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Optimized Platforms for Proper Glycosylation and Sialylation of Recombinant Human Butyrylcholinesterase (rBChE)

Description:

TECHNOLOGY AREA(S): Chem Bio_defensebio Medical 

OBJECTIVE: Develop optimized protein/glycoengineered platform technologies to produce gram quantities of purified, biologically active rBChE protein that can be utilized as an effective medical countermeasure against organophosphorus (OP) Chemical Warfare Agent (CWA) exposure. The optimized rBChE, in a fully glycosylated, sialylated, tetrameric form analogous to the human plasma wild-type configuration, could be utilized as a broad spectrum, prophylactic or therapeutic treatment after intravenous delivery. 

DESCRIPTION: Butyrylcholinesterase (BChE) is a naturally occurring human-derived plasma protein that is efficacious as a prophylactic and as a post-exposure, pre-symptomatic treatment against OP CWA exposure. BChE acts as a stoichiometric bioscavenger that binds to and inactivates OP CWA in the bloodstream, thereby preventing the CWA from reaching critical target organs. Optimally active BChE is a tetrameric (mediated by a proline–rich attachment domain or PRAD protein) enzyme that requires glycosylation and sialylation for prolonged circulatory half-life in vivo (1-4). Purification of sufficient quantities of active, natural BChE from blood plasma is inefficient, low yield, and expensive. Multiple alternative recombinant, molecular biology approaches (e.g., PER.C6, CHO, transgenic goat milk, Nicotiania, and Baculovirus) have produced active rBChE; however, the recombinant proteins have had limited efficacy and short in vivo circulating half-lives. These shortcomings have primarily been due to inappropriate configuration of glycans, lack of proper sialylation, and production of short half-life, monomeric and dimeric forms. Long circulating, active rBChE can now be optimally and efficiently produced by novel cellular and molecular platforms that employ modern, targeted protein and glycoengineering technologies. Specifically, cellular platforms can be engineered to over express optimized proline-rich chaperones, specific branching glycosyltransferases, in combination with α 2,6 sialyltransferases to produce correctly configured BChE. These novel protein/glycoengineering technologies can thus be utilized to produce gram quantities of glycosylated and sialylated, tetrameric rBChE for non-clinical testing and evaluation as a precursor to clinical development. 

PHASE I: Genetically-engineer DNA constructs that express secreted, active monomers/dimers of enzymatically active rBChE in selected cellular platform. Develop a tetramerization strategy via genetically engineered proline-rich attachment domain (PRAD)-like protein peptide or polyproline peptides. Alternatively, evaluate tetramerization employing external extracellular addition of PRAD like peptides or polyproline (2-4). The objective is to produce tetrameric rBChE with enzymatic activity. 

PHASE II: Employ a specific protein/glycoengineering strategy to optimize the correct expression of proline-rich chaperones, mannose N-glycan, and α-2,6 linked sialylation (N-acetylneuraminic acid). The optimized configuration would duplicate the configuration of human wild-type plasma rBChE (correctly glycosylated, sialylated, tetramer) (5). Approaches might include overexpression of sialyltransferases and other glycosyltransferases, manipulation of sialic acid biosynthetic pathways and inhibition of sialidases, or glycosylation site insertion and manipulation of glycan heterogeneity to produce the desired glycoforms (6-8). The platform objective yield is grams of product. Demonstrate optimized enzymatic (> 621 U/mg) and functional binding activity in vitro of the optimized tetrameric rBChE (with CWA or surrogates). 

PHASE III: PHASE III: Evaluate pharmacokinetic and pharmacodynamic blood plasma profile in guinea pigs and/or other appropriate species administered properly glycosylated, tetrameric BChE. Compare to published studies or reports of human-derived BChE administered intravenously to various species, including human. Develop scale-up process for pilot-lot production of tetrameric rBChE. Develop a current Good Manufacturing Practices (cGMP) manufacturing strategy. PHASE III DUAL USE APPLICATIONS: Successful development of the optimized protein/glycoengineering platform technologies can be readily adapted to other recombinant proteins of clinical interest to both the military and civilian communities. These could include vaccines and other therapeutic recombinant proteins. 

REFERENCES: 

1: Involvement of oligomerization, N-glycosylation and sialylation in the clearance of cholinesterases from circulation. Kronman C, Velan B, Marcus D, Ordetlich A, Reuveny S, Shafferman, A. Biochem J. (1995) 311, 959-967.

2:  Polyproline promotes tetramerization of recombinant human butyrylcholinesterase. Larson M, Lockridge O, Hinrichs S. Biochem. J. (2014) 462, 329-335.

3:  Lamellipodin proline rich peptides associated with native plasma butyrylcholinesterase tetramers. Li H, Schopfer L, Masson P, Lockridge O. Biochem J. (2008) 411, 425-432.

4:  Proline-Rich Chaperones Are Compared Computationally and Experimentally for Their Abilities to Facilitate Recombinant Butyrylcholinesterase Tetramerization in CHO Cells. Wang Q, Chen CH, Chung CY, Priola J, Chu JH, Tang J, Ulmschneider MB, Betenbaugh MJ. Biotechnol J. 2018 Mar

5:  13(3):e1700479. Epub 2017 Nov 17

6:  Glycoproteomics characterization of butyrylcholinesterase from human plasma. Kolarich D, Weber A, Pabst M, Stadlmann J, Teschner, W, Ehrlich H, Schwarz H, Altmann, F. Proteomics 2008, 8, 254-263.

7:  Glycoengineering of CHO Cells to Improve Product Quality. Wang Q, Yin B, Chung CY, Betenbaugh MJ. Methods Mol Biol. 2017

8: 1603:25-44.

9:  Strategies for Engineering Protein N-Glycosylation Pathways in Mammalian Cells. Wang Q, Stuczynski M, Gao Y, Betenbaugh MJ. Methods Mol Biol. 2015

10: 1321:287-305.

11:  A novel sugar analog enhances sialic acid production and biotherapeutic sialylation in CHO cells. Yin B, Wang Q, Chung CY, Bhattacharya R, Ren X, Tang J, Yarema KJ, Betenbaugh MJ. Biotechnol Bioeng. 2017 Aug

12: 114(8):1899-1902.

KEYWORDS: Butyrylcholinesterase, Bioscavenger, Nerve Agent Poisoning, Chemical Countermeasure, Glycosylation, Sialylation, Recombinant Human Butyrylcholinesterase, RBChE 

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