CHO supernatants and whole leaf extracts are prepared for purification.

responses and rapid treatment or booster administrations.

attachment domain (PRAD) of ColQ gene (Altamirano & Lockridge, 1999).

symptoms of Gulf War illness in soldiers exposed to these compounds (US DOD, Pesticides-Final Report, 2003).

Currently, the standard (approved) treatment for acute OP pesticide poisoning involves administration of intravenous (iv) atropine and an oxime e.g. obidoxime, pralidoxime to reactivate inhibited AChE (Worek et al., 2010). However, the effectiveness and safety of oxime administration in acute OP pesticide-poisoned patients has been challenged and a recent clinical trial showed no clinical benefits and a trend towards harm in all sub-groups, despite clear evidence that these doses reactivated AChE in the blood (Buckley et al., 2011).

An efficacious prophylactic therapeutic treatment for preventing insecticide poisoning that can bind and scavenge the OP before it reaches and targets AChE in neuromuscular junctions is therefore a high priority. The leading candidate of this type is native (plasma) butyrylcholinesterase (BChE) whose potent OP bioscavenging ability has been demonstrated in many animal models and against varied OP neurotoxins (Doctor et al., 2001; Lenz et al., 2001). While several new catalytic and other stoichiometric enzymes also exhibit this ability (Lenz et al., 2007), based on availability, broad spectrum efficacy, stability and safety (Sun et al., 2005), BChE is the most advanced in terms of development of a human treatment. In Turkey, frozen plasma (BChE levels of 3,000 - 5,700 units) given as an alternative or adjunctive treatment with atropine and oximes, has been shown to prevent mortality and intermediate syndrome in acutely insecticide-exposed and hospitalized individuals (Güven et al., 2004). Currently, BChE also finds use as a treatment of cocaine overdose and for the alleviation of succinylcholine-induced apnea.

Structurally, BChE (also known as pseudocholinesterase or non-specific cholinesterase) is a serine esterase (MW=345,000) comprised of four identical subunits each containing 574 amino acids, held together by non-covalent bonds, with 36 carbohyrdrate chains (23.9% by weight) (Lockridge, 1990; Nachon et al., 2002). BChE is found in all species at levels of 1-20 ug/ml in plasma (Rosenberg, unp. data) and is also abundant in liver, intestine and lung. Recombinant (r) human butyryl-cholinesterase (HuBChE), like the native form, is also a potent bioscavenger of OP neurotoxins (Doctor et al., 2001; Lenz et al., 2001; Raveh et al., 1997) but its development as a human treatment for pesticide exposure has been disadvantaged by: (i) poor in vivo stability (bioavailability) of the unmodified forms and the presence of potentially immunogenic glycans using certain expression systems (ii) a 1:1 stoichiometry between the enzyme and OP (Raveh et al., 1997) and (iii) the high LD50 of insecticides (ug-mg/kg levels). This necessitates the delivery of large, costly, rBChE doses to detoxify exposed individuals which is problematic when intramuscular (im) or subcutaneous (sc) injections are the chosen routes of delivery. In this chapter, we shall describe our experience of how the chemistry, glycosylation, chemical modification, animal model and route of administration may reduce or enhance the potential of BChE bioscavengers as prophylactic therapeutic human antidotes for OP insecticide exposure.

#### **2. Production of tetrameric and monomeric forms of rMaBChE and rHuBChE**

Macaque (Ma) and human (Hu) BChE molecules are very similar molecules differing by only 22 amino acids and sharing ~96% DNA sequence identity, critical glycosylation sites, cysteines and disulfide bridging (Boeck et al., 2002; Rosenberg et al., 2010). Thus, most anti-BChE antisera react with both molecules. Native HuBChE and MaBChE in plasma are composed predominantly of tetramers (98%) with the tetramerization domain being located within the last 40 C-terminal residues of each monomeric subunit (534-574) (Blong et al.,

symptoms of Gulf War illness in soldiers exposed to these compounds (US DOD, Pesticides-

Currently, the standard (approved) treatment for acute OP pesticide poisoning involves administration of intravenous (iv) atropine and an oxime e.g. obidoxime, pralidoxime to reactivate inhibited AChE (Worek et al., 2010). However, the effectiveness and safety of oxime administration in acute OP pesticide-poisoned patients has been challenged and a recent clinical trial showed no clinical benefits and a trend towards harm in all sub-groups, despite clear evidence that these doses reactivated AChE in the blood (Buckley et al., 2011). An efficacious prophylactic therapeutic treatment for preventing insecticide poisoning that can bind and scavenge the OP before it reaches and targets AChE in neuromuscular junctions is therefore a high priority. The leading candidate of this type is native (plasma) butyrylcholinesterase (BChE) whose potent OP bioscavenging ability has been demonstrated in many animal models and against varied OP neurotoxins (Doctor et al., 2001; Lenz et al., 2001). While several new catalytic and other stoichiometric enzymes also exhibit this ability (Lenz et al., 2007), based on availability, broad spectrum efficacy, stability and safety (Sun et al., 2005), BChE is the most advanced in terms of development of a human treatment. In Turkey, frozen plasma (BChE levels of 3,000 - 5,700 units) given as an alternative or adjunctive treatment with atropine and oximes, has been shown to prevent mortality and intermediate syndrome in acutely insecticide-exposed and hospitalized individuals (Güven et al., 2004). Currently, BChE also finds use as a treatment of cocaine

Structurally, BChE (also known as pseudocholinesterase or non-specific cholinesterase) is a serine esterase (MW=345,000) comprised of four identical subunits each containing 574 amino acids, held together by non-covalent bonds, with 36 carbohyrdrate chains (23.9% by weight) (Lockridge, 1990; Nachon et al., 2002). BChE is found in all species at levels of 1-20 ug/ml in plasma (Rosenberg, unp. data) and is also abundant in liver, intestine and lung. Recombinant (r) human butyryl-cholinesterase (HuBChE), like the native form, is also a potent bioscavenger of OP neurotoxins (Doctor et al., 2001; Lenz et al., 2001; Raveh et al., 1997) but its development as a human treatment for pesticide exposure has been disadvantaged by: (i) poor in vivo stability (bioavailability) of the unmodified forms and the presence of potentially immunogenic glycans using certain expression systems (ii) a 1:1 stoichiometry between the enzyme and OP (Raveh et al., 1997) and (iii) the high LD50 of insecticides (ug-mg/kg levels). This necessitates the delivery of large, costly, rBChE doses to detoxify exposed individuals which is problematic when intramuscular (im) or subcutaneous (sc) injections are the chosen routes of delivery. In this chapter, we shall describe our experience of how the chemistry, glycosylation, chemical modification, animal model and route of administration may reduce or enhance the potential of BChE bioscavengers as prophylactic therapeutic human antidotes for OP insecticide exposure.

**2. Production of tetrameric and monomeric forms of rMaBChE and rHuBChE**  Macaque (Ma) and human (Hu) BChE molecules are very similar molecules differing by only 22 amino acids and sharing ~96% DNA sequence identity, critical glycosylation sites, cysteines and disulfide bridging (Boeck et al., 2002; Rosenberg et al., 2010). Thus, most anti-BChE antisera react with both molecules. Native HuBChE and MaBChE in plasma are composed predominantly of tetramers (98%) with the tetramerization domain being located within the last 40 C-terminal residues of each monomeric subunit (534-574) (Blong et al.,

overdose and for the alleviation of succinylcholine-induced apnea.

Final Report, 2003).

1997). In human serum, the association of lamellipodin proline rich peptides with the monomeric chains results in the formation of BChE tetramers (Li et al., 2008). Recombinant BChE produced in mammalian cells, in contrast, has only 10-20% tetrameric forms and therefore optimal tetramerization requires the addition of either poly(L-proline) to the culture medium or co-expression of the full length BChE monomers with the proline-rich attachment domain (PRAD) of ColQ gene (Altamirano & Lockridge, 1999).

To date, rHuBChE and rMaBChE molecules have been produced in transgenic mammalian cells (Chilukuri et al., 2008; Rosenberg et al, 2010), goat milk (Huang et al., 2007) and in plants (Geyer et al., 2010; Jiang, unpub. data). Our approach has been to utilize two expression systems for the production of rMaBChE and rHuBChE. Initially, Chinese hamster ovary cells (CHO) were used because of their human-like glycosylation. More recently, a transient plant expression platform was adopted to increase the yield and reduce the time and cost of producing rBChE. Although CHO cells and plants are able to produce significant levels of tetrameric BChE molecules (Li et al., 2008; Geyer et al., 2010), in the present studies, co-transfection of the BChE and PRAD genes has been shown to increase both levels of tetramerization and yields in each expression system. While the CHO cell expression of recombinant proteins is very well established, recent innovations in transient plant expression systems e.g. Bayer's Magnifection system (Gleba et al., 2005) and the Cow Pea Mosaic Virus Hyper-translatable Protein Expression System (PBL Technology) (Sainsbury et al., 2008) have been shown to be some of the most rapid, cost effective and productive expression systems in existence; capable of producing grams of recombinant proteins in weeks (Goodin et al., 2008).


\*All tobacco plants and leaves from *Nicotiana tobacum* and *N. benthamiana* were transfected using Agrobacterium-mediated infiltration
