Long-Acting Cocaine Hydrolase for Addiction Therapy

Long-acting cocaine hydrolase for addiction therapy

Xiabin Chena,b, Liu Xueb, Shurong Houb, Zhenyu Jina,b, Ting Zhanga,b, Fang Zhenga,b,1, and Chang-Guo Zhana,b,1

aMolecular Modeling and Biopharmaceutical Center, College of Pharmacy, University of Kentucky, Lexington, KY 40536; and bDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536

Edited by Joanna S. Fowler, Brookhaven National Laboratory, Upton, NY, and approved November 30, 2015 (received for review September 4, 2015) Cocaine abuse is a world-wide public health and social problem BChE mutants, recognized as true cocaine hydrolases (CocHs) in without a US Food and Drug Administration-approved medication. the literature (9) when they have at least 1,000-fold improved cat- An ideal anticocaine medication would accelerate cocaine metabo- alytic efficiency against (-)-cocaine compared with wild-type hu- lism, producing biologically inactive metabolites by administration of man BChE (11–14). The first of our designed CocHs, known as an efficient cocaine-specific exogenous enzyme. Our recent studies “CocH1” (the A199S/S287G/A328W/Y332G mutant of human BChE) have led to the discovery of the desirable, highly efficient cocaine (11, 15), truncated after amino acid 529, was fused with human hydrolases (CocHs) that can efficientlydetoxifyandinactivate cocaine serum albumin (HSA) to prolong the biological t1/2 (9). This HSA- without affecting normal functions of the CNS. Preclinical and clinical fused BChE mutant is also known as “Albu-CocH,”“Albu-CocH1,” data have demonstrated that these CocHs are safe for use in humans “AlbuBChE,” or “TV-1380” (Fig. 1B) in the literature (7–9, 16). and are effective for accelerating cocaine metabolism. However, the TV-1380 has been proven safe and effective for use in animals and actual therapeutic use of a CocH in cocaine addiction treatment is humans (7, 8), but its actual therapeutic value for cocaine addiction limited by its short biological half-life (e.g., 8 h or shorter in rats). Here treatment is still limited by the moderate biological t1/2,whichis we demonstrate a novel CocH form, a catalytic antibody analog, ∼8hinrats(9)and43–77 h in humans (7). [In general, the biological which is a fragment crystallizable (Fc)-fused CocH dimer (CocH-Fc) t1/2 of a therapeutic protein is significantly longer in humans than in constructed by using CocH to replace the Fab region of human IgG1. rats (9).] A biological t1/2 of 43–77 h in humans might be adequate The CocH-Fc not only has a high catalytic efficiency against cocaine for a twice-weekly therapy, depending on the dose of the enzyme but also, like an antibody, has a considerably longer biological half- used. In addition, our more recently designed and identified CocHs life (e.g., ∼107 h in rats). A single dose of CocH-Fc was able to accel- (12–14) are significantly more active than TV-1380 against

erate cocaine metabolism in rats even after 20 d and thus block (-)-cocaine. Further engineering a more active CocH with a bio- NEUROSCIENCE cocaine-induced hyperactivity and toxicity for a long period. Given logical t1/2 longer than that of TV-1380 is highly desired. the general observation that the biological half-life of a protein Our current design strategy for enzyme engineering is based on the drug is significantly longer in humans than in rodents, the CocH-Fc observation that an antibody such as human IgG has a very long reported in this study could allow dosing once every 2–4 wk, or biological t1/2, because the fragment crystallizable (Fc) region of IgG longer, for treatment of cocaine addiction in humans. can bind with the neonatal Fc receptor (FcRn) in the acidic environment of the endosome and later is transported to the cell surface enzyme therapy | cocaine addiction | drug abuse | protein engineering where, upon exposure to a neutral pH, IgG is released back into the main bloodstream (17). In comparison, a recombinant enzyme such s is well known, cocaine is one of the most reinforcing as BChE usually has a very short biological t1/2, and an antibody Aabused drugs, stimulating the reward pathway of the brain usually has no catalytic activity. Using a stable analog of the transition and teaching the user to take it again (1–3). Despite decades of effort, state for cocaine hydrolysis, Landry et al. (1) successfully generated the classical pharmacodynamic approach of using small molecules to the first anticocaine catalytic antibody (CAb). Further effort gener- block or counteract the drug’s neuropharmacological actions has not ated the most active anticocaine CAb (Fig. 1C), known as “15A10” proven successful for cocaine, because it would be extremely difficult (kcat = 2.3/min and Km = 220 μM) (18, 19), whose catalytic activity to antagonize cocaine’s physiological effects without affecting normal functions of the CNS (4). In principle, pharmacological treatment for Significance a drug of abuse can be pharmacodynamic or pharmacokinetic (5). Most current medications for other drugs of abuse use the classical It is essential for a truly effective addiction medication to block the pharmacodynamics approach, using small molecules to block or drug’s physiological effects effectively without affecting normal counteract the drug’s neuropharmacological actions at one or more functions of the brain and other critical organs such as the heart and neuronal binding sites. The inherent difficulties of antagonizing co- while still preventing relapse during abstinence. Most popularly caine in the CNS led to the development of protein-based pharma- used pharmacological approaches to addiction treatment, including cokinetic approaches with biologics such as monoclonal antibodies, all currently available addiction therapies, either affect normal vaccines that produce antibodies in the body, and enzymes (4, 6). The functions of brain receptors/transporters or are unable to prevent pharmacokinetic approach with an efficient enzyme is recognized as relapse. The long-acting enzyme approach may provide a novel, the most promising treatment strategy for cocaine overdose and ad- truly promising therapy capable of effectively blocking the physi- diction (4, 7–9). Unlike the stoichiometric binding of an antibody with ological and toxic effects of cocaine without affecting normal drug, one enzyme molecule can degrade multiple drug molecules, functions of the brain and other critical organs and prevent relapse during abstinence. New insights obtained in this study also may be depending on the turnover number (catalytic rate constant, kcat)and valuable in guiding development of other therapeutic proteins. Michaelis–Menten constant (Km). In humans, the principal metabolic enzyme of cocaine is a plasma enzyme known as butyrylcholinesterase (BChE), producing biologically inactive metabolites. Unfortunately, Author contributions: F.Z. and C.-G.Z. designed research; X.C., L.X., S.H., Z.J., and T.Z. performed research; X.C., T.Z., F.Z., and C.-G.Z. analyzed data; and X.C., F.Z., and C.-G.Z. the catalytic efficiency (kcat/Km) of wild-type BChE against (-)-cocaine wrote the paper. (which is the naturally occurring enantiomer of cocaine) is too low The authors declare no conflict of interest. k = K = μ ( cat 4.1/min and m 4.5 M) (10) to be effective for cocaine This article is a PNAS Direct Submission. metabolism. A mutant of human BChE with a considerably improved 1To whom correspondence may be addressed. Email: [email protected] or fzhen2@email. catalytic efficiency against (–)-cocaine is greatly desired. uky.edu. Through structure- and mechanism-based computational modeling This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and simulation, we have successfully designed and identified human 1073/pnas.1517713113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1517713113 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Compared with the CocH3-Fc structure, CocH3(FL)-Fc has 45 extra amino acid residues between CocH3 and Fc. These 45 residues increase the length of the linker between CocH3 and Fc; thus, as shown in SI Appendix,Fig.S3, the CocH3 region is far away from the Fc region in CocH3(FL)-Fc. The longer linker may be proteolyzed more easily because it is flexible; therefore CocH3(FL)- Fc has a shorter biological t1/2 than CocH3-Fc. The CocH3-Fc forms discussed below are those with the truncated CocH3 region. To optimize the Fc region of the CocH3-Fc construct, we tested the use of wild-type Fc, denoted “Fc(WT),” and a triple mutant (i.e., A1V/D142E/L144M), denoted “Fc(M3).” In addition, we also tested the use of known Fc mutants [A1Q/C6S/C12S/C15S/P24S mutant Fc (22) and A1V/E58Q/E69Q/E80Q/D98N/N101D/D142E/ L144M mutant Fc (23), for convenience here denoted “Fc(M1)” and “Fc(M2),” respectively; see Methods for details], that have been used to prolong significantly the half-lives of other types of therapeutic proteins, i.e., abatacept (22) and alefacept (23). As seen in Fig. 2B, within the CocH3-Fc forms examined, CocH3-Fc(M3) showed the longest biological t1/2 (∼107 h) in rats. The A1V/D142E/ L144M mutations on the Fc region prolonged the biological t1/2 of ∼ Fig. 1. Protein structures and their catalytic parameters for cocaine hydrolysis. this CocH3-Fc by 21 h. The other two CocH3-Fc forms, i.e., (A) Human BChE. (B) Albu-CocH1 (or TV-1380). (C)CAb15A10.(D)CocH3-Fc. CocH3-Fc(M1) and CocH3-Fc(M2), actually shortened the biological t1/2 of CocH3-Fc compared with Fc(WT), although Fc(M1) and Fc(M2) successfully prolonged the biological half-lives of other against (-)-cocaine is still significantly lower than that of wild-type fusion proteins (22, 23) compared with Fc(WT). Further, we note BChE. It has been difficult to improve the CAb activity further that the enzyme fusion with Fc (or Fc mutant) does not necessarily because a CAb, unlike an enzyme, can help stabilize the transition prolong the biological t1/2 of an enzyme. In fact, we also tested state only for nonenzymatic cocaine hydrolysis; a mechanistic study fusing a thermally stable mutant of bacterial cocaine esterase (20) indicates there is no formation of a covalent bond or breaking (CocE) (24) with Fc(M3) and found no significant improvement in between the substrate and CAb during the reaction process. the biological t1/2 (within the measurement errors), with t1/2 still We aimed to design a long-acting CocH form that has both the being only within a few minutes (SI Appendix,Fig.S4). The un- − e long biological t1/2 of an antibody and the high catalytic activity of a usually large number of negative charges ( 34 ) possessed by a CocH against (-)-cocaine. For this purpose, starting from human bacterial CocE protein could significantly affect the interaction of IgG1 (a dimer), which has both the Fc region (constant) and Fab Fc in the CocE-Fc fusion protein with FcRn and, potentially, with region (variable), as seen in Fig. 1C, we may replace the Fab region other proteins in the body. by a CocH for each subunit of the dimeric IgG1 to construct a catalytic antibody analog. Technically, the C terminus of CocH3 Dose Dependence of the Enzyme Activity in Comparison with TV-1380. [the A199S/F227A/S287G/A328W/Y332G mutant (12) of human To characterize further the most promising CocH3-Fc form, BChE, full-length or truncated after amino acid 529 to delete the CocH3-Fc(M3), we developed a stable cell line using a lentiviral vector (25) for large-scale production of CocH3-Fc(M3). The pu- tetramerization domain] was fused with the N terminus of the hinge SI Appendix region linked with Fc. CocH3 has a significantly higher catalytic rified CocH3-Fc(M3) is indeed a dimer, as expected ( , Fig. S1). The in vitro kinetic parameters of the fused CocHs are efficiency against (-)-cocaine (12) than does CocH1 or TV-1380. essentially the same as the corresponding unfused CocHs (see Fig. The obtained dimeric CocH3-Fc fusion enzyme depicted in Fig. 1D 1 B and D and SI Appendix,Fig.S2for the determined k and is highly efficient for cocaine hydrolysis. Obviously, the CocH3-Fc cat K values). is different from but is structurally similar to antibody IgG1, con- m As shown in Fig. 2C, the biological t of CocH3-Fc(M3) in- taining Fc that can bind with FcRn in the acidic environment to 1/2 t jected i.v. in rats is independent of the dose, and therefore the prolong the biological 1/2. Further possible mutations on the Fc CocH3-Fc(M3) concentration at a given time point is linearly pro- region of CocH3-Fc also were examined so that the CocH3-Fc portional to the dose. Fig. 2D shows the time-dependent maximum enzyme would have the longest possible biological t1/2. Various CocH activity against cocaine: Vmax ≡ kcat[E] where [E] represents CocH3-Fc forms, including Fc mutants, were proposed, prepared, the concentration of the enzyme [CocH3-Fc(M3) or Albu-CocH1] andtestedfortheiractualinvitroandinvivoprofiles.Inaddition, in rat plasma. Vmax = 1 U/L means that the enzyme is capable Albu-CocH1 or TV-1380 also was prepared and tested for com- of hydrolyzing up to 1 μmol cocaine in 1 L of plasma per minute parison. As shown below, the optimized CocH3-Fc indeed has both (i.e., 1 μM/min). The data in Fig. 2D indicate that CocH3-Fc(M3) a significantly higher catalytic activity against cocaine and a much is far superior to Albu-CocH1 (TV-1380). In particular, after in- t longer biological 1/2 than TV-1380. jection of 5 mg/kg Albu-CocH1 (TV-1380), the rat plasma had a Vmax ≥ 50 U/L within ∼48 h. In comparison, the Vmax was 50 U/L Results at ∼100 h after the injection of 0.2 mg/kg CocH3-Fc(M3) and at Optimization of the CocH3-Fc Entity. The key in vivo data obtained ∼275 h after the injection of 0.6 mg/kg CocH3-Fc(M3). Even a – are depicted in Figs. 2 4. All pharmacokinetic (PK) data depicted in tiny dose (0.06 mg/kg) of CocH3-Fc(M3) produced a larger Vmax Fig. 2 were based on i.v. injection of the enzymes in rats. The PK value in plasma than produced by 5 mg/kg Albu-CocH1 (TV- data reveal that a CocH3-Fc form with the truncated CocH3 1380) starting at ∼80 h after the injection of CocH [CocH3-Fc (without the tetramerization domain) has a much longer biological (M3) or Albu-CocH1]; 0.2 mg/kg CocH3-Fc(M3) generated a t ∼ ∼ 1/2 (up to 107 h) than that ( 18 h) of the corresponding CocH3-Fc larger Vmax value in the plasma than generated by 5 mg/kg Albu- containing the full-length CocH3, denoted as “CocH3(FL)” in Fig. CocH1 (TV-1380) starting at ∼38 h after the injection of CocH; A 2 , with a given Fc region. Notably, the only difference between and 0.6 mg/kg CocH3-Fc(M3) led to a larger Vmax value in the CocH3(FL) and CocH3 is that CocH3(FL) has the extra 45 amino plasma than generated by 5 mg/kg Albu-CocH1 (TV-1380) start- acid residues, 530–574, an α-helix (21), on the C terminus. ing at ∼18 h after the CocH injection.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1517713113 Chen et al. Downloaded by guest on September 28, 2021 Fig. 2. Time-dependent normalized CocH activity (A–C)orVmax (U/L) (D) of various CocH3-Fc forms and Albu-CocH1 (TV-1380). For convenience, “CocH3(FL)” represents full-length CocH3, and “CocH3” (without “FL”) refers to truncated CocH3 (amino acid residues 1–529). Rats (n = 3 for each dose condition) were

injected i.v. with the enzyme [CocH3-Fc(M3) or Albu-CocH1] at a dose of 0.06 mg/kg body weight unless explicitly indicated otherwise. Blood samples were NEUROSCIENCE collected at various time points, and the separated plasma samples were analyzed by a sensitive radiometric assay using [3H](-)-cocaine.

Cocaine Clearance Is Accelerated by CocH3-Fc(M3). In a further recording after 1 and 24 h (day 1) and then every other day (days 3, in vivo test, rats were injected with a single dose of CocH3-Fc(M3) 5, 7, 9, 11, 13, and 15). For each session, the hyperactivity recording (0.2mg/kgi.v.onday0),followedbyi.v.injectionofcocaine began 30 min before cocaine injection. Data recorded for these (5 mg/kg) on days 8, 11, 14, and 20. After each cocaine injection, sessions (Fig. 4 A–H) show that CocH3-Fc(M3) completely blocked blood samples were collected at 2, 5, 10, 15, 30, and 60 min and were cocaine-induced hyperactivity 1 d (24 h) after the 0.2 mg/kg CocH3- analyzed for the concentrations of cocaine and benzoic acid (cocaine Fc(M3) injection and still significantly attenuated cocaine-induced metabolite). The control curves in Fig. 3 reflect the overall effects of hyperactivity after 11 d. At a dose of 2 mg/kg, CocH3-Fc(M3) all possible cocaine-elimination pathways (25–27). In the control completely blocked cocaine-induced hyperactivity within 9 d and still rats, the average concentration of cocaine at the first time point significantly attenuated cocaine-induced hyperactivity after 15 d. (2 min) was ∼7.4 μM, whereas the average concentration of benzoic acid was ∼0.2 μM. In the presence of CocH3-Fc(M3) on day 8 after Effectiveness of CocH3-Fc(M3) in Blocking the Toxic Effects of Cocaine. CocH3-Fc(M3) injection, the average concentration of cocaine at It is well known that cocaine is lethal at high doses [LD50 = ∼2 min in the blood sample was below 1 μM(∼0.9 μM) (SI Ap- 14 mg/kg i.v. (28) or 95.1 mg/kg i.p. (29) in mice]. To examine pendix,Fig.S3A), whereas the average concentration of benzoic acid how long CocH3-Fc(M3) can protect mice from the acute tox- at the first time point (2 min) was ∼11 μM(SI Appendix,Fig.S3B). icity of a lethal dose of cocaine, mice were injected with a single Thus CocH3-Fc(M3) hydrolyzed nearly all the cocaine molecules dose of CocH3-Fc(M3) (2 mg/kg, i.v.) on day 0, followed by a within ∼2 min after the cocaine injection on day 8. According to the lethal dose of cocaine (100 mg/kg, i.p.) every day (starting on data in Fig. 3B, the overall enzyme activity in rats decreased grad- day 4) until a convulsion occurred (the end point of the toxicity ually from day 8 to days 11, 14, and 20, as expected. However, even test for a given mouse). All mice in the control group [without at such a low dose (0.2 mg/kg), the CocH3-Fc(M3) activity in rats CocH3-Fc(M3) injection] had a convulsion within minutes after was still significant after 20 d. the first cocaine challenge, and 60% of the mice died soon after It should be noted that CocH3-Fc(M3) is constructed from human the convulsion. For the test group receiving 2 mg/kg CocH3-Fc(M3), protein sequences with mutations on only a few residues. Because the a convulsion occurred only during the final cocaine challenge, human protein sequences associated with CocH3-Fc(M3) are dif- after 10.2 d in average; a convulsion did not occur during the ferent from the corresponding rodent protein sequences, we did not daily cocaine challenge for 9.2 d on average. The average pro- try to examine the potential antigenicity/immunogenicity of CocH3- tection time, tp, provided by 2 mg/kg CocH3-Fc(M3) is 9.7 ± 1.7 d. Fc(M3) in animals in this study. However, according to clinical data Thus, injection of 2 mg/kg CocH3-Fc(M3) can protect mice reported for TV-1380 (7), one can reasonably assume that CocH3-Fc effectively from the acute toxicity of the lethal dose of cocaine (M3) would induce no, a weak, or a transient immune response in (100 mg/kg i.p.) for nearly 10 d. humans. The effectiveness of CocH3-Fc(M3) in these animals for 20 d supports this assumption. Discussion We have successfully designed a novel, long-acting cocaine- Effectiveness of CocH3-Fc(M3) in Blocking the Physiological Effects of metabolizing enzyme form, CocH3-Fc. The optimized CocH3-Fc Cocaine. In animal behavior studies, mice were injected i.v. with a entity, CocH3-Fc(M3), indeed has a long biological t1/2, like an single dose (2, 0.2, or 0 mg/kg) of CocH3-Fc(M3) on day 0, followed antibody, but with an unprecedentedly high catalytic efficiency by multiple sessions of i.p. injection of cocaine (15 mg/kg) with compared with all CAbs known to date. Compared with the most hyperactivity (measured by increased horizontal distance traveled) active anticocaine CAb, 15A10 (kcat = 2.3/min, Km = 220 μM, and

Chen et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 an Fc mutant) of human IgG1 may not necessarily prolong the biological t1/2 of the enzyme, because the Fc fusion strategy did not prolong the biological t1/2 of the bacterial CocE at all. To develop a truly long-acting enzyme form fused with Fc, one must optimize structurally both the enzyme and the Fc portions of the Fc-fused enzyme. Furthermore, even if the protein fusion with Fc really can prolong the biological t1/2 of a therapeutic protein, the data obtained in this investigation demonstrate that certain amino acid mutations on the Fc portion that can prolong the biological t1/2 of a given Fc-fused therapeutic protein may not necessarily prolong the biological t1/2 of another Fc-fused therapeutic protein. Methods Materials. The cDNAs for CocH1 (the A199S/S287G/A328W/Y332G mutant of human BChE) and CocH3 (the A199S/F227A/S287G/A328W/Y332G mutant of human BChE) containing N-terminal signal were generated in our previous studies (11, 12). pFUSE-hIgG1-Fc2 plasmid was purchased from InvivoGen. The cDNA (clone ID: HsCD00005810) for HSA was obtained from the Dana Farber/Harvard Cancer Center DNA Resource Core. The protein expression vector pCMV-MCS was ordered from Agilent, and pCSC-SP-PW lentiviral vector (plasmid 12335), pMDLg/pRRE (plasmid 12251), pRSV-Rev (plasmid 12253), and pCMV-VSV-G (plasmid 8454) were obtained from Addgene. Phusion DNA polymerase, restriction endonucleases, and T4 DNA ligase were ordered from New England BioLabs. DpnI endonuclease was obtained from Thermo Fisher Scientific. All oligonucleotides were synthesized by Eurofins MWG Operon. CHO-S cells, HEK-293FT cells, FreeStyle CHO expression medium, hypoxanthine/thymidine (HT) supplement, L-glutamine, DMEM, FBS, 4–12% Tris-glycine Mini Protein Gel, and SimpleBlue SafeStain were from Invitrogen. The TransIT-PRO Transfection Kit was obtained from Mirus. Reduction- modified protein (rmp) Protein A Sepharose Fast Flow was purchased from GE Healthcare Life Sciences. Centrifugal filter units were from Millipore. (−)-Cocaine was provided by the National Institute on Drug Abuse Drug Supply Program, and [3H](−)-Cocaine (50 Ci/mmol) was ordered from PerkinElmer. Sprague–Darley rats (200–250 g) and CD-1 mice (25–30 g) were ordered from Harlan. All other chemicals were purchased from Thermo Fisher Scientific or Sigma-Aldrich.

Construction of Expression Plasmids. With specific base-pair alterations, site- directed mutagenesis was performed to obtain the cDNAs for Fc(WT), Fc(M1) (i.e., the A1Q/C6S/C12S/C15S/P24S mutant Fc), Fc(M2) (i.e., the A1V/E58Q/ E69Q/E80Q/D98N/N101D/D142E/L144M mutant Fc), and Fc(M3) of human IgG1 using pFUSE-hIgG1-Fc2 plasmid as the template (31). Five expression plasmids, pCMV-CocH3-Fc(WT), pCMV-CocH3-Fc(M1), pCMV-CocH3-Fc(M2), pCMV-CocH3-Fc(M3), and pCMV-CocH3(FL)-Fc(M3), were constructed. Each plasmid contains a sequence encoding the native BChE signal peptide followed by CocH3 (truncated or full length) linked to the N-terminal of Fc (WT Fig. 3. Cocaine clearance accelerated by CocH3-Fc(M3). Saline or 0.2 mg/kg CocH3-Fc(M3) was injected i.v. in rats (n = 4), followed by i.v. injection of or mutant). To fuse CocH3 to Fc, overlap extension PCR with Phusion DNA 5 mg/kg cocaine after 8, 11, 14, and 20 d. Blood samples were collected 2, 5, polymerase was used. The four primers used for each plasmid construction 10, 15, 30, and 60 min after each i.v. injection of 5 mg/kg cocaine and were are listed in SI Appendix, Table S1. Primers 1 and 2 were used to amplify analyzed for the concentrations of (A) cocaine and (B) benzoic acid (a metabolite CocH3 with the N-terminal signal peptide using pRc/CMV-CocH3 as template; of cocaine). primers 3 and 4 were used to amplify Fc using corresponding Fc cDNA as template. Once two DNA fragments were obtained, they were fused to- gether by using another PCR with primers for the far ends (primers 1 and 4). Then the PCR product was digested with restriction endonucleases Hind III t1/2 < 24 h in rats) (30), CocH3-Fc(M3) has an even significantly and Bgl II and was ligated to the pCMV-MCS expression vector using T4 DNA longer biological t1/2 (kcat = 5,700/min, Km = 2.8 μM, and t1/2 = ∼107hinrats)and∼200,000-fold improved catalytic efficiency ligase. The same method also was used to construct plasmids pCSC-CocH3-Fc (M3) and pCSC-Albu-CocH1 for large-scale protein production. (kcat/Km) against cocaine. The long-acting CocH3-Fc(M3) is highly efficient in blocking the physiological and toxic effects of cocaine Small-Scale Protein Expression and Purification. CHO-S cells, incubated at 37 °C

for a long period. Further, it is well known that a protein drug in a humidified atmosphere containing 8% CO2, were transfected with plasmids generally has a significantly longer biological t1/2 in humans than in encoding the proteins using the TransIT-PRO Transfection Kit once cells had rats [e.g., the, Fc(M1)-fused human cytotoxic lymphocyte-associ- grown to a density of 1.0 × 106 cells/mL. The culture medium (FreeStyle CHO ated antigen abatacept has a t1/2 of 3–6 d in rats and 12–23 d in Expression Medium with 8 mM L-glutamine) was harvested 7 d after trans- humans (22)]. Thus, one may reasonably expect that an appro- fection. Enzyme secreted in the culture medium was purified by protein A priately chosen dose of CocH3-Fc(M3) could protect humans affinity chromatography. Pre-equilibrated rmp Protein A Sepharose Fast Flow effectively against both relapse and overdose for 2–4wkorlonger was added into cell-free medium and was incubated overnight with occasional during treatment for cocaine addiction. stirring, and then the suspension was packed in a column. The column was washed with 20 mM Tris·HCl (pH 7.4) until an OD280 < 0.02 was achieved; then The general strategy of developing a long-acting drug-metabolizing the protein was eluted by adjustment of the pH and salt concentration. The enzyme for treatment of cocaineabusealsomaybeusefulinthe eluate was dialyzed in storage buffer (50 mM Hepes, 20% sorbitol, 1 M glycine, development of long-acting forms of other therapeutic enzymes. On pH 7.4) by Millipore Centrifugal Filter Units. The entire purification process was the other hand, one must keep in mind that the simple fusion of an carried out in a cold room at 6 °C. The purified protein was stored at −80 °C enzyme (such as bacterial CocE, mentioned above) with Fc (or with before enzyme activity tests and in vivo studies.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1517713113 Chen et al. Downloaded by guest on September 28, 2021 Fig. 4. Effects of CocH3-Fc(M3) on cocaine-induced hyperactivity and toxicity in mice. (A–H) Hyperactivity test (n = 8 for each dose condition): Saline or CocH3-Fc(M3) (enzyme) was injected i.v., followed by multiple sessions of locomotor activity tests with i.p. injection of 15 mg/kg cocaine or saline after 1 h and 24 h (1 d) and then every other day. A–H, respectively, report data from the locomotor activity sessions on days 1, 3, 5, 7, 9, 11, 13, and 15 after the CocH3-Fc (M3) injection. For each locomotor activity session, locomotor activity recording started 30 min before the cocaine injection. (I) Toxicity test (n = 5). A single NEUROSCIENCE dose of CocH3-Fc(M3) (2 mg/kg, i.v.) was followed by daily dosing of cocaine (100 mg/kg, i.p.) starting on day 4 until convulsion occurred (the end pointofthe toxicity test) for a given mouse). For a given mouse, if convulsion occurred during the final cocaine challenge on day m but did not occur during the cocaine

challenge on and before day m − 1, the protection time (tp) for this mouse was considered to be between days m − 1andm: m − 1 < tp > m or tp = m − 1/2 d. represents the average tp value for all mice in the group.

Large-Scale Protein Expression and Purification. A lentivirus-based method the benzene ring). The reaction was stopped by adding 200 μL 0.1 M HCl, described in our previous report (25) was performed to generate a highly which neutralized the liberated benzoic acid while ensuring a positive efficient stable cell line expressing CocH3-Fc(M3). Briefly, to package the charge on the residual (−)-cocaine. [3H]Benzoic acid was extracted by 1 mL of lentivirus particles carrying the gene of CocH3-Fc(M3), lentivirus was produced toluene and measured by scintillation counting. All measurements were by cotransfecting pCSC-CocH3-Fc(M3) plasmid with the packaging vectors performed at 25 °C. Substrate concentration-dependent data were analyzed (pMDLg/pRRE and pRSV-Rev) and envelope plasmid (pCMV-VSV-G) into HEK- using standard Michaelis–Menten kinetics so that the catalytic parameters

293FT cells by lipofection. The packaged lentivirus particles were transfected to (kcat and Km) were determined. CHO-S cells. After lentiviral transductions, infected cells were allowed to recover

from the infection for 2 d or more and then were transferred to a shake flask Determination of Biological t1/2 in Rats. Rats were used in accordance with the for further culture. The obtained stable CHO-S cell pool was kept frozen before Guide for the Care and Use of Laboratory Animals as adopted and promulgated being used for large-scale protein production. by the National Institutes of Health (32). The experimental protocols were ap- Large-scale protein production was performed in an agitated bioreactor proved by the IACUC at the University of Kentucky. Rats were injected with BioFlo/CelliGen 115 (Eppendorf). Before being transferred into the bioreactor, purified protein via the tail vein [0.06, 0.2, or 0.6 mg/kg for CocH3-Fc(M3); 0.06 or cells were grown at 37 °C in shake flasks to the designated volume and density. 5 mg/kg for Albu-CocH1; and 0.06 mg/kg for all other CocH3-Fc protein forms]. On the day of transfer, cells in shake flasks were centrifuged at a speed of Blood samples were taken by needle puncture of the saphenous vein. Approx- 1,500 × g for 5 min at room temperature, resuspended in fresh culture medium, imately 50–100 μL of blood was collected into a heparin-treated capillary tube at

and transferred into the bioreactor. The CO2/air gas overlay was set so that the various time points after enzyme administration. Collected blood samples were pH of the cell-culture medium was maintained at 7.0–8.0. The bioreactor was centrifuged for 15 min at a speed of 5,000 × g to separate the plasma, which was run in a batch model with a temperature of 32 °C. After 9 d the culture medium kept at 4 °C before analysis. Radiometric assay using 100 μM(−)-cocaine was was harvested, and the protein was purified. CocH3-Fc(M3) was purified using used to measure the enzyme concentration in plasma. The time-dependent

the aforementioned protein A affinity chromatography, which was performed enzyme concentrations ([E]t) were fitted to a well-known double-exponential − − ½ = k1 t + k2 t on an ÄKTA avant 150 system (GE Healthcare Life Sciences). The purified pro- equation (33) by GraphPad Prism 5: ( E t Ae Be ), which accounts for − tein was dialyzed in storage buffer and stored at 80 °C. both the enzyme distribution process (the fast phase, associated with k1)andthe elimination process (the slow phase, associated with k2). The t1/2 associated with Electrophoresis. Purified CocH3-Fc(M3) was analyzed by SDS/PAGE electropho- the enzyme elimination rate constant k2 is known as the elimination t1/2 or resis. Two protein samples, protein in its native state and protein in a denatured biological t1/2. state, were loaded in a 4–12% Tris-Glycine Mini Protein Gel. To break up the quaternary protein structure, the protein sample was mixed with SDS-loading Characterization of Cocaine Clearance Accelerated by CocH3-Fc(M3). Four rats buffer and was heated at 95 °C for 10 min in the presence of a reducing agent, received i.v. saline before the i.v. injection of 5 mg/kg (−)-cocaine, and four other DTT. Electrophoresis was run at 100 V for 2 h. The protein gel was stained with rats received enzyme CocH3-Fc(M3) (0.2 mg/kg, i.v.) on day 0, followed by i.v. SimpleBlue SafeStain. administration of 5 mg/kg (−)-cocaine on days 8, 11, 14, and 20 after the CocH3- Fc(M3) injection. Blood samples (50–100 μL) were collected from the saphenous In Vitro Activity Test Against (−)-Cocaine. A radiometric assay, which was used vein into a heparin-treated capillary tube 2, 5, 10, 15, 30, and 60 min after in our previous studies (12, 14), was used to test the catalytic activities of (−)-cocaine administration and were mixed immediately with 100 μLof25μM enzymes against (−)-cocaine. Briefly, 150 μL of the enzyme solution (100 mM paraoxon (which inactivates the enzymes). Blood samples were stored at −80 °C phosphate buffer, pH 7.4) was added to varying concentrations of 50 μLof until analysis by HPLC. To assay the (−)-cocaine and benzoic acid (the product of [3H]-labeled (−)-cocaine, denoted as “[3H](−)-cocaine” (with 3H labeled on cocaine hydrolysis by the enzyme) concentrations in the blood samples, frozen

Chen et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 blood samples were thawed on ice for more than 3 h. Then 150 μL mobile were returned immediately to the test chamber for the remaining 1 h of the

phase (76% 0.1% trifluoroacetic acid and 24% acetonitrile) and 50 μL7%HClO4 session for activity monitoring. The locomotor tests were performed in high- were added to each sample. The extraction mixture was vortex mixed for 1 min density, nonporous plastic chambers measuring 50 cm (L) × 50 cm (W) × 38 cm (H) and then was centrifuged at 4 °C for 15 min at a speed of 13,200 × g.The in a light- and sound-attenuating behavioral test enclosure (San Diego In- supernatant was decanted into a 1.5-mL tube, labeled, and stored at –80 °C struments). The cumulative distance traveled was recorded by an EthoVision until analysis by HPLC. The chromatographic analysis was carried out on a XT video tracking system (Noldus Information Technology) to represent the Gemini 5-μm C18 column (Phenomenex), using a mobile phase at a flow locomotor activity. rate of 1 mL/min. The (−)-cocaine fluorescence was monitored at 315 nm with excitation at 230 nm (25, 34), and benzoic acid absorbance was monitored at Protection Study in Mice. Cocaine-induced acute toxicity was characterized in 230 nm. The chromatographic system used comprised a Waters 1525 binary this study by the occurrence of a convulsion. A cocaine-induced convulsion was HPLC pump, a 717 plus Autosampler, a 2487 dual λ absorbance detector, and a defined as the loss of righting posture for at least 5 s with the simultaneous 2475 multi λ fluorescence detector (Waters). presence of clonic limb movements (35). Mice received a single dose of CocH3-Fc (M3) (2 mg/kg, i.v.) or saline (i.v.) on day 0, followed by daily dosing of cocaine Locomotor Activity Assay. The effects of CocH3-Fc(M3) on cocaine-induced (100 mg/kg, i.p.) starting on day 4 until the occurrence of a convulsion in a given hyperactivity were evaluated using a video-tracking system at the University of mouse. Following cocaine administration, mice were placed immediately in Kentucky’s Rodent Behavior Core. Mice received a single dose of CocH3-Fc(M3) containers for observation. The presence or absence of a convulsion was (0.2 or 2 mg/kg, i.v.) or saline, followed by multiple doses of cocaine (15 mg/kg, recorded for 60 min following cocaine administration (12). i.v.) or saline after 1 h, 1, 3, 5, 7, 9, 11, 13, and 15 d. Before cocaine or saline administration, mice were allowed to acclimate to the test chambers for 1 h. ACKNOWLEDGMENTS. This work was supported in part by NIH Grants UH2 The total distance traveled during the last 30 min, which was collected in 5-min DA041115, R01 DA035552, R01 DA032910, R01 DA013930, and R01 DA025100 bins, was used as the basal level. After cocaine or saline administration, mice and by National Science Foundation Grant CHE-1111761.

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