SummaryThanks to their potential to bind virtually all types of molecules; monoclonal antibodies are in increasing demand as therapeutics and diagnostics. To overcome the overloading of current production facilities, alternative expression systems have been developed, of which plants appear the most promising. In this review, we focus on the expression of monoclonal IgG or IgM in plant species. We analyse the data for 32 different antibodies expressed in various ways, differing in DNA construction, transformation method, signal peptide source, presence or absence of an endoplasmic reticulum retention sequence, host species and the organs tested, together resulting in 98 reported combinations. A large heterogeneity is found in the quantity and quality of the antibody produced. We discuss in more detail the strategy used to express both chains, the nature of the transcription promoters, subcellular localization and unintended proteolysis, when encountered.

Genes encoding the heavy and light chains of LO-BM2, a therapeutic IgG antibody, were assembled in the tandem or inverted convergent orientation and expressed in Nicotiana tabacum plants and BY-2 suspension cells. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Degradation increased with leaf age or culture time. Antibodies purified from leaf tissues and BY-2 cells were both functional. However, MS analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas, oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted from BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment.

Background. This randomized, open trial compared regimens including 2 doses (2D) of human papillomavirus (HPV) 16/18 AS04-adjuvanted vaccine in girls aged 9–14 years with one including 3 doses (3D) in women aged 15–25 years.Methods. Girls aged 9–14 years were randomized to receive 2D at months 0 and 6 (M0,6; (n = 550) or months 0 and 12 (M0,12; n = 415), and women aged 15–25 years received 3D at months 0, 1, and 6 (n = 482). End points included noninferiority of HPV-16/18 antibodies by enzyme-linked immunosorbent assay for 2D (M0,6) versus 3D (primary), 2D (M0,12) versus 3D, and 2D (M0,6) versus 2D (M0,12); neutralizing antibodies; cell-mediated immunity; reactogenicity; and safety. Limits of noninferiority were predefined as <5% difference in seroconversion rate and <2-fold difference in geometric mean antibody titer ratio.Results. One month after the last dose, both 2D regimens in girls aged 9–14 years were noninferior to 3D in women aged 15–25 years and 2D (M0,12) was noninferior to 2D (M0,6). Geometric mean antibody titer ratios (3D/2D) for HPV-16 and HPV-18 were 1.09 (95% confidence interval, .97–1.22) and 0.85 (.76–.95) for 2D (M0,6) versus 3D and 0.89 (.79–1.01) and 0.75 (.67–.85) for 2D (M0,12) versus 3D. The safety profile was clinically acceptable in all groups.Conclusions. The 2D regimens for the HPV-16/18 AS04-adjuvanted vaccine in girls aged 9–14 years (M0,6 or M0,12) elicited HPV-16/18 immune responses that were noninferior to 3D in women aged 15–25 years.Clinical Trials Registration. NCT01381575.

This observer-blind study (clinicaltrials.gov NCT01462357) compared the immunogenicity and safety of 2 doses of the HPV-16/18 AS04-adjuvanted vaccine (HPV-16/18(2D)) vs. 2 or 3 doses of the HPV-6/11/16/18 vaccine (HPV-6/11/16/18(2D) and HPV-6/11/16/18(3D)) in healthy girls aged 9–14 y. Girls were randomized (1:1:1) to receive HPV-16/18(2D) at months (M) 0,6 (N = 359), HPV-6/11/16/18(2D) at M0,6 (N = 358) or HPV-6/11/16/18(3D) at M0,2,6 (N = 358). The primary objective was non-inferiority/superiority of HPV-16/18 antibodies by ELISA for HPV-16/18(2D) vs. HPV-6/11/16/18(2D) at M7 in the according-to-protocol immunogenicity cohort (ATP-I) and total vaccinated cohort, respectively. Secondary objectives included non-inferiority/superiority of HPV-16/18(2D) vs. HPV-6/11/16/18(3D) at M7, non-inferiority/superiority at M12, HPV-16/18 neutralizing antibodies, frequencies of T-cells/B-cells, reactogenicity and safety. Antibody responses at M7 for HPV-16/18(2D) were superior to those for HPV-6/11/16/18(2D) and HPV-6/11/16/18(3D) (lower limit of 95% confidence interval for geometric mean titer ratio (GMR) was >1): HPV-16/18(2D)/HPV-6/11/16/18(2D) GMRs were 1.69 [1.49–1.91] for anti-HPV-16 and 4.52 [3.97–5.13] for anti-HPV-18; HPV-16/18(2D)/HPV-6/11/16/18(3D) GMRs were 1.72 [1.54–1.93] for anti-HPV-16 and 3.22 [2.82–3.68] for anti-HPV-18; p = 0.0001 for all comparisons. Non-inferiority/superiority was also demonstrated at M12. Among initially seronegative girls in the ATP-I, neutralizing antibody titers were at least 1.8-fold higher for HPV-16/18(2D) vs. HPV-6/11/16/18(2D) and HPV-6/11/16/18(3D) at M7 and M12. Frequencies of HPV-16/18-specific T-cells and B-cells were in similar ranges between groups. Reactogenicity and safety were in line with the known profile of each vaccine. In conclusion, superior HPV-16/18 antibody responses were elicited by 2 doses of the HPV-16/18 AS04-adjuvanted vaccine compared with 2 or 3 doses of the HPV-6/11/16/18 vaccine in girls (9–14 years).

Recombinant proteins secreted from plant suspension cells into the medium are susceptible to degradation by host proteases secreted during growth. Some degradation phenomena are inhibited in the presence of various protease inhibitors, such as EDTA or AEBSF/PMSF, suggesting the presence of different classes of proteases in the medium. Here, we report the results of a proteomic analysis of the extracellular medium of a Nicotiana tabacum bright yellow 2 culture. Several serine proteases belonging to a Solanaceae-specific subtilase subfamily were identified and the genes for four cloned. Their expression at the RNA level during culture growth varied depending on the gene. An in-gel protease assay (zymography) demonstrated serine protease activity in the extracellular medium from cultures. This was confirmed by testing the degradation of an antibody added to the culture medium. This particular subtilase subfamily, therefore, represents an interesting target for gene silencing to improve recombinant protein production. Key message The extracellular medium of Nicotiana tabacum suspension cells contains serine proteases that degrade antibodies.