Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor.

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens.

A synthetic hemagglutinin (HA) gene from the highly pathogenic avian influenza (HPAI) virus A/chicken/Indonesia/7/2003 (H5N1) (Indo/03) was expressed in aquatic plant Lemna minor (rLemna-HA). In Experiment 1, efficacy of rLemna-HA was tested on birds immunized with 0.2µg or 2.3 µg HA and challenged with 10(6) mean chicken embryo infectious doses (EID50) of homologous virus strain. Both dosages of rLemna-HA conferred clinical protection and dramatically reduced viral shedding. Almost all the birds immunized with either dosage of rLemna-HA elicited HA antibody titers against Indo/03 antigen, suggesting an association between levels of anti-Indo/03 antibodies and protection. In Experiment 2, efficacy of rLemna-HA was tested on birds immunized with 0.9 µg or 2.2 µg HA and challenged with 10(6) EID50 of heterologous H5N1 virus strains A/chicken/Vietnam/NCVD-421/2010 (VN/10) or A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Birds challenged with VN/10 exhibited 100% survival regardless of immunization dosage, while birds challenged with PWT/06 had 50% and 30% mortality at 0.9 µg HA and 2.2 µg HA, respectively. For each challenge virus, viral shedding titers from 2.2 µg HA vaccinated birds were significantly lower than those from 0.9µg HA vaccinated birds, and titers from both immunized groups were in turn significantly lower than those from sham vaccinated birds. Even if immunized birds elicited HA titers against the vaccine antigen Indo/03, only the groups challenged with VN/10 developed humoral immunity against the challenge antigen. None (rLemna-HA 0.9 µg HA) and 40% (rLemna-HA 2.2 µg HA) of the immunized birds challenged with PWT/06 elicited pre-challenge antibody titers, respectively. In conclusion, Lemna-expressed HA demonstrated complete protective immunity against homologous challenge and suboptimal protection against heterologous challenge, the latter being similar to results from inactivated whole virus vaccines. Transgenic duckweed-derived HA could be a good alternative for producing high quality antigen for an injectable vaccine against H5N1 HPAI viruses.

An afucosylated anti-CD20 monoclonal antibody with greater antibody-dependent cellular cytotoxicity and B-cell depletion and lower complement-dependent cytotoxicity than rituximab.

The objective of this study was to characterize the in vitro and in vivo activity of a novel afucosylated rituximab (BLX-300) expressed in a Lemna aquatic plant-based system free of zoonotic pathogens. The glycosylation of BLX-300 was shown to be homogeneous, composed of a single major N-glycan species without detectable fucose or xylose. Target cell binding and induction of apoptosis were similar for BLX-300 and rituximab. Antibody-dependent cellular cytotoxicity (ADCC) was increased by BLX-300 versus rituximab in phenylalanine/phenylalanine (F/F), phenylalanine/valine (F/V) and valine/valine (V/V) genotype donors, as indicated by respective log reductions of 0.82, 1.07 and 0.92 in EC(50). BLX-300 also showed greater B-cell depletion than rituximab in whole blood from donors of F/F, F/V and V/V genotype in vitro and cynomolgus monkeys in vivo. Temporal changes in circulating levels of BLX-300 and rituximab were similar in cynomolgus monkeys. Complement-dependent cytotoxicity (CDC) was attenuated by BLX-300 relative to rituximab, as judged by a log increase of 0.51 in EC(50). The higher ADCC and B-cell depletion suggest a potential improvement in effectiveness and potency, while lower CDC may mitigate infusion toxicity.