Production of a tumour-targeting antibody with a human-compatible glycosylation profile in N. benthamiana hairy root cultures.
Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant-type glycans. Here, we expressed the tumor-targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco-engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2-3 mg/L). N-glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant-typical complex structures for N. tabacum-derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT-derived counterpart. Both antibody glyco-formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co-infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor-targeting mAb H10 with a human-compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of 'next generation' human therapeutic antibodies.
Laboratory of Biotechnology ENEA Research Center, Rome, Italy.
Chiara
C
Lonoce
Laboratory of Biotechnology ENEA Research Center, Rome, Italy.
Reda
R
Salem
Laboratory of Biotechnology ENEA Research Center, Rome, Italy.
Carla
C
Marusic
Département de phytologie, Centre de recherche et innovation des végétaux, Université Laval, Québec, Québec, Canada.
Philippe V
PV
Jutras
Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, Napoli, Italy.
Andrea
A
Scaloni
Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, Napoli, Italy.
Anna Maria
AM
Salzano
Laboratory of Biotechnology ENEA Research Center, Rome, Italy.
Sergio
S
Lucretti
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
Herta
H
Steinkellner
Laboratory of Biotechnology ENEA Research Center, Rome, Italy.
Eugenio
E
Benvenuto
Laboratory of Biotechnology ENEA Research Center, Rome, Italy. marcello.donini@enea.it.
Marcello
M
Donini
D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000254:growth & development; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005643:Fucose / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D009369:Neoplasms / Q000378:metabolism; D018517:Plant Roots / Q000235:genetics* / Q000254:growth & development / Q000382:microbiology; D030821:Plants, Genetically Modified / Q000254:growth & development / Q000378:metabolism / Q000382:microbiology; D011134:Polysaccharides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development / Q000382:microbiology
D000911:Antibodies, Monoclonal; D011134:Polysaccharides; D011994:Recombinant Proteins; D005643:Fucose
Glycosylation; Hairy roots; Molecular farming; Proteolysis; Rhizosecretion; mAb