Plant pharming of a full‐sized, tumour‐targeting antibody using different expression strategies

Abstract

SummaryThe 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 n M ), 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.

Antibody proteolysis: a common picture emerging from plants

“…3,4 In the case of antibodies, unintended proteolysis driven by this complex peptidase repertoire can affect the final yield of intact IgG, and may lead to almost complete degradation. 5 The presence of antibody fragments in plants has been ascribed to the following 3 phenomena: i) partial assembly intermediates; ii) extracellular peptidase activity after secretion 6 ; iii) activity of peptidases released during sample homogenization. 7 Additionally, fragments resulting from a first "opening cleavage" might be further processed into smaller fragments by plant proteases.…”

“…7 Additionally, fragments resulting from a first "opening cleavage" might be further processed into smaller fragments by plant proteases. 8 Recent studies have been focused on the identification of cleavage fragments resulting from in planta proteolysis by either mass spectrometry (MS) 5,9 or Nterminal sequencing by Edman degradation. 10 Mass analysis allowed 2 major degradation products to be identified, compatible with a cleavage presumably occurring close to the heavy chain (HC) hinge region of the H10 antibody.…”

“…In previous studies we demonstrated that the HC is specifically cleaved in plants yielding protein fragments of about 15 kDa and 25 kDa on reducing gel electrophoresis. 5,11 For this reason we expected that higher MW spots (»50 kDa) corresponded to the complete HC and the selected spots could represent HC-derived fragments. Spots 1-6 have a molecular weight of »25 kDa, and are distributed within the pI range 4-7.…”

Tobacco plant: A possible key to Ebola vaccine

“…The virus gets entered into body through the gastrointestinal tract, conjunctiva or breaks in the skin the body. The maximum virus concentrations are found in blood while lower levels are present in urine and throat washings [14,15].…”

Developments in Plant-Based Vaccines Against Diseases of Concern in Developing Countries

“…The Agrobacterium-based method, called "agroinfiltration", involves the injection or vacuum infiltration of whole plants or parts of them with a suspension of bacteria harboring the construct of interest. This approach has a wide spectrum of applications and has resulted useful for the study of glycosylation [30], protein-protein interaction [31], conformation, folding, virus-like particles formation [32] and to produce interesting molecules for the therapeutic industry as monoclonal antibodies [25,33] and antigens of human pathogens [34].…”