title: Utility of the P19 suppressor of gene-silencing protein for production of therapeutic antibodies in Nicotiana expression hosts.
abstract: To study how the P19 suppressor of gene-silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different P19 concentrations; compatibility of P19 with various Nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co-expressed with P19 in an RNAi-based glycomodified Nicotiana benthamiana expression host. The coding sequences for the heavy and light chains of trastuzumab were cloned into five plant expression vectors (102-106) containing different 5' and 3' UTRs, designated as vector sets 102-106 mAb. The P19 protein of Tomato bushy stunt virus (TBSV) was also cloned into vector 103, which contained the Cauliflower mosaic virus (CaMV) 35S promoter and 5'UTR together with the terminator region of the nopaline synthase gene of Agrobacterium. Transient expression of the antibody vectors resulted in different levels of trastuzumab accumulation, the highest being 105 and 106 mAb at about 1% of TSP. P19 increased the concentration of trastuzumab approximately 15-fold (to about 2.3% of TSP) when co-expressed with 103 mAb but did not affect antibody levels with vectors 102 and 106 mAb. When 103 mAb was expressed together with P19 in different N. tabacum cultivars, all except Little Crittenden showed a marked discolouring of the infiltrated areas of the leaf and decreased antibody expression. Co-expression of P19 also abolished antibody accumulation in crosses between N. tabacum cv. I-64 and Little Crittenden, indicating a dominant mode of inheritance for the observed P19-induced responses.
authors.affiliation: School of Environmental Sciences, University of Guelph, Guelph, ON, Canada.
authors.forename: Freydoun
authors.initials: F
authors.lastname: Garabagi
authors.forename: Erin
authors.initials: E
authors.lastname: Gilbert
authors.forename: Andreas
authors.initials: A
authors.lastname: Loos
authors.forename: Michael D
authors.initials: MD
authors.lastname: McLean
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Hall
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis*; D005647:Fucosyltransferases / Q000378:metabolism; D020868:Gene Silencing; D010430:Pentosyltransferases / Q000378:metabolism; D046549:Plantibodies / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013045:Species Specificity; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D000068878:Trastuzumab; D014764:Viral Proteins / Q000378:metabolism*; D000097608:UDP Xylose-Protein Xylosyltransferase
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D005647:Fucosyltransferases; D010430:Pentosyltransferases; D046549:Plantibodies; D011994:Recombinant Proteins; D000068878:Trastuzumab; D014764:Viral Proteins; D000097608:UDP Xylose-Protein Xylosyltransferase; C493885:P19 protein, tomato bushy stunt virus