Development of a long-acting Fc-fused cocaine hydrolase with improved yield of protein expression

Abstract

Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a ~2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an Nterminal fusion protein, Fc(M3)-(PAPAP) 2 -CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP) 2 -CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105 ± 7 hours in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP) 2 -CocH3 has a ~21fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP) 2 -CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction.

Structure and therapeutic uses of butyrylcholinesterase: Application in detoxification, Alzheimer's disease, and fat metabolism

“…Therefore, it obtained extended biological half‐life, 3–6 days in rats and 12‐23 days in human. But the direct connection led to decreased anticocaine activity (~30% of the original CocH3), probably owing to that the fused fragment Fc blocked the entrance of cocaine molecules into catalytic sites 123 . Therefore in 2019, they introduced a linker, (PAPAP) 2 , between the two fragments and successfully restored the catalytic activity against cocaine 123 .…”

“…But the direct connection led to decreased anticocaine activity (~30% of the original CocH3), probably owing to that the fused fragment Fc blocked the entrance of cocaine molecules into catalytic sites 123 . Therefore in 2019, they introduced a linker, (PAPAP) 2 , between the two fragments and successfully restored the catalytic activity against cocaine 123 . Moreover, they changed the Fc to the N‐terminus of CocH3, and the fusion enzyme, Fc‐(PAPAP) 2 ‐CocH3, was reported to show an improved expression level by 21‐fold compared to original enzyme in CHO cells.…”

Recovery of dopaminergic system after cocaine exposure and impact of a long‐acting cocaine hydrolase

“…However, despite of decades of effects, there is still no Food and Drug Administration (FDA)‐approved pharmacotherapy specific for CUD, due to difficulty to antagonize cocaine's physiological effects without affecting normal functions of brain. Nevertheless, through computational modelling and simulation‐guided rational design, 17–20 we have developed highly efficient cocaine hydrolases (CocHs) that are rationally designed mutants of human butyrylcholinesterase (BChE) 19,21–24 and derived a long‐acting form of it (denoted as CocH‐Fc) through protein fusion with the Fc fragment (wild‐type or mutant) of human immunoglobulin G1 (IgG1) 25–27 . It has been demonstrated that these proteins are capable of accelerating cocaine metabolism, rapidly detoxifying cocaine and its toxic metabolites 22,27–30 .…”

Clinical potential of a rationally engineered enzyme for treatment of cocaine dependence: Long-lasting blocking of the psychostimulant, discriminative stimulus, and reinforcing effects of cocaine

“…To solve the dosing frequency problem, we have designed and discovered a long-acting form of CocH by using human immunoglobulin G1 (IgG1) Fc fragment, instead of HSA, as an alternative carrier protein to produce Fc-fused CocH protein, denoted as CocH-Fc, with a significantly improved biological half-life (Chen, Xue et al 2016, Chen, Deng et al 2019. Through further structure-based computational design, our recently designed CocH-Fc mutant, known as CocH3-Fc(M6), has a markedly prolonged biological half-life (t 1/2 = ~206±7 hours) in rats .…”