dec 6, 2011Corixa CorporationFusion proteins of mycobacterium tuberculosisUS8084042May 7, 2007Dec 27, 2011Corixa CorporationPolypeptide comprising a soluble Mycobacterium tuberculosis antigen; vaccinesUS8110200Feb 2, 2010Feb 7, 2012Corixa CorporationFusion proteins of Mycobacterium tuberculosisUS8110201Feb 2, 2010Feb 7, 2012Corixa CorporationFusion proteins of mycobacterium tuberculosisUS8138311Apr 26, 2011Mar 20, 2012Research Development FoundationModified proteins, designer toxins, and methods of making thereofUS8143386Oct 29, 2007Mar 27, 2012Corixa CorporationFusion proteins of mycobacterium tuberculosis antigens and their usesUS8163522May 19, 1995Apr 24, 2012Hoffman-Laroche Inc.Human TNF receptorUS8252897Jun 20, 2008Aug 28, 2012Angelica Therapeutics, Inc.Modified toxinsUS8268593Apr 5, 2010Sep 18, 2012Rinat Neuroscience Corp.Polynucleotides encoding antibodies directed against amyloid-beta peptideUS8283127Nov 9, 2007Oct 9, 2012Dimerix Bioscience Pty Ltd.Detection system and uses thereforUS8398978Mar 15, 2010Mar 19, 2013Rinat Neuroscience Corp.Antibodies directed against amyloid-beta peptide and methods using sameUS8470314Dec 15, 2008Jun 25, 2013Angelica Therapeutics, Inc.Modified toxinsUS8481021Sep 2, 2011Jul 9, 2013Amgen Inc.IL-1F5 polypeptidesUS8486414Apr 4, 2008Jul 16, 2013Infectious Disease Research InstituteImmunogenic compositions comprising Mycobacterium tuberculosis polypeptides and fusions thereofUS8530225Oct 25, 2011Sep 10, 2013Research Development FoundationTherapeutic agents comprising pro-apoptotic proteinsUS8568719Aug 13, 2010Oct 29, 2013Crucell Holland B.V.Antibodies against human respiratory syncytial virus (RSV) and methods of useUS8568730Apr 22, 2010Oct 29, 2013Indiana University Research & Technology CorporationCompositions for use in the treatment of chronic obstructive pulmonary diseases and asthmaUS8568997Sep 10, 2012Oct 29, 2013Dimerix Bioscience Pty Ltd.Detection system and uses thereforUS8569015May 30, 2007Oct 29, 2013Pfenex Inc.RPA optimizationUS8697139Sep 21, 2004Apr 15, 2014Frank M. PhillipsMethod of intervertebral disc treatment using articular chondrocyte cellsUS8722033Oct 26, 2010May 13, 2014Amgen Inc.Human IL-23 antigen binding proteinsUSRE36755 *Aug 31, 1998Jun 27, 2000Immunex CorporationDNA encoding tumor necrosis factor-α and -β receptorsEP0815233A1 *Mar 5, 1996Jan 7, 1998Commonwealth Scientific And Industrial Research OrganisationNovel avian cytokines and genetic sequences encoding sameEP1347055A2Aug 30, 1996Sep 24, 2003Corixa CorporationCompounds for immunotherapy and diagnosis of tuberculosisEP1701165A1Mar 7, 2005Sep 13, 2006Johannes Dr. CoyTherapeutic and diagnostic uses of TKTL1 and inhibitors and activators thereofEP1772515A1May 12, 2000Apr 11, 2007Immunex CorporationImmunoregulator from the family designated 'leukocyte immunoglobulin-like receptors' (LIR)EP1961819A2Jun 28, 2001Aug 27, 2008Corixa CorporationComposition and methods for the therapy and diagnosis of lung cancerEP1988097A1May 9, 2002Nov 5, 2008Corixa CorporationCompositions and methods for the therapy and diagnosis of prostate cancerEP1992640A1Dec 22, 1998Nov 19, 2008Corixa CorporationCompounds for immunotherapy and diagnosis of breast cancer and methods for their useEP1998177A2Jan 6, 2004Dec 3, 2008WyethCompositions and methods for diagnosing and treating colon cancersEP2003201A2Mar 17, 1999Dec 17, 2008Corixa CorporationCompounds and methods for therapy and diagnosis of lung cancerEP2011510A2Jul 18, 2003Jan 7, 2009University of WashingtonPharmaceutical compositions comprising immunologically active herpes simplex virus (HSV) protein fragmentsEP2022800A2Jul 17, 2001Feb 11, 2009Corixa CorporationCompositions for the therapy and diagnosis of ovarian cancerEP2028190A1Apr 3, 2000Feb 25, 2009Corixa CorporationCompounds and methods for therapy and diagnosis of lung cancerEP2105502A1Nov 30, 2001Sep 30, 2009Corixa CorporationCompositions and methods for the therapy and diagnosis of lung cancerEP2133100A1Jun 20, 2001Dec 16, 2009Corixa CorporationMTB32A Antigen of mycobacterium tuberculosis with inactivated active site and fusion proteins thereofEP2133362A1Jul 23, 2004Dec 16, 2009Amgen, IncAntagonists and agonists of LDCAM and methods of useEP2143731A1Feb 25, 1998Jan 13, 2010Corixa CorporationCompounds for immunotherapy of prostate cancer and methods for their useEP2154248A1Aug 30, 1996Feb 17, 2010Corixa CorporationCompounds and methods for diagnosis of tuberculosisEP2172476A2Oct 30, 2002Apr 7, 2010Corixa CorporationCompositions and methods for WT1 specific immunotherapyEP2172551A2May 20, 1998Apr 7, 2010Corixa CorporationCompounds for immunotherapy and diagnosis of tuberculosis and methods of their useEP2192128A2Apr 23, 2001Jun 2, 2010Corixa CorporationCompounds and methods for treatment and diagnosis of chlamydial infectionEP2199801A2Jul 14, 2005Jun 23, 2010The Regents Of The University Of CaliforniaBiomarkers for early detection of ovarian cancerEP2218733A1Dec 8, 1999Aug 18, 2010Corixa CorporationCompounds and methods for treatment and diagnosis of chlamydial infectionEP2223935A2Dec 8, 1999Sep 1, 2010Corixa CorporationCompounds and methods for treatment and diagnosis of chlamydial infectionEP2224012A1Dec 16, 2002Sep 1, 2010Corixa CorporationCompositions and methods for the therapy and diagnosis of inflammatory bowel diseaseEP2236519A1Sep 18, 2008Oct 6, 2010Amgen, IncHuman GM-CSF antigen binding proteinsEP2248900A1May 20, 1998Nov 10, 2010Corixa CorporationCompounds for immunotherapy and diagnosis of tuberculosis and methods of their useEP2261672A2Jul 14, 2005Dec 15, 2010The Regents of the University of CaliforniaBiomarkers for early detection of ovarian cancerEP2263686A1Jul 18, 2003Dec 22, 2010University of WashingtonPharmaceutical compositions comprising immunologically active herpes simplex virus (HSV) protein fragmentsEP2272859A2Aug 5, 1999Jan 12, 2011University of WashingtonImmunological herpes simplex virus antigens and methods for use thereofEP2277892A2Dec 8, 1999Jan 26, 2011Corixa CorporationCompounds and methods for treatment and diagnosis of chlamydial infectionEP2277893A2Dec 8, 1999Jan 26, 2011Corixa CorporationCompounds and methods for treatment and diagnosis of chlamydial infectionEP2292665A1May 25, 2001Mar 9, 2011Immunex CorporationUse of interleukin-4 antagonists and compositions thereofEP2298333A2Dec 19, 2000Mar 23, 2011Immunex CorporationTweak receptorEP2298334A2Dec 19, 2000Mar 23, 2011Immunex CorporationTweak receptorEP2298877A1Feb 25, 1998Mar 23, 2011Corixa CorporationCompounds for immunotherapy of prostate cancer and methods for their useEP2305701A1Jun 29, 2006Apr 6, 2011Forsyth Dental Infirmary for ChildrenTuberculosis antigen detection assays and vaccinesEP2316479A2Jul 18, 2003May 4, 2011University of WashingtonPharmaceutical compositions comprising immunologically active herpes simplex virus (HSV) protein fragmentsEP2322550A1Dec 20, 2005May 18, 2011Amgen, IncCompositions comprising anti-IGF-1R Antibodies and Methods for obtaining said AntibodiesEP2322551A2Dec 20, 2005May 18, 2011Amgen, IncCompositions comprising Anti-IGF-1R Antibodies and Methods for their ProductionEP2336177A1Aug 4, 2005Jun 22, 2011Amgen, IncAntibodies to DKK-1EP2363495A1May 30, 2007Sep 7, 2011Pfenex, Inc.Anthrax vaccineEP2386314A1Mar 24, 2006Nov 16, 2011GlaxoSmithKline Biologicals SAVaccines against chlamydial infectionEP2392347A2Mar 24, 2006Dec 7, 2011GlaxoSmithKline Biologicals S.A.Vaccines against chlamydial infectionEP2392348A2Mar 24, 2006Dec 7, 2011GlaxoSmithKline Biologicals S.A.Vaccines against chlamydial infectionEP2392349A2Mar 24, 2006Dec 7, 2011GlaxoSmithKline Biologicals S.A.Vaccines against chlamydial infectionEP2426141A2Apr 27, 2006Mar 7, 2012GlaxoSmithKline Biologicals S.A.Method for preventing or treating M tuberculosis infectionEP2441846A2Jan 9, 2007Apr 18, 2012The Regents Of the University of CaliforniaImmunostimulatory combinations of TNFRSF, TLR, NLR, RHR, purinergic receptor, and cytokine receptor agoinsts for vaccines and tumor immunotherapyEP2444418A2Sep 10, 2007Apr 25, 2012Amgen, IncIL-1 Family VariantsEP2457577A1Nov 24, 2004May 30, 2012Anjin CorporationDiphtheria toxin variantEP2457926A1Apr 27, 2006May 30, 2012GlaxoSmithKline Biologicals S.A.Novel method for preventing or treating m tuberculosis infectionEP2463299A2Mar 5, 2004Jun 13, 2012Dendreon CorporationCompositions and methods employing alternative reading frame polypeptides for the treatment of cancer and infectious diseaseEP2497831A1May 25, 2005Sep 12, 2012Oregon Health and Science UniversityTB vaccination using HCMV-based vaccine vectorsEP2559705A2Sep 7, 2007Feb 20, 2013Amgen Inc.Anti-activin a antibodies and uses thereofEP2562186A1Jun 26, 2008Feb 27, 2013Amgen Inc.Antigen binding proteins that bind PAR-2EP2589610A1Aug 19, 2008May 8, 2013Amgen, IncHuman c-fms antigen binding proteinsEP2592093A1Aug 19, 2008May 15, 2013Amgen, IncHuman c-fms antigen binding proteinsEP2605015A1Nov 9, 2007Jun 19, 2013Dimerix Bioscience Pty.Ltd.Kits for determining the interaction of a test compound with two associated receptorsEP2711375A1Dec 3, 2010Mar 26, 2014Amgen Inc.Human Antigen Binding Proteins that bind Beta-Klotho, FGF Receptors and complexes thereofWO1992006116A1 *Sep 26, 1991Mar 29, 1992Ortho Pharma CorpHybrid growth factorsWO1992006117A1 *Sep 27, 1991Apr 16, 1992Seragen IncInhibiting unwanted immune responsesWO1992013548A1 *Jan 31, 1992Aug 20, 1992Immunex CorpMethod of inhibiting replication of hiv in macrophagesWO1992016221A1 *Mar 13, 1992Sep 16, 1992Synergen IncPegylation of polypeptidesWO1993005169A1 *Aug 24, 1992Mar 18, 1993Hutchinson Fred Cancer ResHybrid cytokinesWO1995013393A1 *Nov 7, 1994May 18, 1995Cell Therapeutics IncHybrid cytokinesWO1998033914A1 *Jan 30, 1998Aug 6, 1998Univ CaliforniaChimeric antibody fusion proteins for the recruitment and stimulation of an antitumor immune responseWO1998037093A2Feb 25, 1998Aug 27, 1998Corixa CorpCompounds for immunotherapy of prostate cancer and methods for their useWO1998053075A2May 20, 1998Nov 26, 1998Corixa CorpCompounds for immunotherapy and diagnosis of tuberculosis and methods of their useWO2000008051A2Aug 5, 1999Feb 17, 2000Univ WashingtonImmunological herpes simplex virus antigens and methods for use thereofWO2000034483A2Dec 8, 1999Jun 15, 2000Ajay BhatiaCompounds and methods for treatment and diagnosis of chlamydial infectionWO2000060076A2Feb 15, 2000Oct 12, 2000Corixa CorpCompositions for the treatment and diagnosis of breast cancer and methods for their useWO2001045730A2Dec 19, 2000Jun 28, 2001Immunex CorpTweak receptorWO2001092340A2May 25, 2001Dec 6, 2001Immunex CorpUse of interleukin-4 antagonists and compositions thereofWO2001098460A2Jun 20, 2001Dec 27, 2001Corixa CorpFusion proteins of mycobacterium tuberculosisWO2003053220A2Dec 16, 2002Jul 3, 2003Corixa CorpCompositions and methods for the therapy and diagnosis of inflammatory bowel diseaseWO2004019866A2Aug 21, 2003Mar 11, 2004Immunex CorpCompositions and methods for treating cardiovascular diseaseWO2006117240A2Apr 27, 2006Nov 9, 2006Glaxosmithkline Biolog SaNovel method for preventing or treating m tuberculosis infectionWO2008031061A2Sep 7, 2007Mar 13, 2008Amgen IncAnti-activin a antibodies and uses thereofWO2008054603A2Oct 1, 2007Jul 3, 2008Amgen IncIl-17 receptor a antigen binding proteinsWO2009005726A1Jun 26, 2008Jan 8, 2009Amgen IncAntigen binding proteins that bind par-2WO2009014835A2Jun 20, 2008Jan 29, 2009Angelica Therapeutics IncModified toxinsWO2010045002A2Sep 26, 2009Apr 22, 2010Tocagen Inc.Gene therapy vectors and cytosine deaminasesWO2010047515A2Oct 20, 2009Apr 29, 2010Gwangju Institute Of Science And TechnologyBipodal peptide binderWO2010075238A1Dec 18, 2009Jul 1, 2010Amgen Inc.Human cgrp receptor binding proteinsWO2010075440A1Dec 22, 2009Jul 1, 2010Targeted Growth, Inc.Modified photosynthetic microorganisms with reduced glycogen and their use in producing carbon-based productsWO2010107752A2Mar 16, 2010Sep 23, 2010Amgen Inc.Alpha-4-beta-7 heterodimer specific antagonist antibodyWO2010132609A2May 12, 2010Nov 18, 2010The Regents Of The University Of CaliforniaMethods and compositions for treating neurodegenerative disorders and alzheimer's disease and improving normal memoryWO2011009624A1Jul 22, 2010Jan 27, 2011Cenix Bioscience GmbhDelivery system and conjugates for compound delivery via naturally occurring intracellular transport routesWO2011014671A1Jul 29, 2010Feb 3, 2011Amgen Inc.Polypeptides that bind tissue inhibitor of metalloproteinase type three (timp-3), compositions and methodsWO2011020079A1Aug 13, 2010Feb 17, 2011Calmune CorporationAntibodies against human respiratory syncytial virus (rsv) and methods of useWO2011046958A1Oct 12, 2010Apr 21, 2011Amgen Inc.Use of il-17 receptor a antigen binding proteinsWO2011056600A1Oct 26, 2010May 12, 2011Amgen Inc.Human il-23 antigen binding proteinsWO2011071783A1Dec 3, 2010Jun 16, 2011Amgen Inc.Human antigen binding proteins that bind beta-klotho, fgf receptors and complexes thereofWO2011072265A1Dec 10, 2010Jun 16, 2011Atyr Pharma, Inc.Aminoacyl trna synthetases for modulating inflammationWO2011072266A2Dec 10, 2010Jun 16, 2011Atyr Pharma, Inc.Aminoacyl trna synthetases for modulating hematopoiesisWO2011092253A1Jan 27, 2011Aug 4, 2011Glaxosmithkline Biologicals S.A.Modified tuberculosis antigensWO2011127069A1Apr 5, 2011Oct 13, 2011Targeted Growth, Inc.Modified photosynthetic microorganisms for producing lipidsWO2011127418A1Apr 8, 2011Oct 13, 2011Amgen Inc.Btnl9 proteins, nucleic acids, and antibodies and uses thereofWO2011130417A2Apr 13, 2011Oct 20, 2011Amgen Inc.HUMAN FGF RECEPTOR AND β-KLOTHO BINDING PROTEINSWO2012006596A2Jul 8, 2011Jan 12, 2012Calmune CorporationAnti-human respiratory syncytial virus (rsv) antibodies and methods of useWO2012024242A1Aug 15, 2011Feb 23, 2012Amgen Inc.Antibodies that bind myostatin, compositions and methodsWO2012057904A1Aug 16, 2011May 3, 2012Infectious Disease Research InstituteMycobacterium tuberculosis antigens and combinations thereof having high seroreactivityWO2012058393A2Oct 27, 2011May 3, 2012Amgen Inc.Dkk1 antibodies and methods of useWO2012064659A1Nov 7, 2011May 18, 2012Infectious Disease Research InstituteVaccines comprising non-specific nucleoside hydrolase and sterol 24-c-methyltransferase (smt) polypeptides for the treatment and diagnosis of leishmaniasisWO2012087963A1Dec 19, 2011Jun 28, 2012Targeted Growth, Inc.Modified photosynthetic microorganisms for producing lipidsWO2012087982A2Dec 19, 2011Jun 28, 2012Targeted Growth, Inc.Modified photosynthetic microorganisms for producing lipidsWO2012101235A1Jan 26, 2012Aug 2, 2012Cenix Bioscience GmbhDelivery system and conjugates for compound delivery via naturally occurring intracellular transport routesWO2012106556A2Feb 2, 2012Aug 9, 2012Amgen Inc.Methods and compositons relating to inhibition of igf-1rWO2012118903A2Feb 29, 2012Sep 7, 2012Amgen Inc.Bispecific binding agentsWO2012177595A1Jun 19, 2012Dec 27, 2012Oncofactor CorporationCompositions and methods for the therapy and diagnosis of cancerWO2013015821A1Sep 19, 2011Jan 31, 2013The Research Foundation Of State University Of New YorkAntibodies to the b12-transcobalamin receptorWO2013043933A2Sep 20, 2012Mar 28, 2013Amgen Inc.Cd27l antigen binding proteinsWO2013063155A2Oct 24, 2012May 2, 2013Halozyme, Inc.Companion diagnostic for anti-hyaluronan agent therapy and methods of use thereofWO2013086443A1Dec 7, 2012Jun 13, 2013Amgen Inc.Agonistic human lcat antigen binding proteins and their use in therapyWO2013128027A1Mar 1, 2013Sep 6, 2013Amgen Research (Munich) GmbhLong life polypeptide binding moleculesWO2013134577A2Mar 8, 2013Sep 12, 2013Detectogen, Inc.Leishmaniasis antigen detection assays and vaccinesWO2013134743A1Mar 8, 2013Sep 12, 2013Halozyme, Inc.Conditionally active anti-epidermal growth factor receptor antibodies and methods of use thereofWO2013163377A1Apr 25, 2013Oct 31, 2013Novo Nordisk A/SHuman cd30 ligand antigen binding proteinsWO2013169734A1May 7, 2013Nov 14, 2013Amgen Inc.Anti-erythropoietin antibodiesWO2014022515A1Jul 31, 2013Feb 6, 2014Bioasis Technologies, Inc.Dephosphorylated lysosomal storage disease proteins and methods of use thereofWO2014062856A1Oct 16, 2013Apr 24, 2014Halozyme, Inc.Hypoxia and hyaluronan and markers thereof for diagnosis and monitoring of diseases and conditions and related methodsWO2014062963A1Oct 17, 2013Apr 24, 2014Amgen Inc.Methods and compositions relating to anti-il-21 receptor antibodiesWO2014089335A2Dec 5, 2013Jun 12, 2014Amgen Inc.Bcma antigen binding proteins* Cited by examinerClassifications U.S. Classification424/85.5, 435/69.51, 424/85.6, 530/351, 424/85.7International ClassificationC12N1/21, C07K14/52, C12N15/00, A61P35/00, A61K38/00, A61P37/00, C07K14/565, C07K14/00, C07K14/57, C12P21/00, A61P31/12, C07K14/555, C07H21/04, C07K14/54, C12R1/19, C12N15/70, C07K14/475, C07K14/525, A61K38/22, C12N15/09, A61K38/21, C12P21/02, C12N15/62, C07K19/00Cooperative ClassificationY10S930/142, Y10S930/143, C07K2319/00, C07K2319/55, C07K2319/75, C07K14/52, C12N15/70, A61K38/00, C07K14/57, C07K14/475, C12N15/62, C07K14/565European ClassificationC07K14/565, C07K14/57, C07K14/475, C12N15/70, C07K14/52, C12N15/62Legal EventsDateCodeEventDescriptionJan 9, 2002REMIMaintenance fee reminder mailedDec 18, 2001FPAYFee paymentYear of fee payment: 12Sep 30, 1997FPAYFee paymentYear of fee payment: 8Dec 7, 1993CCCertificate of correctionDec 3, 1993FPAYFee paymentYear of fee payment: 4Feb 26, 1991ASAssignmentOwner name: G.D. 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