A fast and easy one-step purification strategy for plant-made antibodies using Protein A magnetic beads

A major difficulty to reach commercial- scale production for plant-made antibodies is the complexity and cost of their purification from plant extracts. Here, using Protein A magnetic beads, two monoclonal antibodies are purified in a one-step procedure directly from non-clarified crude plant extracts. This technique provides significant savings in terms of resources, operation time, and equipment

Exploring the Potentiality of a Plant Platform for Monoclonal Antibody Production in Veterinary Medicine

Canine atopic dermatitis (CAD) is an allergic, inflammatory, and pruritic skin disease associated with the production of IgE antibodies against environmental allergens and mainly house dust mite allergens. This complex dermatological pathology involves Interleukin 31 (IL-31) as a central itch mediator. One of the most effective CAD treatments is a caninized monoclonal antibody (mAb) called Lokivetmab. It is produced in CHO cells and targets specifically canine IL-31 (cIL-31) and blocks its cellular messaging. This treatment has undoubtedly contributed to a breakthrough in dermatitis-related pruritus. However, its production in mammalian cells requires time-consuming procedures, high production costs, and investment. Plants are considered an emerging protein production platform for recombinant biopharmaceuticals due to their cost-effectiveness and rapidity for production. Here, we use transient expression in Nicotiana benthamiana plants to produce recombinant canine Interleukin 31 (cIL-31) and an anti-IL-31 monoclonal antibody (M1). First, we describe the production and characterization of M1 and then its activity on an IL-31-induced pruritic model in dogs compared to its commercial homolog. Dogs treated with the plant-made M1 mAb have shown similar improvements to Lokivetmab-treated ones after different challenges using canine IL-31. Furthermore, M1 injections were not associated with any side effects. These results demonstrate the safety and efficacy of this plant-made Lokivetmab biosimilar to control dogs’ pruritus in a well-established model. Finally, this study shows that the plant-production platform can be utilized to produce rapidly functional mAbs and bring hope to the immunotherapy field of veterinary medicine

Targeting of proConA to the Plant Vacuole depends on its Nine Amino-acid C-terminal Propeptide

Concanavalin A (ConA) is a well characterized and extensively used lectin accumulated in the protein bodies of jack bean cotyledons. ConA is synthesized as an inactive precursor proConA. The maturation of inactive proConA into biologically active ConA is a complex process including the removal of an internal glycopeptide and a C-terminal propeptide (CTPP), followed by a head-to-tail ligation of the two largest polypeptides. The cDNA encoding proConA was cloned and expressed in tobacco BY-2 cells. ProConA was slowly transported to the vacuole where its maturation into ConA was similar to that in jack bean cotyledons, apart from an incomplete final ligation. To investigate the role of the nine amino acid CTPP, a truncated form lacking the propeptide (proConAΔ9) was expressed in BY-2 cells. In contrast to proConA, proConAΔ9 was rapidly chased out of the endoplasmic reticulum (ER) and secreted into the culture medium. The CTPP was then fused to the C-terminal end of a secreted form of green fluorescent protein (secGFP). When expressed in tobacco BY-2 cells and leaf protoplasts, the chimaeric protein was located in the vacuole whereas secGFP was located in the culture medium and in the vacuole. Altogether, our results show we have isolated a new C-terminal vacuolar sorting determinan

Transport et rétention d une protéine membranaire de type II dans le réticulum endoplasmique d une cellule végétale

ROUEN-BU Sciences (764512102) / SudocROUEN-BU Sciences Madrillet (765752101) / SudocSudocFranceF

Identification et caractérisation de l alpha-glucosidase II d Arabidopsis thaliana

Les rôles de la maturation des N-glycannes sont peu connus chez les plantes. Notre étude s est focalisée sur les étapes de maturation des N-glycannes, catalysées par l a -glucosidase II, qui permet de cliver les 2 résidus glucose liés en a (1,3) sur l oligosaccharide précurseur. Cette enzyme est la seule enzyme de maturation des N-glycannes à être soluble, composée d une sous-unité a catalytique et d une sous-unité b de rétention de l hétérodimère dans le RE. L a glucosidase II végétale n avait jamais été caractérisée. Deux gènes codant un homologue de lasous-unité alpha et un, un homologue de la sous-unité b, ont été identifiés chez A. thaliana. Nous avons montré qu At5g63840 code une sous-unité a d a -glucosidase II nommée AtGCSIIa. Malheureusement, il a été impossible de définir l activité de la sous-unité b potentielle. Cette étude a été complétée par l étude du mutant rsw3 d A. thaliana, confirmant le rôle majeur d AtGCSIIa dans la maturation des N-glycannes chez A. thaliana.The roles of N-glycan maturation in plants remain to be established. This study is focused on the early steps of N-glycan maturation catalyzed by -glucosidase II. It hydrolyzes the a(1,3) glucose residues. a-glucosidase II is the only soluble heterodimeric N-glycan maturation enzyme, composed of a catalytic a subunit and an ER retention b subunit. No plant a glucosidase II was characterized before. We identified 2 potential a subunit-encoding gene and 1 potential b subunit-encoding gene in A. thaliana genome. We have characterized these genes and the activity of the proteins they encode through their expression in S. pombe mutants devoid of a-glucosidase II activity. We have shown that At5g63840 encodes an a subunit of a glucosidase II that we named AtGCSIIa. Unfortunately, we were unable to define the potential b subunit activity. This study was completed by the analysis of the A. thaliana rsw3 mutant and confirmed that AtGCSIIa is essential in N-glycans maturation in A. thalianaROUEN-BU Sciences (764512102) / SudocSudocFranceF

Modifications post-traductionnelles d une serpine humaine recombinante exprimée chez les plantes

ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Inactivation de l'a(1.3)-fucosyltransférase et de la b(1.2)-xylotransférase, en vue de la production de protéines recombinantes d'intérêt thérapeutique chez la luzerne

Ces travaux développent la stratégie de PTGS dans l'optique d'inhiber les activités de l'a(1.3)-fucosyltransférase et de la b(1.2)-xylotransférase chez Medicago sativa. Dans cette perspective, nous avons cloné et caractérisé les ADNc codant ces deux glycosyltransférases. Pour induire chez M. sativa le PTGS, nous avons transformé des plantes avec des constructions sens, antisens et intron-hairpin ARN (ihpARN) ciblant soit les a(1.3)-FucT, soit la b(1.2)-XylT. Nous avons sélectionné un transformant qui présente une forte diminution du niveau de l'a(1.3)-fucosylation de ses glycoprotéines et deux plantes présentant une diminution de la b(1.2)-xylosylation. Chez ces plantes, nous avons montré une forte diminution des transcrits correspondant aux glycotransférases ciblées. Enfin, nous avons démontré que les constructions " inhibitrices " réalisées à partir de l'ADNc de la b(1.2)-XylT de M. sativa, sont capables d'induire le PTGS dans un système hétérologue : les cellules de tabac BY-2.This work develops the strategy of PTGS with the aim of inhibiting a(1.3)-fucosyltransferase (FucT) and b(1.2)-xylotransferase (XylT) activities in Medicago sativa. From this perspective, the cDNAs coding for these two glycoltransferases have been cloned and characterized. To induce the PTGS in M. sativa, we have transformed plants with sens, antisens and intron-hairpin ARN (ihpARN) constructs targeting either a(1.3)-FucT or b(1.2)-XylT. Then, we have selected one transformant presenting a decrease in a(1.3)-fucosylation of its glycoproteins and two plants presenting a reduction in the b(1.2)-xylosylation. In addition, in these plants, we have shown a strong diminution in the amount of a(1.3)-FucT and b(1.2)-XylT transcripts respectively. Furthermore, we have demonstrated the inhibiting constructs realized from the M. sativa b(1.2)-XylT cDNA are able to induce the PTGS in a heterologous system: tobacco BY-2 cells.ROUEN-BU Sciences (764512102) / SudocSudocFranceF