The Arabidopsis thaliana ortholog of a purported maize cholinesterase gene encodes a GDSL-lipase.

Acetylcholinesterase is an enzyme that is intimately associated with regulation of synaptic transmission in the cholinergic nervous system and in neuromuscular junctions of animals. However the presence of cholinesterase activity has been described also in non-metazoan organisms such as slime molds, fungi and plants. More recently, a gene purportedly encoding for acetylcholinesterase was cloned from maize. We have cloned the Arabidopsis thaliana homolog of the Zea mays gene, At3g26430, and studied its biochemical properties. Our results indicate that the protein encoded by the gene exhibited lipase activity with preference to long chain substrates but did not hydrolyze choline esters. The At3g26430 protein belongs to the SGNH clan of serine hydrolases, and more specifically to the GDS(L) lipase family.

Antibodies to watch in 2020.

This 2020 installment of the annual 'Antibodies to Watch' series documents the antibody therapeutics approved in 2019 and in regulatory review in the United States or European Union, as well as those in late-stage clinical studies, as of November 2019*. At this time, a total of 5 novel antibody therapeutics (romosozumab, risankizumab, polatuzumab vedotin, brolucizumab, and crizanlizumab) had been granted a first approval in either the US or EU, and marketing applications for 13 novel antibody therapeutics (eptinezumab, teprotumumab, enfortumab vedotin, isatuximab, [fam-]trastuzumab deruxtecan, inebilizumab, leronlimab, sacituzumab govitecan, satralizumab, narsoplimab, tafasitamab, REGNEB3 and naxituximab) were undergoing review in these regions, which represent the major markets for antibody therapeutics. Also as of November 2019, 79 novel antibodies were undergoing evaluation in late-stage clinical studies. Of the 79 antibodies, 39 were undergoing evaluation in late-stage studies for non-cancer indications, with 2 of these (ublituximab, pamrevlumab) also in late-stage studies for cancer indications. Companies developing 7 (tanezumab, aducanumab, evinacumab, etrolizumab, sutimlimab, anifrolumab, and teplizumab) of the 39 drugs have indicated that they may submit a marketing application in either the US or EU in 2020. Of the 79 antibodies in late-stage studies, 40 were undergoing evaluation as treatments for cancer, and potentially 9 of these (belantamab mafodotin, oportuzumab monatox, margetuximab, dostarlimab, spartalizumab, 131I-omburtamab, loncastuximab tesirine, balstilimab, and zalifrelimab) may enter regulatory review in late 2019 or in 2020. Overall, the biopharmaceutical industry's clinical pipeline of antibody therapeutics is robust, and should provide a continuous supply of innovative products for patients in the future. *Note on key updates through December 18, 2019: 1) the US Food and Drug Administration granted accelerated approval to enfortumab vedotin-ejfv (Padcev) on December 18, 2019, bringing the total number of novel antibody therapeutics granted a first approval in either the US or EU during 2019 to 6; 2) the European Commission approved romosozumab on December 9, 2019; 3) the European Medicines Agency issued a positive opinion for brolucizumab; 4) Sesen Bio initiated a rolling biologics license application (BLA) on December 6, 2019; 5) GlaxoSmithKline submitted a BLA for belantamab mafodotin; and 6) the status of the Phase 3 study (NCT04128696) of GSK3359609, a humanized IgG4 anti-ICOS antibody, in patients with head and neck squamous cell carcinoma was updated to recruiting from not yet recruiting.

Plant-derived human acetylcholinesterase-R provides protection from lethal organophosphate poisoning and its chronic aftermath.

Therapeutically valuable proteins are often rare and/or unstable in their natural context, calling for production solutions in heterologous systems. A relevant example is that of the stress-induced, normally rare, and naturally unstable "read-through" human acetylcholinesterase variant, AChE-R. AChE-R shares its active site with the synaptic AChE-S variant, which is the target of poisonous organophosphate anticholinesterase insecticides such as the parathion metabolite paraoxon. Inherent AChE-R overproduction under organophosphate intoxication confers both short-term protection (as a bioscavenger) and long-term neuromuscular damages (as a regulator). Here we report the purification, characterization, and testing of human, endoplasmic reticulum-retained AChE-R(ER) produced from plant-optimized cDNA in Nicotiana benthamiana plants. AChE-R(ER) purified to homogeneity showed indistinguishable biochemical properties, with IC50 = 10(-7) M for the organophosphate paraoxon, similar to mammalian cell culture-derived AChE. In vivo titration showed dose-dependent protection by intravenously injected AChE-R(ER) of FVB/N male mice challenged with a lethal dose of paraoxon, with complete elimination of short-term clinical symptoms at near molar equivalence. By 10 days postexposure, AChE-R prophylaxis markedly limited postexposure increases in plasma murine AChE-R levels while minimizing the organophosphate-induced neuromuscular junction dismorphology. Our findings present plant-produced AChE-R(ER) as a bimodal agent, conferring both short- and long-term protection from organophosphate intoxication.

Characterizing pea acetylcholinesterase.

Although the best-defined role of acetylcholinesterase (AChE) is to terminate cholinergic transmission signals, the presence of the enzyme and its substrate is not limited to the metazoan nervous system. Here, we describe the presence of cholinesterase and its initial characterization in pea roots.

Purification of transgenic plant-derived recombinant human acetylcholinesterase-R.

Nicotiana benthamiana plants were engineered to express a codon-optimized gene encoding the human acetylcholinesterase-R (AChE) isoform. The transgenic plants expressed the protein at >0.4% of total soluble protein, and the plant-produced enzyme was purified to homogeneity. Following lysis, procainamide affinity chromatography and anion-exchange chromatography, more than 400-fold purification was achieved and electrophoretic purity was obtained. This pure protein is kinetically indistinguishable from the only commercially available source of human acetylcholinesterase, which is produced in mammalian cell culture. Thus, we have demonstrated a model system for the production of acetylcholinesterase, which is not susceptible to the quantitative limitations or mammalian pathogens associated with purification from mammalian cell culture or human serum.

A mucosally targeted subunit vaccine candidate eliciting HIV-1 transcytosis-blocking Abs.

A vaccine that would engage the mucosal immune system against a broad range of HIV-1 subtypes and prevent epithelial transmission is highly desirable. Here we report fusing the mucosal targeting B subunit of cholera toxin to the conserved galactosylceramide-binding domain (including the ELDKWA-neutralizing epitope) of the HIV-1 gp41 envelope protein, which mediates the transcytosis of HIV-1 across the mucosal epithelia. Chimeric protein expressed in bacteria or plants assembled into oligomers that were capable of binding galactosyl-ceramide and G(M1) gangliosides. Mucosal (intranasal) administration in mice of the purified chimeric protein followed by an i.p. boost resulted in transcytosis-neutralizing serum IgG and mucosal IgA responses and induced immunological memory. Plant production of mucosally targeted immunogens could be particularly useful for immunization programs in developing countries, where desirable product traits include low cost of manufacture, heat stability, and needle-free delivery.