Production of a tumour‐targeting antibody with a human‐compatible glycosylation profile in N. benthamiana hairy root cultures

Abstract

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.

Enhancing the Secretion of a Glyco-Engineered Anti-CD20 scFv-Fc Antibody in Hairy Root Cultures

“…Interestingly, a recent work showed that the treatment of roots expressing mAb M12 with NAA over a prolonged period caused an altered root phenotype, which was characterized by root hair proliferation, elongation and formation of nodular structures . This phenotype was more pronounced in roots induced with a higher concentration of NAA; the same phenomenon was also observed in HR cultures expressing the mAbH10 . Given these evidences, we have generated HR cultures expressing a secretory version of RFP, which then were used as a model system to study protein secretion from roots.…”

“…The A. rhizogenes A4 strain carrying the pBIΩ‐2B8‐Fc or pBIΩ‐RFP expression vector was used to transform wild type (WT) N. benthamiana (in the case of RFP) or ΔXTFT N. benthamiana (for 2B8‐Fc) leaf segments as described before . Briefly, recombinant A. rhizogenes A4 was grown in YEB medium to OD600 = 0.5, at 28 °C and 160 rpm.…”

“…Previously, we showed that it is possible to rapidly establish HR cultures secreting full size mAbs with a targeted glycosylation profile through a simple A. rhizogenes infection protocol applied to ΔXTFT N. benthamiana leaf segments . In the present study, we have exploited the advantages offered by these glyco‐engineered HR cultures as well as their stimulation with 2,4‐D in efficiently producing 2B8‐FcΔXF with a human compatible glycosylation profile in the culture medium.…”

“…The latter is the first antibody‐based drug approved for the treatment of patients with B‐cell lymphomas . The scFv‐Fc format has several advantages: i) it can be easily engineered for plant transient expression; ii) it generally retains its full antigen‐binding activity; iii) it maintains the Fc portion of human IgG1 immunoglobulins, which can activate different arms of human immune responses and can be efficiently expressed in plants or plant tissues at high levels . In a previous work, the recombinant 2B8‐Fc antibody was efficiently engineered in our laboratory and produced in N. benthamiana plants .…”

Expression of Single Chain Variable Fragment (scFv) Molecules in Plants: A Comprehensive Update

“…Well-characterized in vitro propagation, transformation, and maintenance methods of these plants are the factors behind their selection. The protein was expressed in whole plant tissue [66,68,69], leaves [70][71][72][73][74][75], flower [76], seeds [77][78][79][80][81], hairy root culture [82] or tobacco BY-2 (tobacco cultivar Bright Yellow) cells [8,83,84] (Fig. 4).…”