Recombinant production of authentic human proteins using human cell expression systems

Abstract

The present invention relates to novel expression cassettes and vectors for efficiently producing authentic recombinant human proteins from stable cultures of novel human cell lines, the authentic recombinant proteins produced therefrom, and antibodies raised against those authentic recombinant proteins.

1 .- 65 . (canceled) 
     
     
         66 . An isolated recombinant authentic human cytokine, wherein the cytokine comprises LEFTY1 or LEFTY2. 
     
     
         67 . The isolated recombinant authentic human cytokine of  claim 66 , wherein the authentic human cytokine is more similar to a native version of the cytokine than to a version of the cytokine expressed in a non-human cell with respect to one or more of size, structure, molecular weight, glycosylation pattern, and post-transcriptional modifications. 
     
     
         68 . The isolated recombinant authentic human cytokine of  claim 66 , wherein the cytokine comprises LEFTY1. 
     
     
         69 . The isolated recombinant authentic human cytokine of  claim 66 , wherein the cytokine comprises LEFTY2. 
     
     
         70 . An isolated recombinant authentic human cytokine, wherein the cytokine is produced by a human cell comprising an expression vector comprising a cassette comprising the following operably linked expression elements:
 (A) a human cytomegalovirus immediate early enhancer element;   (B) a functional human beta-actin promoter;   (C) a human globin gene intron; and   (D) a human signal peptide consisting of a immunoglobulin superfamily 8 signal peptide, or an alpha-fibrinogen signal peptide,   wherein the cytokine is encoded by and expressed from the vector, and   wherein the cytokine comprises LEFTY1 or LEFTY2.   
     
     
         71 . The isolated recombinant authentic human cytokine of  claim 70 , wherein the authentic human cytokine is more similar to a native version of the cytokine than to a version of the cytokine expressed in a non-human cell with respect to one or more of size, structure, molecular weight, glycosylation pattern, and post-transcriptional modifications. 
     
     
         72 . The isolated recombinant authentic human cytokine of  claim 70 , wherein the cytokine comprises LEFTY1. 
     
     
         73 . The isolated recombinant authentic human cytokine of  claim 70 , wherein the cytokine comprises LEFTY2. 
     
     
         74 . A composition comprising an isolated recombinant authentic human cytokine produced by a human cell comprising an expression vector comprising a cassette comprising the following operably linked expression elements:
 (A) a human cytomegalovirus immediate early enhancer element;   (B) a functional human beta-actin promoter;   (C) a human globin gene intron; and   (D) a human signal peptide consisting of a immunoglobulin superfamily 8 signal peptide, or an alpha-fibrinogen signal peptide,   wherein the cytokine is encoded by and expressed from the vector, and   wherein the cytokine comprises LEFTY1 or LEFTY2.   
     
     
         75 . The composition of  claim 74 , wherein the authentic human cytokine is more similar to a native version of the cytokine than to a version of the cytokine expressed in a non-human cell with respect to one or more of size, structure, molecular weight, glycosylation pattern, and post-transcriptional modifications. 
     
     
         76 . The composition of  claim 74 , wherein the cytokine comprises LEFTY1. 
     
     
         77 . The composition of  claim 74 , wherein the cytokine comprises LEFTY2. 
     
     
         78 . The composition of  claim 74 , further comprising one or more additional cytokines. 
     
     
         79 . The composition of  claim 78 , wherein the one or more additional cytokines comprises a cytokine selected from the group consisting of Activin A, Activin B, Activin A/2xINHbA, Activin B/2xINHbB, AMH/MIS, Artemin, BDNF, BMP2, BMP15/GDF9B, BMP2/BMP2A, BMP3/Osteogenin, BMP4, BMP4/BMP2B, BMP5, BMP7/OP-1, BMP1, BMP10, BMP15/GDF9B, β-NGF, Cystatin C, Delta 1, EGF, Erythropoietin (EPO), FGF acidic, FGF basic, FGF10, FGF5, FGF7, FGF8b, FLT3 ligand, G-CSF, GDF15, GDF2/BMP9, GDF3, GDF5/BMP14, GDF8/myostatin, GDF9, GDNF, GM-CSF, HGF, HGH, IFN-α2A, IFN-α2B, IFN-γ, IFN-β1, IGF I, IGF II, IGF IIv1, IGF IIv2, IL10, IL11, IL12, IL15, IL17/IL17A, IL17F, IL1 β IL2, IL23, IL27, IL28A/IFN-lambda-2, IL28B/IFN-lambda-3, IL29/IFN-lambda-1, IL3, IL32, IL35, IL4, IL5, IL6, IL7, IL8, IL9, Inhibin A/INHa&INHbA, Inhibin B/INHa&INHbB, Inhibin C/INHa&INHbC, Inhibin E/INHa&INHbE, M-CSF, mouse CSF, mouse SCF, NODAL, Noggin, NT3 (neurotrophin3), Oncostatin M, PDGFα, PDGFβ, Persephin, SCF, SDFla, SHH, Somatotropin, TGF β1, TGF β2, TGF β3, TNF α, TPOα, VEGF121aa, VEGF165aa, WIF1, WNT1, Wnt10A, Wnt10B/12, Wnt11, Wnt16, Wnt2, Wnt2B/13, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8B, and Wnt9A/14. 
     
     
         80 . The composition of  claim 74 , further comprising an anti-tumor drug. 
     
     
         81 . The composition of  claim 80 , wherein the anti-tumor drug comprises one or more anti-tumor drugs selected from the group consisting of Panorex® (edrecolomab), Rituxan® (rituximab), Herceptin® (traztuzumab), Mylotarg® (gentuzumab), Campath® (alemtuzumab), Zevalin™ (ibritumomab), Erbitux™ (cetuximab), and Avastin™ (bevicizumab). 
     
     
         82 . A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising the isolated recombinant human cytokine of  claim 66 . 
     
     
         83 . The method of  claim 82 , wherein the authentic human cytokine is more similar to a native version of the cytokine than to a version of the cytokine expressed in a non-human cell with respect to one or more of size, structure, molecular weight, glycosylation pattern, and post-transcriptional modifications. 
     
     
         84 . The method of  claim 82 , wherein the cytokine comprises LEFTY1. 
     
     
         85 . The method of  claim 82 , wherein the cytokine comprises LEFTY2. 
     
     
         86 . The method of  claim 82 , further comprising administering to the subject one or more additional cytokines. 
     
     
         87 . The method of  claim 86 , wherein the one or more additional cytokines comprises a cytokine selected from the group consisting of Activin A, Activin B, Activin A/2xINHbA, Activin B/2xINHbB, AMH/MIS, Artemin, BDNF, BMP2, BMP15/GDF9B, BMP2/BMP2A, BMP3/Osteogenin, BMP4, BMP4/BMP2B, BMP5, BMP7/OP-1, BMP1, BMP10, BMP15/GDF9B, β-NGF, Cystatin C, Delta 1, EGF, Erythropoietin (EPO), FGF acidic, FGF basic, FGF10, FGF5, FGF7, FGF8b, FLT3 ligand, G-CSF, GDF15, GDF2/BMP9, GDF3, GDF5/BMP14, GDF8/myostatin, GDF9, GDNF, GM-CSF, HGF, HGH, IFN-α2A, IFN-α2B, IFN-γ, IFN-β 1 , IGF I, IGF II, IGF IIv1, IGF IIv2, IL10, IL11, IL12, IL15, IL17/IL17A, IL17F, IL1 β IL2, IL23, IL27, IL28A/IFN-lambda-2, IL28B/IFN-lambda-3, IL29/IFN-lambda-1, IL3, IL32, IL35, IL4, IL5, IL6, IL7, IL8, IL9, Inhibin A/INHa&INHbA, Inhibin B/INHa&INHbB, Inhibin C/INHa&INHbC, Inhibin E/INHa&INHbE, M-CSF, mouse CSF, mouse SCF, NODAL, Noggin, NT3 (neurotrophin3), Oncostatin M, PDGFα, PDGFβ, Persephin, SCF, SDF1α, SHH, Somatotropin, TGF β1, TGF β2, TGF β3, TNF α, TPOα, VEGF121aa, VEGF165aa, WIF1, WNT1, Wnt10A, Wnt10B/12, Wnt11, Wnt16, Wnt2, Wnt2B/13, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8B, and Wnt9A/14. 
     
     
         88 . The method of  claim 82 , further comprising administering to the subject an anti-tumor drug. 
     
     
         89 . The method of  claim 88 , wherein the anti-tumor drug comprises one or more anti-tumor drugs selected from the group consisting of Panorex® (edrecolomab), Rituxan® (rituximab), Herceptin® (traztuzumab), Mylotarg® (gentuzumab), Campath® (alemtuzumab), Zevalin™ (ibritumomab), Erbitux™ (cetuximab), and Avastin™ (bevicizumab).