Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies.
The aims of this work were to obtain a human antibody against the tumour-associated antigen tenascin-C (TNC) and to compare the yield and quality of plant-produced antibody in either stable transgenics or using a transient expression system. To this end, the characterization of a full-sized human immunoglobulin G (IgG) [monoclonal antibody H10 (mAb H10)], derived from a selected single-chain variable fragment (scFv) and produced in plants, is presented. The human mAb gene was engineered for plant expression, and Nicotiana tabacum transgenic lines expressing both heavy (HC) and light (LC) chain were obtained and evaluated for antibody expression levels, in vivo assembly and functionality. Affinity-purified H10 from transgenics (yield, 0.6-1.1 mg/kg fresh weight) revealed that more than 90% of HC was specifically degraded, leading to the formation of functional antigen-binding fragments (Fab). Consequently, H10 was transiently expressed in Nicotiana benthamiana plants through an Agrobacterium-mediated gene-transfer system. Moreover, the use of the p19 silencing suppressor gene from artichoke mottled crinkle virus raised antibody expression levels by an order of magnitude (yields of purified H10, 50-100 mg/kg fresh weight). Approximately 75% of purified protein consisted of full-sized antibody functionally binding to TNC (K(D) = 14 nm), and immunohistochemical analysis on tumour tissues revealed specific accumulation around tumour blood vessels. The data indicate that the purification yields of mAb H10, using a transient expression system boosted by the p19 silencing suppressor, are exceptionally high when compared with the results reported previously, providing a technique for the over-expression of anticancer mAbs by a rapid, cost-effective, molecular farming approach.
Villani
Maria Elena
ME
ENEA, Dipartimento BAS, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, Via Anguillarese 301, I-00123, Rome, Italy.
Morgun
Bogdan
B
Brunetti
Patrizia
P
Marusic
Carla
C
Lombardi
Raffaele
R
Pisoni
Ivan
I
Bacci
Camilla
C
Desiderio
Angiola
A
Benvenuto
Eugenio
E
Donini
Marcello
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015870:Gene Expression; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009374:Neoplasms, Experimental / Q000276:immunology; D030821:Plants, Genetically Modified / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D019063:Tenascin / Q000037:antagonists & inhibitors*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D019063:Tenascin