US008735124B2

(12) United States Patent (10) Patent No.: US 8,735,124 B2 Tawfik et al. (45) Date of Patent: May 27, 2014

(54) ISOLATED PON1 POLYPEPTIDES, FOREIGN PATENT DOCUMENTS POLYNUCLEOTDESENCODING SAME AND WO WO 2004/078991 9, 2004 SERENERSFSR WO WO 2007/105223 9, 2007 WO WO 2011/033506 3, 2011 EXPOSURE ASSOCATED DAMAGE WO WO 2013/136335 9, 2013 (75) Inventors: Dan S. Tawfik, Jerusalem (IL); Rinkoo OTHER PUBLICATIONS Devi Gupta, Rehovot (IL); Moshe Goldsmith, Rehovot (IL); Yaacov Broun et al., Catalytic plasticity of fatty acid modification enzymes Ashani, Rehovot (IL) underlying chemical diversity of plant lipids. Science, 1998, vol. 282: 1315-1317. (73) Assignee: Yeda Research and Development Co. Chica et al., Semi-rational approaches to engineering enzyme activ Ltd., Rehovot (IL) ity: combining the benefits of directed evolution and rational design. Curr. Opi. Biotechnol., 2005, vol. 16: 378-384.* ( c ) Notice: Subj ect to any disclaimer, the term of this Devos et al., Practical limits of function prediction. Proteins: Struc patent is extended or adjusted under 35 ture, Function, and Genetics. 2000, vol. 41: 98-107. U.S.C. 154(b) by 43 days. Kisselev L., Polypeptide release factors in prokaryotes and eukaryotes: same function, different structure. Structure, 2002, vol. 10:8-9. (21) Appl. No.: 13/420,920 Sen et al., Developments in directed evolution for improving enzyme functions. Appl. Biochem. Biotechnol., 2007, vol. 143: 212-223.* (22) Filed: Mar 15, 2012 Whisstock et al., Prediction of protein function from protein sequence. Q. Rev. Biophysics., 2003, vol. 36 (3): 307-340.* (65) Prior Publication Data Wishart et al. A single mutation converts a novel phosphotyrosine US 2012/0213834A1 Aug. 23, 2012 binding1995, sdomain 270(45): into E"a dual-specificity typhosp phosphatase. J. Biol. Chem. Witkowski et al., Conversion of b-ketoacyl synthase to a Malonyl Decarboxylase by replacement of the active cysteine with glutamine. Related U.S. Application Data Biochemistry, 1999, vol. 38: 11643-1 1650.* (63) Continuation-in-part of application No. Communication Relating to the Results of the Partial International PCT/IL2010/000754, filed on Sep. 15, 2010. Search Dated Mar. 22, 2011 From the International Searching Authority Re. Application No. PCT/IL2010/000754. (60) Provisional application No. 61/272,363, filed on Sep. International Search Report and the Written Opinion Dated Jun. 7. 17, 2009. 2011 From the International Searching Authority Re. Application No. PCT, IL2010.000754. (51) Int. Cl. Aharoni et al. “Directed Evolution of Mammalian Paraoxonases CI2N 9/16 (2006.01) PON1 and PON3 for Bacterial Expression and Catalytic Specializa CI2N 9/14 (2006.01) tion”. Proc. Natl. Acad. Sci. USA, PNAS, XP002438727, 101(2): CI2P2/06 (2006.015 482-487, Jan. 13, 2004. CI2P 19/34 (2006.015 Ashani et al. “Stereo-Specific Synthesis of Analogs of Nerve Agents and Their Utilization for Selection and Characterization of CI2N IS/00 (2006.01) Paraoxonase (PON1) Catalytic Scavengers',gerS, Chemico-BiologicalUnemico-Slologica C7H 2L/04 (2006.01) Interactions, XP027174373, 187(1-3): 362-369, Sep. 6, 2010. C07K L/00 (2006.01) Bajgar “Organophosphates/Nerve Agent Poisoning: Mechanism of AOIN 25/34 (2006.01) Action, Diagnosis, Prophylaxis, and Treatment'. Advances in Clini A6 IK9/00 (2006.01) cal Chemistry, XP009 109278, 38: 151-216, Jan. 1, 2004. A6 IK38/46 (2006.01) Gupta et al. “Directed Evolution of Hydrolases for Prevention of (52) U.S. Cl. G-Type Nerve Agent Intoxication', Nature Chemical Biology, USPC ...... 435/196; 435/195; 435/69.1; 435/91.1; XP002627100, 7(2): 120-125, Feb. 2011. 435/320.1536/23.1536/23.2:530/350; 424/402; 424/400; 424/94.6 (Continued) (58) Field of Classification Search USPC ...... 435/196, 195, 69.1, 91.1, 320.1; Primary Examiner — Ganapathirama Raghu 536/23.1, 23.2; 530/350: 424/402,400, 424/94.6 See application file for complete search history. (57) ABSTRACT An isolated polypeptide comprising the amino acid sequence (56) References Cited of serum paraoxonase (PON1) having catalytic efficiency of U.S. PATENT DOCUMENTS k/K-10°-5-107 M'min' for a G-type organophosphate.

7,786,071 B2 * 8/2010 Tawfik et al...... 424/94.63 2006/0205933 A1* 9, 2006 Tawfik et al...... 536,232 16 Claims, 27 Drawing Sheets 2011/0171197 A1* 7, 2011 Tawfik et al...... 424/94.6 (17 of 27 Drawing Sheet(s) Filed in Color) US 8,735,124 B2 Page 2

(56) References Cited DiTargiani et al. “In Search of a Catalytic Bioscavenger for the Prophylaxis of Nerve Agent Toxicity'. Chemo-Biological Interac OTHER PUBLICATIONS tions, 187: 349-354, 2010. Ghanem et al. “Detoxification of Organophosphate Nerve Agents by Bacterial Phosphotriesterase'. Toxicology and Applied Pharmacol Harel et al. “Structure and Evolution of the Serum Paraoxonase ogy, 207: S459-S470, 2005. Family of Detoxifying and Anti-Atherosclerosis Enzymes', Nature Hill et al. “Enhanced Degradation of Chemical Warfare Agents Structural & Molecular Biology, XP002627099, 11(5): 412-419, Thorugh Molecular Engineering of the Phosphotriesterase Active May 2004. Site”, Journal of the American Society, JACS, 125(30): 8990-8991, Rochu et al. “Human Paraoxonase: A Promising Approach for Pre Jul. 8, 2003. Kassa et al. “The Influence of Combinations of Oximes on the Reac Treatment and Therapy of Organophosphorus Poisoning. Toxicol tivating and Therapeutic Efficacy of Antidotal Treatment of Soman ogy, XPO220 12211, 233(1-3): 47-59, Mar. 31, 2007. Table 3. Poisoning in Rats and Mice'. Toxicology Mechanisms and Methods, Alcolombri et al. “Directed Evolution of Sulfotransferases and 19(9): 547-551, Nov. 2009. Paraoxonases by Ancestral Libraries”,Journal of Molecular Biology, Lenz et al. “Stoichimetric and Catalytic Scavengers as Protection 411: 837-853, 2011. Against Nerve Agent Toxicity: A Mini Review”. Toxicology, 233: Ashani et al. “In Vitro Detoxification of Cyclosarin in Human Blood 31-39, 2007. Pre-Incubated Ex Vivo With Recombinant Serum Paraoxonases', Li et al. “Paraoxonase Protects Against Chlorpyrifos Toxicity in Toxicology Letters, 206: 24-28, 2011. Mice". Toxicology Letters, 76: 219-226, 1995. International Preliminary Report on Patentability Dated Apr. 4, 2012 Li et al. “Stereoselective Detoxification of Chiral Sarin and Soman From the International Preliminary Examining Authority Re. Appli Analogues by Phosphotriesterase'. Bioorganic & Medicinal Chem cation No. PCTIL2010.000754. istry, 9: 2083-2091, 2001. International Search Report and the Written Opinion Dated May 22, Luo et al. “Mechanism for Potent Reactivation Ability of HOximes 2013 From the International Searching Authority Re. Application Analyzed by Reactivation Kinetic Studies With Cholinesterases No. PCT, IL2012/05O239. From Different Species”. Chemico-Biological Interactions, 187: Fairchild et al. “Computational Characterization of How the VX 185-190, 2010. Nerve Agent Binds Human Serum Paraoxonase 1’, Journal of Masson et al. "Engineering of Catalytic Bioscavengers of Molecular Modeling, XPO19856245, 17(1): 97-100, Apr. 9, 2010. Organophosphorus Compounds’, Bulletin de l'Academie Nationale Abstract. de Medicine, 191(1): 95-111, Jan. 2007. Abstract. Amitai et al. “Asymmetric Fluorogenic Organophosphatases for the Mastrobattista et al. “High-Througput Screening of Enzyme Librar Development of Active Organophosphates Hydrolases With ies: In Vitro Evolution of a Beta-Galactosidase by Fluorescence Reversed Stereoselectivity”. Toxicology, 233: 187-198, 2007. Activated Sorting of Double Emulsions”, Chemistry & Biology, 12: Ashanietal. "Prophylaxis Against Organophosphate Poisoning by an 1291-1300, Dec. 2005. Enzyme Hydrolysis Organophosphorus Compounds in Mice'. Life Melzer et al. “Reversed Enantioselectivity of Diisopropyl Sciences, 49: 367-374, 1991. Fluorophosphatase Against Organophosphorus Nerve Agents by Blumetal. “Inhibitory Potency Against Human Acetylcholinesterase Rational Design”, Journal of the American Chemical Society, JACS, and Enzymatic Hydrolysis of Fluorogenic Nerve Agent Mimics by 131: 17226-17232, 2009. Human Paraoxonase 1 and Squid Diisopropyl Fluorophosphatase'. Segallet al. “Direct Observation and Elucidation of the Structures of Biochemistry, 47(18): 5216-5224, Apr. 9, 2008. Aged and Nonaged Phosphorylated Cholinesterases by 31P NMR Broomfield "A Purified Recombinant Organophosphorus Acid Spectroscopy”. Biochemistry, 32(40): 13441-13450, 1993. Anhydrase Protects Mice Against Soman'. Pharmacology & Toxi cology, 70: 65-66, 1992. * cited by examiner U.S. Patent May 27, 2014 Sheet 1 of 27 US 8,735,124 B2

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E::::::::::::::iii says s s , s s X Aime ties easy, US 8,735,124 B2 1. 2 ISOLATED PON1 POLYPEPTIDES, The G-agents cyclosarin (GF) and Soman (GD) comprise a POLYNUCLEOTDESENCODING SAME AND prime target for, Scavenger-based prophylaxis due to the low USES THEREOF INTREATING OR efficacy of pharmacological drugs used to counteract their PREVENTING ORGANOPHOSPHATE toxicity Kassa, J., Karasova, J. Z. Caisberger, F. & Bajgar, J. EXPOSURE ASSOCATED DAMAGE The influence of combinations of oximes on the reactivating and therapeutic efficacy of antidotal treatment of Soman poi RELATED APPLICATIONS soning in rats and mice. Toxicol Mech Methods 19, 547-51 (2009)). Although applied as racemates, their S isomers comprise the tangible threat (FIG. 5). Unfortunately, This application is a Continuation-In-Part (CIP) of PCT enzymes tested thus far primarily hydrolyze less toxic R. Patent Application No. PCT/IL2010/000754 having Interna isomer Harvey, S. P. et al. Stereospecificity in the enzymatic tional filing date of Sep.15, 2010, which claims the benefit of hydrolysis of cyclosarin (GF). Enzyme and Microbial Tech priority of U.S. Provisional Patent Application No. 61/272, nology 37, 547-555 (2005); Li, W. S., Lum, K. T., Chen 363 filed Sep. 17, 2009. The contents of the above applica Goodspeed, M., Sogorb, M. A. & Raushel, F. M. Stereose tions are all incorporated herein by reference. 15 lective detoxification of chiral Sarin and Soman analogues by phosphotriesterase. Bioorg Med Chem 9, 2083-91 (2001). FIELD AND BACKGROUND OF THE Additional background art includes: INVENTION WO2004/O78991 Alcolombri, U., Elias, M., and Tawfik. D. S. (2011). The present invention, in some embodiments thereof, Directed evolution of Sulfotransferases and paraoXonases by relates to isolated PON1 polypeptides, polynucleotides ancestral libraries. Journal of molecular biology 411, 837 encoding same and uses thereof in treating or preventing 853; Ashani, Y., Goldsmith, M., Leader, H., Silman, I., Sus organophosphate exposure associated damage. sman, J. L., and Tawfik. D. S. (2011). In vitro detoxification of Inhibitors of acetylcholinesterase (AChE), including orga cyclosarin in human blood pre-incubated ex vivo with recom nophosphate (OP)-based pesticides and nerve agents, 25 binant serum paraoXonases. Toxicology letters 206, 24-28; threaten both military and civilian populations. A timely and Gupta, R. D., Goldsmith, M., Ashani.Y., Simo.Y., Mullo pharmacological treatment with atropine and oxime AChE kandov, G., Bar, H., Ben-David, M., Leader, H., Margalit, R., reactivators can save lives but in many cases does not prevent Silman, I., et al. (2011). Directed evolution of hydrolases for cholinergic crisis and the resulting onset of secondary toxic prevention of G-type nerve agent intoxication. Nat Chem Biol manifestations induced by OP intoxication. Side effects asso 30 7, 120-125. ciated with drugs such as pyridostigmine used as protective treatment prior to OP exposure have also prompted the search SUMMARY OF THE INVENTION for effective prophylactics and antidotes. Rather than mini mizing the damages caused by the OP, the goal of prophylac According to an aspect of some embodiments of the tic drugs is to intercept the OPs before they even reach their 35 present invention there is provided an isolated polypeptide target organs. A stoicheiometric bioscavenger based on comprising an amino acid sequence of serum paraoXonase human butyrylcholinesterase has been recently developed. (PON1) having catalytic efficiency of 10°-5-107 M'min' However, owing to the daunting mass ratio of OP to protein, for a G-type organophosphate. hundreds of mgs of protein are required to confer protection According to an aspect of some embodiments of the against exposure to doses beyond a single LDso dose Ashani, 40 present invention there is provided an isolated polypeptide Y. & Pistinner, S. Estimation of the upper limit of human comprising an amino acid sequence of serum paraoXonase butyrylcholinesterase dose required for protection against (PON1) having catalytic efficiency of k/K-10°-5-107 organophosphates toxicity: a mathematically based toxicoki M'min' for a G-type organophosphate and further having a netic model. Toxicol Sci 77,358-67 (2004). Catalytic scav catalytic efficiency of greater than 10 M'min' for a VX engers, namely enzymes displaying multiple turnovers, may 45 type organophosphate. allow rapid and efficient protection against high OP doses According to an aspect of some embodiments of the using low protein amounts Ditargiani, R. C., Chandraseka present invention there is provided an isolated polypeptide ran, L., Belinskaya, T. & Saxena, A. In search of a catalytic comprising an amino acid sequence of serum paraoXonase bioscavenger for the prophylaxis of nerve agent toxicity. (PON1) having catalytic efficiency of k/K-10°-5-107 Chem Biol Interact Epub ahead of print (2010. However, 50 M'min' for a G-type organophosphate, wherein the amino with few exceptions, xenobiotics such as OPs are promiscu acid sequence comprises the mutations L69V, H115A, ous Substrates for natural enzymes and are degraded with low H134R, F222M and I291L and T332S, wherein amino acid catalytic efficiencies. Improved OPhydrolyzing enzyme vari coordinates correspond to the G3C9 PON1 variant. ants have been engineered (e.g. PTE, DFPase, Hill, C.M., Li, According to an aspect of some embodiments of the W. S., Thoden, J. B., Holden, H. M. & Raushel, F. M. 55 present invention there is provided an isolated polypeptide Enhanced degradation of chemical warfare agents through comprising an amino acid sequence of serum paraoXonase molecular engineering of the phosphotriesterase active site. J (PON1) having catalytic efficiency of ki/K-10°-5-107 Am ChemSoc. 125, 8990-1 (2003), Mee-Hie Cho, C., Mul M'min' for a G-type organophosphate, wherein the amino chandani, A. & Chen, W. Functional analysis of organophos acid sequence comprises the mutations L69V, H115A, phorus hydrolase variants with high degradation activity 60 H134R, F222M, I291L, L55I, D136Q and T332S, wherein towards organophosphate pesticides. Protein Eng Des Sel 19, amino acid coordinates correspond to the G3C9 PON1 vari 99-105 (2006), Melzer, M. et al. Reversed enantioselectivity ant. of diisopropyl fluorophosphatase against organophosphorus According to an aspect of some embodiments of the nerve agents by rational design. JAm ChemSoc. 131, 17226 present invention there is provided an isolated polypeptide 32 (2009)), but prophylactic protection from >1XLDso doses 65 comprising an amino acid sequence of serum paraoXonase at reasonable protein amounts requires catalytic scavengers (PON1) having catalytic efficiency of k/K-10°-5-107 whose efficiencies ink/K terms are >10 M'min'. M'min' for a G-type organophosphate, wherein the amino US 8,735,124 B2 3 4 acid sequence comprises the mutations L69V, H115A, According to some embodiments of the invention, the pro H134R, F222M, L55I, H197R, I291L and T332S, wherein viding is effected by inhalation administration. amino acid acid coordinates correspond to the G3C9 PON1 According to some embodiments of the invention, the pro variant. viding is effected 10 hours prior to the exposure until 7 days According to an aspect of some embodiments of the following exposure. present invention there is provided an isolated polypeptide According to some embodiments of the invention, the pro comprising an amino acid sequence of serum paraoXonase viding is effected by inhalation and injection. (PON1) having catalytic efficiency of ki/K-10°-5-107 According to some embodiments of the invention, the M'min' for a G-type organophosphate, wherein the amino method further comprises administering to the Subject atro acid sequence is selected from the group consisting of 140 10 pine and optionally oxime. 142. According to some embodiments of the invention, the pro According to some embodiments of the invention, the viding is effected by topical application. nerve-agent Substrate comprises an Sp isomer. According to an aspect of some embodiments of the According to some embodiments of the invention, the iso present invention there is provided an article of manufacture lated polypeptide has catalytic efficiency of ki/K-10 15 for treating or preventing organophosphate exposure associ M'min' for Sp nerve-agent substrates. ated damage, the article of manufacture comprising the iso According to some embodiments of the invention, the Sp lated polypeptide immobilized on to a Solid Support. isomer comprises each of Soman (GD), cyclosarin (GF) and According to Some embodiments of the invention, the solid sarin (GB). Support is for topical administration. According to some embodiments of the invention, the iso According to Some embodiments of the invention, the solid lated polypeptide has catalytic efficiency ofk/K-10°-107 Support for topical administration is selected from the group M'min' for the Rp isomer of tabun (GA). consisting of a sponge, a wipe and a fabric. According to some embodiments of the invention, the iso According to Some embodiments of the invention, the solid lated polypeptide has a catalytic efficiency of greater than 10° Support is selected from the group consisting of a filter, a M'min' for a VX type organophosphate. 25 fabric and a lining. According to some embodiments of the invention, the According to an aspect of some embodiments of the amino acid sequence of serum paraoXonase (PON1) com present invention there is provided a method of detoxifying a prises a mutation selected from the group consisting of Surface, the method comprising contacting the Surface with L69G/A/L/V/S/M, K70A/S/Q/N.Y71/F/C/A/L/I, H115W/L/ the isolated polypeptide, thereby detoxifying the surface. V/C, H134R/N, F222S/M/C, F292S/V/L, T332S/M/C/A, 30 According to some embodiments of the invention, the M196V/L/F, V97A, V346A, N41D, Y293S, V97A, V276A, method further comprises contacting the Surface with a T326S, S111T, S11 OP, P135A, N41D, N324D, M289I, decontaminating foam, a combination of baking condition L240S/V, L14M, L10S, K233E, H285R, H243R, F28Y. heat and carbon dioxide, or a combination thereof. F264L, D309N/G, A6E, N227L, F178V, D49N, wherein the According to some embodiments of the invention, the amino acid coordinates correspond to the G3C9 PON1 vari 35 polypeptide is comprised in a coating, a paint, a non-film ant. forming coating, an elastomer, an adhesive, an Sealant, a According to some embodiments of the invention, the iso material applied to a textile, or a wax. lated polypeptide is expressible in bacteria. Unless otherwise defined, all technical and/or scientific According to some embodiments of the invention, the terms used herein have the same meaning as commonly amino acid sequence is selected from the group consisting of 40 understood by one of ordinary skill in the art to which the the sequences set forth in SEQID NO: 129, 2-54 and 120 invention pertains. Although methods and materials similar 128. or equivalent to those described herein can be used in the According to some embodiments of the invention, the practice or testing of embodiments of the invention, exem amino acid sequence is selected from the group consisting of plary methods and/or materials are described below. In case the sequences set forth in SEQID NO: 129, 2, 4, 7, 9, 12, 24, 45 of conflict, the patent specification, including definitions, will 47, 53, 120-128. control. In addition, the materials, methods, and examples are According to an aspect of some embodiments of the illustrative only and are not intended to be necessarily limit present invention there is provided an isolated polynucleotide 1ng. comprising a nucleic acid sequence encoding the polypep tide. 50 BRIEF DESCRIPTION OF THE DRAWINGS According to an aspect of some embodiments of the present invention there is provided a pharmaceutical compo The patent or application file contains at least one drawing sition comprising as an active ingredient the isolated polypep executed in color. Copies of this patent or patent application tide and a pharmaceutically acceptable carrier. publication with color drawing(s) will be provided by the According to an aspect of some embodiments of the 55 Office upon request and payment of the necessary fee. present invention there is provided a nucleic acid construct Some embodiments of the invention are herein described, comprising the isolated polynucleotide and a cis-regulatory by way of example only, with reference to the accompanying element driving expression of the polynucleotide. drawings. With specific reference now to the drawings in According to an aspect of some embodiments of the detail, it is stressed that the particulars shown are by way of present invention there is provided a method of treating or 60 example and for purposes of illustrative discussion of preventing organophosphate exposure associated damage in a embodiments of the invention. In this regard, the description Subject in need thereof, the method comprising providing the taken with the drawings makes apparent to those skilled in the subject with a therapeutically effective amount of the isolated art how embodiments of the invention may be practiced. polypeptide to thereby treat the organophosphate exposure In the drawings: associated damage in the Subject. 65 FIG. 1 is a scheme illustrating the pET32PON1 plasmid. According to some embodiments of the invention, the pro This plasmid was used for the expression of PON1 variants viding is effected prior to the organophosphate exposure. with a C-terminal His-tag and no GFP. The plasmid was US 8,735,124 B2 5 6 derived from pET32b(+) from which the thioredoxin fusion and R for GA). The chiral carbon of GD is indicated by an protein and peptide tags were truncated using the Not/XhoI asterix. B. Structures of the S isomers of the coumarin ana sites. The recombinant PON1 variant G3C9, and library vari logues of GD (PMP-coumarin) and GF (CMP-coumarin). ants, were inserted using the NcoI/NotI sites. The NotI FIGS. 8A-B are tables illustrating sequences and fold restriction site was inserted upstream to the His tag to enable improvement of selected variants from round 1 with Sarin the cloning of various PON1 variants with no alterations to (FIG. 8A) and Soman (FIG. 8B). the tag. FIG. 8A: FIGS. 2A-C are graphs of FACS detection and sorting of * First row—Fold Improvement in activity of each variant PON1-carrying E. coli cells in w/o/w emulsion droplets. E. relative to control variant 2D8 as measured in cleared cell coli BL21 (DE3) cells possessing GFPuv gene in the genome 10 were used for expression of the PON1 under the T7 promoter. lysates. Numbers represent an average of 2 measurements Cells were emulsified, together with the fluorogenic substrate with S.D.<10% of value. (DEPCyC). Briefly, filtered cells were compartmentalized in ** Second row—variant names. the first emulsion (water-in-oil), and 100 mM solutions of FIG.8B DEPCyC was added to the oil phase (0.8 ul, to a final con 15 * First row—Fold Improvement in activity of each variant centration of 50 uM). relative to control variant 2D8 as measured in cleared cell The production of the second emulsion (water-in-oil-in-wa lysates. Numbers represent an average of 2 measurements ter) and sorting were performed as described. More than 10° with S.D.<10% of value. events, at 2000 events/sec. were sorted using FACSAria (Bec ** Second row—variant names. ton-Dickinson). Events corresponding to single E. coli cells FIG.9 is a table illustrating sequences of improved variants were gated by GFP emission (at 530 nm, using blue laser for from round 1 used for shuffling. excitation). FIG.2A-Representative density plot FSC-H (for * First row—variant names. ward scatter) and SSC-H (side scatter) analysis of the double ** Sequence of rePON1 and variant 2D8 shown for refer emulsion. FIG.2B-Histogram of the GFP emission for the R1 CCC. population of droplets. Events gated in R2 correspond to 25 FIG. 10 is a table illustrating sequences of improved vari droplets that contain GFP expressing cells. FIG. 2C. The ants from round 2. R1+R2 gated events were analyzed for the hydrolytic activity. * First row—The number of times a clone with the same Events gated in R3 representactive variants that were present genotype was independently selected is indicated as “Times as 0.5–1% of total population; these were sorted into liquid repeated' (No number indicated one time). growth media. 30 ** Second row—variant names. FIG. 3 is a graph illustrating kinetic parameters. Shown is *** Last rows—Fold Improvement in activity of each vari a representative Michaelis-Menten plot for rePON1 variants ant relative to round 1 variant PG 11 as measured in cleared 8C8, OC9, and 3D8, evolved towards S-CMP-MeCyC cell lysates. Numbers represent an average of 3 measure hydrolysis. Enzyme concentrations were 0.65 uM for 8C8, ments with S.D.<10% of value. and 12.5 nM for OC9 and 3D8. Substrate concentrations were 35 FIG. 11 is a table illustrating sequences of improved vari varied from 0.4 uM up to 1000 uM.: ants from round 2 used for shuffling. FIG. 4 is a graph showing the effect of excess of free * First row—variant names coumarin on the hydrolysis of CMP-F by variant 4E9. The ** Sequence of rePON1 and variant 2D8 shown for refer kinetics of CMP-F (40 nM) hydrolysis by 4E9 (16 nM) were CC determined with and without the addition of a 4-fold excess of 40 FIGS. 12A-B are tables summarizing sequences and fold free coumarin (64 nM). improvement of selected variants from round 3 with Sarin FIG. 5 shows some organophosphates (Ops) used herein. (FIG. 12A) and Soman (FIG. 12B). Shown are the two enantiomers of G-agents: cyclosarin (GF, FIG 12 A: R=cyclohexyl), sarin (GB, R=iso-propyl) and soman (GD, * First row—Fold Improvement in activity of each variant R pinacolyl). For consistency, the fluorogenic analogues 45 relative to round 2 variants IA4 and VIID11 as measured in (X=3-cyano-7-hydroxy-4-methylcoumarin) are dubbed cleared cell lysates. Numbers represent an average of 2 mea CMP-coumarin, IMP-coumarin, and Pin-coumarin, respec surements with S.D.<10% of value. tively, and the actual agents (X=F) CMP-F, IMP-F, and Pin-F, ** Second row—variant names (names starting with “1” respectively. are from library 1, with "2 are from library 2) FIGS. 6A-C shows the hydrolysis of CMP-coumarin and 50 FIG. 12 B: CMP-F by rePON1 variants. Enzyme concentrations were * First row—Fold Improvement in activity of each variant varied depending on the variant's activity, and are noted in the relative to round 2 variants IA4 and VIID11 as measured in figure. FIG. 6A. Hydrolysis of racemic CMP-coumarin (12 cleared cell lysates. Numbers represent an average of 2 mea uM) in the presence of variants 4E9, 3D8, 3B3 (plus addition surements with S.D.<10% of value. of 0.03 uM 4E9 after 6 mins; indicated by the black arrow), 55 ** Second row—variant names (names starting with “1” and wild-type-like rePON1 (plus addition of 0.03 uM 4E9 are from library 1, with "2 are from library 2). after 20 mins). FIG. 6B. Hydrolysis of S-CMP-coumarin (6 FIG. 13 is a table illustrating the Sequences of improved uM) in the presence of variants 4E9,3D8, 3B3, and rePON1. variants from round 3 used for shuffling. FIG. 6C. Residual AChE activity was assayed following the * First row—variant names incubation of in-situ generated CMP-F (40 nM) and 4E9, 60 ** Sequence of rePON1 and variant 2D8 shown for refer 3D8, 3B3, and rePON1; the data were fitted to a first-order CCC. rate equation to derive the apparent rate constant for hydroly FIG. 14 is a table illustrating the sequences and fold SiS of CMP-F. improvement of selected variants from round 4. FIGS. 7A-B illustrate the structures of the nerve agents and * First row—The number of times a clone with the same their analogues used in Example 7. A. Structures of GA, GB, 65 genotype was independently selected is indicated as “Times GD, GF and VX. Shown are the toxic isomers, i.e. the more repeated' (No number indicated one time). potent AChE inhibiting isomers (S, for GB, GD, GF and VX. ** Second row—variant names. US 8,735,124 B2 7 8 *** Last rows—Fold Improvement in activity of each vari function in order to visualize the stereo-preference of the ant relative to round 3 variant 1-I-F11, as measured in cleared tested PON1s. Inset: expansion of the abscise from t=0 to 20 cell lysates. Numbers represent an average of 3 measure minutes. ments with S.D.<10% of value. FIGS. 19A-B illustrate activity of variants in blood FIG. 15 is a graph illustrating the hydrolysis of GD by 5 samples. evolved variants. Residual AChE activity was assayed and A. Protection by evolved variants of blood cholinesterases plotted as % of inhibitor activity after the incubation of in situ from inhibition by GF. Evolved variants (0.5-2.1 uM) were generated GD (50-100 nM) with 2D8, VIID11, IIG1 and incubated in whole blood samples (37°C.) for 24 h. GF was rePON1 at the concentrations noted in the figure (data were added (0.1 uM) and the samples were assayed for residual 10 acetylcholinesterase activity. Cholinesterase protection activ fitted to a 2"-order rate equation to derive the apparent rate ity was compared to the initial protection levels observed after constants for hydrolysis of the two toxic isomers of GD). 5 min of incubation (41-45%). B. Hydrolytic activity in whole FIGS. 16A-B are graphs illustrating the hydrolysis of S blood samples. Evolved variants (0.3-1.1 uM) were incubated CMP-coumarin (FIG. 15A) and R-CMP-coumarin (FIG. in whole blood samples (37° C.) for 24 hours. Incubated 15B). 15 samples were diluted in buffer and residual enzyme activities FIG. 16A is a Michaelis-Menten plot for rePON1 variants were assayed with CMP-coumarin. The percent activity was PG11, VIID11, 1-I-F11 and VIID2, with S-CMP-coumarin. determined relative to the hydrolytic activity observed after 5 All variants were at 10 nM concentration. Substrate concen min incubation. trations were varied from 3 uM up to 240 uM. Initial veloci FIG. 20 is a graph illustrating the Stability of evolved ties (Vo) were measured at 405 nm. variants in buffer at 37° C. Evolved variants: PG 11 (0.2 uM), FIG. 16B is a Michaelis-Menten plot for rePON1 variants VIID11 (0.13 uM), 1-I-F11 (0.23 uM), VIID2 (0.12 uM) were PG11, VIID11, 1-I-F11 and VIID2, with R-CMP-coumarin. incubated in buffer (Tris 50 mM pH7.4, CaCl, 1 mM, NaCl50 Variant concentrations were: PG 11 0.3 uM, VIID1 1 0.2 LM, mM, Tergitol 0.1%) at 37°C. for 24 hours. Incubated samples 1-I-F1 1 0.4 uM and VIID2 0.6 uM. Substrate concentrations were assayed for CMP-coumarin hydrolysis using 0.3 mM were varied from 4 uM up to 700 uM. Initial velocities (Vo) 25 CMP-coumarin at 400 nm in buffer (Tris 50 mM pH 8, CaCl, were measured at 405 nm. 1 mM, NaCl 50 mM, Tergitol 0.1%) at 25° C. Percent hydro FIGS. 17A-B are graphs illustrating hydrolysis of PMP lytic activity by each variant was determined relative to the coumarin by evolved variants. initial hydrolytic activity derived following only 5 min of A. The hydrolysis of a racemic mixture of PMP-coumarin incubation in buffer. (10 uM) by various PON1 variants (0.5uM) was monitored at 30 400 nm. Complete hydrolysis of all isomers was observed DESCRIPTION OF SPECIFIC EMBODIMENTS upon addition of NaF (0.25M). Partial hydrolysis, restricted OF THE INVENTION to R, isomers (R.S., R.R.), was displayed by the previously described rePON1 variant 3B3 (Ashani, et al., 2010). Round The present invention, in Some embodiments thereof, 1 variant PG 11 hydrolyzed both sets of R, and S isomers, 35 relates to isolated PON1 polypeptides, polynucleotides although at different rates resulting in two distinct phases. In encoding same and uses thereof in treating or preventing contrast, Round 2-4 variants VIID1 1, 1-1-F11 and VIID2 organophosphate exposure associated damage. exclusively hydrolyzed the S isomer pair (S.R., S.S.). B. Before explaining at least one embodiment of the invention The hydrolysis of S, PMP-coumarin isomers (S.R.S.S.) by in detail, it is to be understood that the invention is not nec evolved variants was monitored under similar conditions. 40 essarily limited in its application to the details set forth in the Prior to the addition of these variants (indicated by a dashed following description or exemplified by the Examples. The arrow), samples were pre-incubated with the R specific vari invention is capable of other embodiments or of being prac ant 3B3. Complete hydrolysis was monitored by addition of ticed or carried out in various ways. NaF. Organophosphates (OPs), including pesticides and nerve FIG. 18A is a readout of a 'P NMR analysis of GA 45 agents, comprise a prime target for detoxification. Albeit, no hydrolysis by rePON. The hydrolysis of GA by rePON1 was natural enzymes are available that proficiently degrade most monitored at different time intervals using 'P NMR. GA (1 of these xenobiotics. Obtaining highly proficient OP hydro mg/ml) was incubated with rePON1 (1.25 uM) in activity lases, and in particular for the more toxic stereoisomer Sp of buffer for up to 75 min. The 'PNMR spectra of the mixture, the G-type nerve agents remains a challenge. containing also 10% aceton and 2 mg/ml internal standard of 50 The present inventors generated through laborious experi O.O.-diisopropyl methylphosphonate, was recorded at the mentation and Screening a series of variants of mammalian indicated times. The traces of 'P NMR show the intact GA serum paraoXonase (PON1)—an enzyme that is potentially (1; 6, -4.5 ppm), the hydrolysis product N,N-di-methyla applicable in vivo, with sufficiently high catalytic efficiency mido-O-ethyl phosphate (2: 6, 14.5 ppm), and the internal for detoxification (k/Ke107 M'min'). Directed evolu standard (3; 8,36.0). A control sample without PON1 did not 55 tion of PON1 using structure-based as well as random exhibit any detectable GA hydrolysis for at least 75 min (data mutagenesis, and combining low-throughput methodologies not shown). (96-well plate screening) with high-throughput screens using FIG. 18B is a graph illustrating the kinetics of GA hydroly compartmentalization in emulsions, enabled taking wild sis by PON1 variants. The hydrolysis of GA was monitored at type-like PON1 that has no detectable activity with Sp G-type different time intervals using 'P NMR. GA (1 mg/ml) was 60 OPs, and generating variants with catalytic efficiency of>107 incubated with either rePON1 (1.25 uM); open square, vari M'min'. While the directed evolution used model OPs with ant 2D8 (2.5 M); open circle or variant VIID2 (0.36 uM); a fluorogenic leaving group, a final Screen was done using an filled square in activity buffer containing 10% aceton and acetylcholinesterase inhibition assay and in-situ generated 20% DO (for signal locking) for up to 75 min. The integrated nerve agents to identify highly proficient variants that can area under the GA peak was normalized against the area 65 hydrolyse the actual nerve agents. under the intact O.O-diisopropyl methylphosphonate (2 The present detoxification model was also validated by mg/ml). Data points were fitted to a bi-exponential decay demonstrating prophylactic protection in an animal model. US 8,735,124 B2 9 10 The differences in Survival and intoxication symptoms paraoxon. More recently, in addition to its role in lipid between mice pretreated with the evolved variant 4E9 and metabolism and, hence, in cardiovascular disease and arterio mice pretreated with the conventional atropine-oxime treat sclerosis, PON1 has also been shown to be involved in the ment probably relate to the very different effects of these metabolism of lactones and cyclic carbonates. Early studies treatments—atropine plus 2-PAM aims to minimize the dam of enzymatic activity in serum indicated a bimodal or trimo ages of the OP, whereas rePON-4E9 neutralizes the agent dal distribution in Caucasian populations. Two main poly before it even reaches its target. In conclusion, there is a direct morphisms in the coding region, as well as five in the 5' correlation between the catalytic efficiency of evolved PON1 regulating region, have been characterized. The Q192R poly variants at OP hydrolysis in-vitro and the ability of these morphism determines the catalytic efficiency of hydrolysis of variants to act as effective prophylactics in-vivo. 10 The newly isolated rePON1 variants, and the methodolo Some Substrates, and certain promoter polymorphisms, in gies described here, also provide the basis for further engi particular C-108T, contribute to the level of expression of neering of PON1 towards other G-type nerve agents, e.g. PON1. Recently, additional polymorphisms in the coding sarin, and soman. The evolved variants hydrolyze these G region, 5' regulatory region, and PON1 introns have been type nerve agents, and Soman (GD) in particular, at relatively 15 reported. high rates (4E9's apparent k/K value for sarin (IMP-F) is Any PON1 may be used e.g., human PON1, rabbit PON1. s3x10 M' min', and for soman (Pin-F), 7.4x10 M' Others are listed below (Table 1a1). TABLE 1a1

Human Organism Gene Locus Description Similarity NCBI accessions dog PON1 paraoxonase 89.11(n) 475234XM 845126.1 XP 850219.1 (Canis 1 87.89(a) familiaris) chimpanzee PON1 — paraoxonase 99.44(n) 463547 XM 51921.1.2 XP 519211.1 (Pan 1 99.15(a) troglodytes) COW PON1 paraOXonase 85.4(n) 523798 NM OO1046269.1 NP OO1039734.1 (Bos tattrits) 1 82.49(a) rat Pon1 paraoxonase 82.54(n) 84024 NM 032077.1 NP 114466.1 (Rattus 1 80.56(a) norvegicus) OSC PO1 6 (0.50 cM) paraoxonase 83.1(n)" 18979 NM O11134.2 NP O35264.1 (Mus 1 81.97(a) BCO12706 L40488 musculus) min', and 0.58x10 M' min', for the two toxic isomers In a specific embodiment the enzyme is expressible in E. respectively). 1-I-F11 exhibits ki/K-4*10° (M' min') Colisuch as the PON1 variant G3C9 having GenBank Acces with the toxic isomer of Sarin (GB) and a catalytic rate of sion AY499193 (see e.g., WO2004/078991, which describes this variant and other equivalent variants and is hereby incor k/K-4.4*107 (M'min') for all toxic isomers of Soman 40 porated by reference in its entirety). (GD). A catalytic efficiency ofk/K-4.6*10 (M'min') As used herein, a “nerve agent” refers to an organophos for the more toxic isomer of cyclosparin (GF) is exhibited phate (OP) compound Such as having an acetylcholinesterase with the 1-I-F11 enzyme. inhibitory activity. The toxicity of an OP compound depends Using exemplary variants, the present inventors showed on the rate of its inhibition of acetylcholinesterase with the that they were able to protect human blood cholinesterases 45 concomitant release of the leaving group Such as fluoride, ex-vivo from inhibition by GF for at least 24 hours, thus alkylthiolate, cyanide oraryoxy group. The nerve agent may Supporting the possibility of utilizing them for in-vivo pro be a racemic composition orapurified enantiomer (e.g., Sp or phylaxis. Finally, it was found that the novel variants had Rp). improved by >150-fold relative to wild-type PON1 for According to a specific embodiment, the nerve agent Sub hydrolysis of the toxic isomer of VX, thus providing a starting 50 strate comprises an Sp isomer. point for the directed evolution of PON1 for neutralization of It will be appreciated that a single variant of this aspect of V-type agents. the present invention may be able to efficiently hydrolyse (i.e. Thus, according to an aspect of the invention there is pro having a ki/K-10°-5-107 M'min') more than one Sp vided an isolated polypeptide comprising an amino acid isomer of G agents, for example Sp isomers of two different sequence of serum paraoxonase (PON1) having catalytic effi 55 Gagents, three different G agents, or even four different G ciency of k/K-10°-5-107 M'min' for a nerve-agent agents and may therefore serve as a broad range G-type substrate. prophylactic. Thus for example the present inventors have As used herein the term "serum paraoxonase (PON1) shown that VII-D11, 1-1-F11 and IIG1 has a catalytic activity refers to a naturally occurring or man-made sequence. PON1 for the Sp isomer of each of GD, GB and GF in this range. 60 Further, the present invention conceives of variants which (EC 3.1.8.1 or EC 3.1.1.2 e.g., PON1 HUMAN, P27169) is a have high catalytic activities (i.e. having ak/K-10°-5-107 high-density lipoprotein (HDL)-associated serum enzyme M'min') towards the Spisomers of GD, GB and GF, and in whose primary physiological role is to protect low-density addition having a high catalytic activity towards the Rp iso lipoproteins (LDLs) from oxidative modifications. PON1 can mer of GA. also hydrolyze organophosphorus (OP) compounds, includ 65 Certain OP compounds are so toxic to humans that they ing commonly used insecticides, and its name derives from have been adapted for use as chemical warfare agents one of its most commonly used in vitro Substrates— (CWAS). Such as tabun, Soman, Sarin, cyclosarin, VX, and US 8,735,124 B2 11 12 R-VX. A CWA may be in airborne form and such a formula Amino acid coordinates should be adapted easily to PON1 tion is known herein as an “OP-nerve gas.” Examples of variants of the same or other species by amino acid sequence airborne forms include a gas, a vapor, an aerosol, a dust, or a alignments which may be done manually or using specific combination thereof. Examples of an OP compounds that bioinformatic tools such as FASTA, L-ALIGN and protein may be formulated as an OP nerve gas include tabun, Sarin, 5 Blast. Soman, cyclosarin, VX. GX or a combination thereof. An example of an organophosphate which is close to, albeit not similar in its properties to those of the nerve gases is that of DFP. diisopropylfluorophosphonate, which is considerably less Volatile than certain members of this group. 10 In addition to the initial inhalation route of exposure com mon to Such agents, CWAS, especially persistent agents such as VX and thickened Soman, pose threats through dermal absorption. In “Chemical Warfare Agents: Toxicity at Low Levels.” (Satu M. Somani and James A. Romano, Jr., Eds.) p. 15 414, 2001. Such persistent CWA agents remain as a solid or liquid while exposed to the open air for more than three hours. Often after release, a persistent agent may convert from an Thus the present teachings provide for an isolated polypep airborne dispersal form to a solid or liquid residue on a Sur tide comprising an amino acid sequence of serum paraoXo face, thus providing the opportunity to contact the skin of a nase (PON1) having catalytic efficiency between the range of human. k/K-10°-10 M'min', or specifically of k/K-10 Examples of an OP pesticide include bromophos-ethyl, M'min', k/K-5x10 M'min', k/K-107 M' chlorpyrifos, chlorfenvinphos, chlorothiophos, chlorpyrifos min', k/K-5x10'M'min', k/K-10M'min' for methyl, coumaphos, crotoxyphos, crufomate, cyanophos, nerve-agent Substrates (e.g., Sp isomers). diazinon, dichlofenthion, dichlorvos, dursban, EPN, ethop 25 The polypeptides of the present invention are preferably rop, ethyl-parathion, etrimifos, famphur, fensulfothion, expressible in bacteria such as E. coli e.g., BL21, BL21 fenthion, fenthrothion, isofenphos, jodfenphos, leptophos (DE3), Origami B (DE3), available from Novagen (wwwdot oxon, malathion, methyl-parathion, mevinphos, paraoxon, calbiochemdotcom) and RIL (DE3) available from Strat parathion, parathion-methyl, pirimiphos-ethyl, pirimiphos agene, (www.dotstratagenedotcom). Essentially, at least 2%, methyl, pyrazophos, quinalphos, ronnel, Sulfopros, Sul 30 at least 5%, at least 10%, at least 20%, at least 30%, at least fotepp, trichloronate, or a combination thereof. 40%, at least 50%, at least 60%, at least 70%, at least 80%, at Methods of selecting PON1 polypeptides with the desired least 90%, at least 95% or more, say 100%, of bacterially activity are provided in the Examples section below. Typi expressed protein remains soluble (i.e., does not precipitate cally, these methods involve directed evolution of PON1 into inclusion bodies). using structure-based as well as random mutagenesis, and 35 According to some embodiments of the invention, the combining low-throughput methodologies (96-well plate amino acid sequence of the polypeptide is selected from the screening) with high-throughput screens e.g., using compart group consisting of the sequences set forth in SEQ ID NO: mentalization in emulsions, 129, 2-54, 120-128 and 140-142. As used herein the phrase “in vitro evolution process” (also According to a specific embodiment, the isolated polypep referred to as “a directed evolution process') refers to the 40 tide is selected from the list below (Table 1a2). Other manipulation of genes and selection or screening of a desired polypeptides are listed in the Examples section which fol activity. A number of methods, which can be utilized to effect lows. in vitro evolution, are known in the art. One approach of executing the in-vitro evolution process is provided in the TABLE 1a2 Examples section. 45 General outline of directed evolution is provided in Round Name of clone SEQID NO: Tracewell C A, Arnold F H “Directed enzyme evolution: Round O 8C8 47 climbing fitness peaks one amino acid at a time Curr Opin OC9 53 Chem Biol. 2009 February; 13(1):3-9. Epub 2009 Feb. 25; 1A4 12 Gerlt JA, Babbitt PC, Curr Opin Chem Biol. 2009 February: 50 2D8 2 4E9 4 13(1): 10-8. Epub 2009 Feb. 23 and WO2004/078991 (either Round 1 called SH8 7 of which is hereby incorporated by reference in its entirety). G1-2D8 2G11 24 Methods of producing recombinant proteins are well 9C3 9 known in the art. Round 2 called VI-D2 124 According to a specific embodiment, mutations which may 55 G2-2D8 MG2-I-A4 120 IV-D11 122 be employed to improve the hydrolytic efficiency of PON1 to II-A1 121 nerve agent Substrates comprise mutations in at least one of VB3 123 the following residues, F28, N41, E53, D54, L69, K70,Y71, VII-D11 125 P72, G73, I74, M75, H115, G116, H134, V167, N168, D169, Round 3 called 2-II-D12 126 also -G3-2D8 1-I-D10 127 T181, D183, H184, M196, H197, F222, A223, N224, G225, 60 1-IV-H9 128 L240, L241, L267, V268, D269, N270, C284, H285, N287, Round 4 1-I-F11 129 G288, R290, I291, F292, F293, Y294, N309, G330, S331, IIG1 140 T332, V346, F347 V436 Y293, V276, T326, S111, S110, VH3 141 P135, N41, N324, M289, L240, L14, L10, L55, K233, H285, VIID2 142 H243, F28, F264, D309, A6, N227, F178, R136Q and D49, 65 where the coordinates corresponds to the PON1 variant G3C9 As used herein the term "isolated’ refers to isolated from (SEQ ID NO: 1) having GenBank Accession AY499.193. the natural environment e.g., serum. US 8,735,124 B2 13 14 The term “polypeptide' as used herein encompasses native Peptide bonds ( CO. NH ) within the peptide may be polypeptides (synthetically synthesized polypeptides or substituted, for example, by N-methylated bonds ( N recombinant polypeptides) and peptidomimetics, as well as peptoids and semipeptoids which are peptide analogs, which (CH3)-CO ), ester bonds ( C(R)H-C-O-O C(R)- may have, for example, modifications rendering the polypep N ), ketomethylen bonds ( CO-CH2-), C.-aza bonds tides more stable while in a body or more capable of penetrat ( NH N(R)—CO ), wherein R is any alkyl, e.g., methyl, ing into cells. Such modifications include, but are not limited carba bonds (—CH2-NH ), hydroxyethylene bonds ( CH to N terminus modification, C terminus modification, peptide (OH)—CH2-), thioamide bonds (—CS NH ), olefinic bond modification, including, but not limited to, CH2-NH. double bonds (—CH=CH-), retro amide bonds ( NH CH2-S, CH2-S-O, O–C NH, CH2-O, CH2-CH2, 10 CO ), peptide derivatives ( N(R)—CH2-CO—), wherein S—C NH, CH=CH or CF=CH, backbone modifications, R is the “normal side chain, naturally presented on the car and residue modification. Methods for preparing peptidomi bon atom. metic compounds are well known in the art and are specified, Synthetic amino acid Substitutions may be employed to for example, in Quantitative Drug Design, C. A. Ramsden improve stability and bioavailability. Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which 15 Table la3 below lists non-conventional or modified amino is incorporated by reference as if fully set forth herein. Fur acids e.g., synthetic, which can be used with the present ther details in this respect are provided hereinunder. invention. TABLE 1 a.3

Non-conventional amino acid Code Non-conventional amino acid Code C-aminobutyric acid Abu L-N-methylalanine Nimala C-amino-O-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine NmaSn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgin carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisoleucine Nmile D-alanine Dal L-N-methyleucine Nimleu D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine Dgln L-N-methylnorvaline Nminva D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline Nmpro D-leucine Deu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nimthr D-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nwa D-tyrosine Dtyr C.-methyl-aminoisobutyrate Maib D-valine Dval C-methyl-y-aminobutyrate Mgabu D-O-methylalanine Dmala C.-methylcyclohexylalanine Mchexa D-O-methylarginine Dmarg C.-methylcyclopentylalanine Mcpen D-O-methylasparagine DmaSn C.-methyl-C-napthylalanine Manap D-O-methylaspartate Dmasp C.-methylpenicillamine Mpen D-O-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-O-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-O-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-O-methylisoleucine Dmile N-amino-C.-methylbutyrate Nmaabu D-O-methylleucine Dmleu C-napthylalanine Anap D-O-methyllysine Dmlys N-benzylglycine Nphe D-O-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln D-O-methylornithine Dmorn N-(carbamylmethyl)glycine Nasin D-O-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu D-O-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-O-methylserine Dmser N-cyclobutylglycine Nicbut D-O-methylthreonine Dmthr N-cycloheptylglycine Nchep D-O-methyltryptophan Dmitrp N-cyclohexylglycine Nchex D-O-methyltyrosine Dmity N-cyclodecylglycine Nicdec D-O-methylvaline Dmval N-cyclododeclglycine Nicdod D-O-methylalnine Dnimala N-cyclooctylglycine Nicoct D-O-methylarginine Dnmarg N-cyclopropylglycine Nicpro D-O-methylasparagine DnmaSn N-cycloundecylglycine Ncund D-O-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm D-O-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe D-N-methyleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen US 8,735,124 B2 15 16 TABLE 1a3-continued

Non-conventional amino acid Code Non-conventional amino acid Code N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnimpro N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Neu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nwa D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen Y-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-C.-methylalanine Mala L-C.-methylarginine Marg L-C.-methylasparagine Masin L-C.-methylaspartate Masp L-C.-methyl-t-butylglycine Mtbug L-C.-methylcysteine Mcys L-methylethylglycine Metg L-C.-methylglutamine Mglin L-C.-methylglutamate Mglu L-C-methylhistidine Mhis L-C.-methylhomo phenylalanine Mhphe L-C.-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet D-N-methylglutamine Dnmgn N-(3-guanidinopropyl)glycine Narg D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine Nser D-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine Nhis D-N-methyleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnimpro N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Neu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen Y-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-C.-methylalanine Mala L-C.-methylarginine Marg L-C.-methylasparagine Masin L-C.-methylaspartate Masp L-C.-methyl-t-butylglycine Mtbug L-C.-methylcysteine Mcys L-methylethylglycine Metg L-C.-methylglutamine Mglin L-C.-methylglutamate Mglu L-C-methylhistidine Mhis L-C.-methylhomophenylalanine Mhphe L-C.-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-C.-methyleucine Meu L-C-methyllysine Mlys L-C.-methylmethionine Mmet L-C.-methylmorleucine Minle L-C.-methylnorvaline Mnva L-C-methylornithine Morn L-C.-methylphenylalanine Mphe L-C-methylproline Mpro L-C.-methylserine Se L-C-methylthreonine Mthr L-C-methylvaline Mtrp L-C-methyltyrosine Mtyr L-C.-methyleucine Mval Nnbhm L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl) carbamylmethyl-glycine Nnbhm carbamylmethyl(1)glycine Nnbhe -carboxy-1-(2,2-diphenyl Nmbc ethylamino)cyclopropane

The present teachings also provide for nucleic acid 50 sequence can be Subsequently amplified in vivo or in vitro sequences encoding Such PON1 polypeptides. using a DNA dependent DNA polymerase. Thus, according to an aspect of the present invention there As used herein the phrase "genomic polynucleotide is provided an isolated polynucleotide including a nucleic sequence” refers to a sequence derived (isolated) from a chro acid sequence, which encodes the isolated polypeptide of the mosome and thus it represents a contiguous portion of a present invention. 55 As used herein the phrase “an isolated polynucleotide' chromosome. refers to a single or a double stranded nucleic acid sequence As used herein the phrase “composite polynucleotide which is isolated and provided in the form of an RNA sequence” refers to a sequence, which is at least partially sequence, a complementary polynucleotide sequence complementary and at least partially genomic. A composite (cDNA), a genomic polynucleotide sequence and/or a com 60 sequence can include Some exonal sequences required to posite polynucleotide sequences (e.g., a combination of the encode the polypeptide of the present invention, as well as above). Some intronic sequences interposing therebetween. The As used herein the phrase “complementary polynucleotide intronic sequences can be of any source, including of other sequence” refers to a sequence, which results from reverse 65 genes, and typically will include conserved splicing signal transcription of messenger RNA using a reverse transcriptase sequences. Such intronic sequences may further include cis or any other RNA dependent DNA polymerase. Such a acting expression regulatory elements. US 8,735,124 B2 17 18 According to an exemplary embodiment the polynucle tides of the present invention. Such a medium typically otide is selected from the group consisting of 56-108 and includes an aqueous Solution having assimilable carbon, 130-139. nitrogen and phosphate sources, and appropriate salts, min Polypeptides of the present invention can be synthesized erals, metals and other nutrients, such as vitamins. Cells of the using recombinant DNA technology or solid phase technol 5 present invention can be cultured in conventional fermenta Ogy. tion bioreactors, shake flasks, test tubes, microtiter dishes, Recombinant techniques are preferably used to generate and petri plates. Culturing can be carried out at a temperature, the polypeptides of the present invention. Such recombinant pH and oxygen content appropriate for a recombinant cell. techniques are described by Bitter et al., (1987) Methods in Such culturing conditions are within the expertise of one of Enzymol. 153:516-544, Studier et al. (1990) Methods in 10 Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511 ordinary skill in the art. 514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et Depending on the vector and host system used for produc al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) tion, resultant proteins of the present invention may either Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. remain within the recombinant cell; be secreted into the fer 6:559-565 and Weissbach & Weissbach, 1988, Methods for 15 mentation medium; be secreted into a space between two Plant Molecular Biology, Academic Press, NY. Section VIII, cellular membranes, such as the periplasmic space in E. coli: pp. 421-463. or be retained on the outer surface of a cellor viral membrane. To produce a polypeptide of the present invention using Following a certain time in culture, recovery of the recom recombinant technology, a polynucleotide encoding a binant protein is effected. The phrase “recovering the recom polypeptide of the present invention is ligated into a nucleic binant protein’ refers to collecting the whole fermentation acid expression construct, which includes the polynucleotide medium containing the protein and need not imply additional sequence under the transcriptional control of a cis-regulatory steps of separation or purification. Proteins of the present (e.g., promoter) sequence Suitable for directing constitutive invention can be purified using a variety of Standard protein or inducible transcription in the host cells, as further purification techniques, such as, but not limited to, affinity described hereinbelow. 25 chromatography, ion exchange chromatography, filtration, Other than containing the necessary elements for the tran electrophoresis, hydrophobic interaction chromatography, Scription and translation of the inserted coding sequence, the gel filtration chromatography, reverse phase chromatogra expression construct of the present invention can also include phy, concanavalin A chromatography, chromatofocusing and sequences (i.e., tags) engineered to enhance stability, produc differential solubilization. tion, purification, yield or toxicity of the expressed polypep 30 Polypeptides of the present invention can be used for treat tide. Such a fusion protein can be designed so that the fusion ing an organophosphate exposure associated damage. protein can be readily isolated by affinity chromatography: Thus according to an aspect of the invention there is pro e.g., by immobilization on a column specific for the heterolo vided a method of treating or preventing organophosphate gous protein. Where a cleavage site is engineered between the exposure associated damage in a subject in need thereof, the peptide moiety and the heterologous protein, the peptide can 35 method comprising providing the Subject with a therapeuti be released from the chromatographic column by treatment cally effective amount of the isolated polypeptide described with an appropriate enzyme or agent that disrupts the cleav above to thereby treat the organophosphate exposure associ age site e.g., see Booth et al. (1988) Immunol. Lett. 19:65 ated damage in the Subject. 70; and Gardella et al., (1990) J. Biol. Chem. 265:15854 As used herein the term “treating refers to preventing, 15859. 40 curing, reversing, attenuating, alleviating, minimizing, Sup A variety of prokaryotic or eukaryotic cells can be used as pressing or halting the deleterious effects of the immediate host-expression systems to express the polypeptide coding life-threatening effects of organophosphate intoxication and sequence. These include, but are not limited to, microorgan its long-term debilitating consequences. isms, such as bacteria transformed with a recombinant bac As used herein the phrase “organophosphate exposure teriophage DNA, plasmid DNA or cosmid DNA expression 45 associated damage” refers to short term (e.g., minutes to vector containing the polypeptide coding sequence; yeast several hours post-exposure) and long term damage (e.g., one transformed with recombinant yeast expression vectors con week up to several years post-exposure) to physiological taining the polypeptide coding sequence; plant cell systems function (e.g., motor and cognitive functions). Organophos infected with recombinant virus expression vectors (e.g., cau phate exposure associated damage may be manifested by the liflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or 50 following clinical symptoms including, but not limited to, transformed with recombinant plasmid expression vectors, headache, diffuse muscle cramping, weakness, excessive Such as Ti plasmid, containing the polypeptide coding secretions, nausea, vomiting and diarrhea. The condition may sequence. Mammalian expression systems can also be used to progress to seizure, coma, paralysis, respiratory failure, express the polypeptides of the present invention. Bacterial delayed neuropathy, muscle weakness, tremor, convulsions, systems are preferably used to produce recombinant polypep 55 permanent brain dismorphology, social/behavioral deficits tides, according to the present invention, thereby enabling a and general cholinergic crisis (which may be manifested for high production volume at low cost. instance by exacerbated inflammation and low blood count. Other expression systems such as insects and mammalian Extreme cases may lead to death of the poisoned Subjects. host cell systems, which are well known in the art can also be As used herein the term “organophosphate compound used by the present invention. 60 refers to a compound comprising a phosphoryl center, and In any case, transformed cells are cultured under effective further comprises two or three esterlinkages. In some aspects, conditions, which allow for the expression of high amounts of the type of phosphoester bond and/or additional covalent recombinant polypeptides. Effective culture conditions bond at the phosphoryl center classifies an organophosphorus include, but are not limited to, effective media, bioreactor, compound. In embodiments wherein the phosphorus is linked temperature, pH and oxygen conditions that permit protein 65 to an oxygen by a double bond (PdbdO), the OP compound is production. An effective medium refers to any medium in known as an “oxon OP compound' or “oxon organophospho which a cell is cultured to produce the recombinant polypep rus compound. In embodiments wherein the phosphorus is US 8,735,124 B2 19 20 linked to a sulfur by a double bond (PdbdS), the OP com abrogate the biological activity and properties of the admin pound is known as a “thion OP compound' or “thion orga istered compound. An adjuvant is included under these nophosphorus compound.” phrases. Additional examples of bond-type classified OP com Herein the term “excipient” refers to an inert substance pounds include a phosphonocyanidate, which comprises a added to a pharmaceutical composition to further facilitate P CN bond; a phosphoroamidate, which comprises a P N administration of an active ingredient. Examples, without bond; a phosphotriester, which comprises a P( O—R1) limitation, of excipients include calcium carbonate, calcium bond; a phosphodiester, which comprises a P(-O-R) phosphate, various Sugars and types of starch, cellulose bond, where R is alkyl or aryl moieties; a phosphonofluori derivatives, gelatin, vegetable oils and polyethylene glycols. 10 Techniques for formulation and administration of drugs date, which comprises a P-F bond; and a phosphonothiolate, may be found in “Remington's Pharmaceutical Sciences.” which comprises a P S-alkyl or P S-alkyl-N(R'), bond, Mack Publishing Co., Easton, Pa., latest edition, which is where R is any alkyl group. A “dimethoxy OP compound incorporated herein by reference. comprises two methyl moieties covalently bonded to the Suitable routes of administration may, for example, phosphorus atom, such as, for example, malathion. A "diethyl 15 include oral, rectal, dermal, transmucosal, especially trans OP compound comprises two ethoxy moieties covalently nasal, intestinal or parenteral delivery, including intramuscu bonded to the phosphorus atom, Such as, for example, diazi lar, Subcutaneous and intramedullary injections as well as non or paraoxon. intrathecal, direct intraventricular, intravenous, inrtaperito In general embodiments, an OP compound comprises an neal, intranasal, intrabone or intraocular injections. organophosphorus nerve agent or an organophosphorus pes Alternately, one may administer the pharmaceutical com ticide. position in a local rather than systemic manner, for example, As used herein the phrase “a subject in need thereof refers via injection of the pharmaceutical composition directly into to a human or animal subject who is sensitive to OP toxic a tissue region (e.g., skin) of a patient. Topical administration effects. Thus, the subject may be exposed or at a risk of is also contemplated according to the present teachings. exposure- to OP. Examples include civilians contaminated by 25 Pharmaceutical compositions of the present invention may a terrorist attack at a public event, accidental spills in industry be manufactured by processes well known in the art, e.g., by and during transportation, field workers subjected to pesti means of conventional mixing, dissolving, granulating, dra cidefinsecticide OP poisoning, truckers who transport pesti gee-making, levigating, emulsifying, encapsulating, entrap cides, pesticide manufacturers, dog groomers who are over ping or lyophilizing processes. exposed to flea dip, pest control workers and various domestic 30 Pharmaceutical compositions for use in accordance with and custodial workers who use these compounds, military the present invention thus may be formulated in conventional personnel exposed to nerve gases. manner using one or more physiologically acceptable carriers As mentioned, in Some embodiments of the invention the comprising excipients and auxiliaries, which facilitate pro method is effected by providing the subject with a therapeu cessing of the active ingredients into preparations which, can tically effective amount of the PON1 polypeptide of the 35 be used pharmaceutically. Proper formulation is dependent invention. upon the route of administration chosen. As OP can be rapidly absorbed from lungs, skin, gastro For injection, the active ingredients of the pharmaceutical intestinal (GI) tract and mucous membranes, PON1 may be composition may be formulated in aqueous solutions, prefer provided by various administration routes or direct applica ably in physiologically compatible buffers such as Hank’s tion on the skin. 40 Solution, Ringer's Solution, or physiological salt buffer. For For example, PON1 may be immobilized on a solid support transmucosal administration, penetrants appropriate to the e.g., a porous Support which may be a flexible sponge-like barrier to be permeated are used in the formulation. Such substance or like material, wherein the PON1 is secured by penetrants are generally known in the art. immobilization. The support may be formed into various For oral administration, the pharmaceutical composition shapes, sizes and densities, depending on need and the shape 45 can be formulated readily by combining the active com of the mold. For example, the porous support may be formed pounds with pharmaceutically acceptable carriers well into a typical household sponge, wipe or a towelette. known in the art. Such carriers enable the pharmaceutical For example, such articles may be used to clean and decon composition to be formulated as tablets, pills, dragees, cap taminate wounds, while the immobilized PON1 will not leach Sules, liquids, gels, syrups, slurries, Suspensions, and the like, into a wound. Therefore, the Sponges can be used to decon 50 for oral ingestion by a patient. Pharmacological preparations taminate civilians contaminated by a terrorist attack at a pub for oral use can be made using a solid excipient, optionally lic event. grinding the resulting mixture, and processing the mixture of Alternatively or additionally, PON1 may be administered granules, after adding Suitable auxiliaries if desired, to obtain to the Subject perse or in a pharmaceutical composition where tablets or dragee cores. Suitable excipients are, in particular, it is mixed with suitable carriers or excipients. 55 fillers such as Sugars, including lactose, Sucrose, mannitol, or As used herein a “pharmaceutical composition” refers to a Sorbitol; cellulose preparations such as, for example, maize preparation of one or more of the active ingredients described starch, wheat starch, rice starch, potato starch, gelatin, gum herein with other chemical components such as physiologi tragacanth, methyl cellulose, hydroxypropylmethyl-cellu cally Suitable carriers and excipients. The purpose of a phar lose, sodium carbomethylcellulose; and/or physiologically maceutical composition is to facilitate administration of a 60 acceptable polymers such as polyvinylpyrrolidone (PVP). If compound to an organism. desired, disintegrating agents may be added, such as cross Herein the term “active ingredient” refers to the PON1 linked polyvinyl pyrrolidone, agar, or alginic acid or a salt accountable for the biological effect. thereof Such as Sodium alginate. Hereinafter, the phrases “physiologically acceptable car Dragee cores are provided with suitable coatings. For this rier and “pharmaceutically acceptable carrier which may 65 purpose, concentrated Sugar Solutions may be used which be interchangeably used refer to a carrier or a diluent that does may optionally contain gum arabic, talc, polyvinyl pyrroli not cause significant irritation to an organism and does not done, carbopol gel, polyethylene glycol, titanium dioxide, US 8,735,124 B2 21 22 lacquer Solutions and Suitable organic solvents or solvent Determination ofatherapeutically effective amount is well mixtures. Dyestuffs or pigments may be added to the tablets within the capability of those skilled in the art, especially in or dragee coatings for identification or to characterize differ light of the detailed disclosure provided herein. ent combinations of active compound doses. For any preparation used in the methods of the invention, Pharmaceutical compositions which can be used orally, the therapeutically effective amount or dose can be estimated include push-fit capsules made of gelatin as well as Soft, initially from in vitro and cell culture assays. For example, a sealed capsules made of gelatin and a plasticizer, Such as dose can be formulated in animal models to achieve a desired glycerol or Sorbitol. The push-fit capsules may contain the concentration or titer (see the Examples section which fol active ingredients in admixture with filler such as lactose, lows). Such information can be used to more accurately deter binders such as starches, lubricants such as talc or magnesium 10 mine useful doses in humans. Stearate and, optionally, stabilizers. In soft capsules, the Toxicity and therapeutic efficacy of the active ingredients active ingredients may be dissolved or Suspended in Suitable described herein can be determined by standard pharmaceu liquids, such as fatty oils, liquid paraffin, or liquid polyethyl tical procedures in vitro, in cell cultures or experimental ene glycols. In addition, stabilizers may be added. All formu animals. The data obtained from these invitro and cell culture lations for oral administration should be in dosages Suitable 15 assays and animal studies can be used in formulating a range for the chosen route of administration. of dosage for use in human. The dosage may vary depending For buccal administration, the compositions may take the upon the dosage form employed and the route of administra form of tablets or lozenges formulated in conventional man tion utilized. The exact formulation, route of administration . and dosage can be chosen by the individual physician in view For administration by nasal inhalation, the active ingredi of the patient’s condition. (See e.g., Fingl, et al., 1975, in “The ents for use according to the present invention are conve Pharmacological Basis of Therapeutics' Ch. 1 p. 1). niently delivered in the form of an aerosol spray presentation Dosage amount and interval may be adjusted individually from a pressurized pack or a nebulizer with the use of a to provide plasma or brain levels of the active ingredient are Suitable propellant, e.g., dichlorodifluoromethane, trichlorof Sufficient to induce or Suppress the biological effect (minimal luoromethane, dichloro-tetrafluoroethane or carbon dioxide. 25 effective concentration, MEC). The MEC will vary for each In the case of a pressurized aerosol, the dosage unit may be preparation, but can be estimated from in vitro data. Dosages determined by providing a valve to deliver a metered amount. necessary to achieve the MEC will depend on individual Capsules and cartridges of, e.g., gelatin for use in a dispenser characteristics and route of administration. Detection assays may beformulated containing a powder mix of the compound can be used to determine plasma concentrations. and a suitable powder base Such as lactose or starch. 30 Depending on the severity and responsiveness of the con The pharmaceutical composition described herein may be dition to be treated, dosing can be of a single or a plurality of formulated for parenteral administration, e.g., by bolus injec administrations, with course of treatment lasting from several tion or continuos infusion. Formulations for injection may be days to several weeks or until cure is effected or diminution of presented in unit dosage form, e.g., in ampoules or in multi the disease state is achieved. dose containers with optionally, an added preservative. The 35 The amount of a composition to be administered will, of compositions may be suspensions, Solutions or emulsions in course, be dependent on the Subject being treated, the severity oily or aqueous vehicles, and may contain formulatory agents of the affliction, the manner of administration, the judgment Such as Suspending, stabilizing and/or dispersing agents. of the prescribing physician, etc. Pharmaceutical compositions for parenteral administra PON1 may be administered prior to the OP exposure (pro tion include aqueous Solutions of the active preparation in 40 phylactically, e.g., 10 or 8 hours before exposure), and alter water-soluble form. Additionally, suspensions of the active natively or additionally administered post exposure, even ingredients may be prepared as appropriate oily or water days after (e.g., 7 days) in a single or multiple-doses. based injection Suspensions. Suitable lipophilic Solvents or Embodiments of the invention also contemplate the use of vehicles include fatty oils such as Sesame oil, or synthetic other agents in combination with PON-1 for the treatment or fatty acids esters such as ethyl oleate, triglycerides or lipo 45 prevention of OP damage. The following regimen is intended Somes. Aqueous injection Suspensions may contain Sub to encompass treatment with PON1 alone or in combination stances, which increase the Viscosity of the Suspension, Such with other agents. as sodium carboxymethyl cellulose, sorbitol or dextran. Thus, according to an exemplary embodiment, PON1 may Optionally, the Suspension may also contain Suitable stabiliz be administered by inhalation to protect the lungs and injec ers or agents which increase the Solubility of the active ingre 50 tion (i.v.) to protect the circulation up to 2 hours post expo dients to allow for the preparation of highly concentrated Sure. Atropine may be added 2-4 hours post exposure. Daily Solutions. injections of PON1 may be administered up to 7 days post Alternatively, the active ingredient may be in powder form poisoning. Oximes like H1-6 and mono-bisquaternary oximes for constitution with a Suitable vehicle, e.g., Sterile, pyrogen such as pralidoxime chloride (2-PAM) may be added to free water based solution, before use. 55 improve treatment efficacy. The pharmaceutical composition of the present invention Compositions of the present invention may, if desired, be may also be formulated in rectal compositions such as Sup presented in a pack or dispenser device, such as an FDA positories or retention enemas, using, e.g., conventional Sup approved kit, which may contain one or more unit dosage pository bases such as cocoa butter or other glycerides. forms containing the active ingredient. The pack may, for Pharmaceutical compositions suitable for use in context of 60 example, comprise metal or plastic foil. Such as ablisterpack. the present invention include compositions wherein the active The pack or dispenser device may be accompanied by instruc ingredients are contained in an amount effective to achieve tions for administration. The pack or dispenser may also be the intended purpose. More specifically, a therapeutically accommodated by a notice associated with the container in a effective amount means an amount of active ingredients form prescribed by a governmental agency regulating the (nucleic acid construct) effective to prevent, alleviate orame 65 manufacture, use or sale of pharmaceuticals, which notice is liorate symptoms of a disorder (e.g., ischemia) or prolong the reflective of approval by the agency of the form of the com survival of the subject being treated. positions or human or veterinary administration. Such notice, US 8,735,124 B2 23 24 for example, may be of labeling approved by the U.S. Food ingly, the description of a range should be considered to have and Drug Administration for prescription drugs or of an specifically disclosed all the possible Subranges as well as approved product insert. Compositions comprising a prepa individual numerical values within that range. For example, ration of the invention formulated in a compatible pharma description of a range such as from 1 to 6 should be consid ceutical carrier may also be prepared, placed in an appropriate ered to have specifically disclosed Subranges Such as from 1 container, and labeled for treatment of an indicated condition, to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 as if further detailed above. to 6 etc., as well as individual numbers within that range, for The ability of PON1 to sequester OP molecules, suggests example, 1, 2, 3, 4, 5, and 6. This applies regardless of the use of same in the decontamination of OP contaminated Sur breadth of the range. faces and detoxification of airborne OP. 10 Whenever a numerical range is indicated herein, it is meant Thus, an aspect of the invention further provides for a to include any cited numeral (fractional or integral) within the method of detoxifying a surface contaminated with an OP indicated range. The phrases “ranging/ranges between a first molecule; or preventing contamination of the Surface with indicate number and a second indicate number and “ranging/ OP. The method is effected by contacting the surface with ranges from a first indicate number “to a second indicate PON1. 15 number are used herein interchangeably and are meant to Thus, synthetic and biological Surfaces contemplated include the first and second indicated numbers and all the according to embodiments of the invention include, but are fractional and integral numerals therebetween. not limited to, equipment, laboratory hardware, devices, fab As used herein the term “method’ refers to manners, rics (clothes), skin (as described above) and delicate mem means, techniques and procedures for accomplishing a given branes (e.g., biological). The mode of application will very task including, but not limited to, those manners, means, much depend on the target Surface. Thus, for example, the techniques and procedures either known to, or readily devel surface may be coated with foam especially when the surface oped from known manners, means, techniques and proce comprises cracks, crevices, porous or uneven Surfaces. Appli dures by practitioners of the chemical, pharmacological, bio cation of Small quantities may be done with a spray-bottle logical, biochemical and medical arts. equipped with an appropriate nozzle. If a large area is con 25 It is appreciated that certain features of the invention, taminated, an apparatus that dispenses a large quantity of which are, for clarity, described in the context of separate foam may be utilized. embodiments, may also be provided in combination in a Coatings, linings, paints, adhesives sealants, waxes, single embodiment. Conversely, various features of the sponges, wipes, fabrics which may comprise the PON1 may invention, which are, for brevity, described in the context of a be applied to the Surface (e.g., in case of a skin Surface for 30 single embodiment, may also be provided separately or in any topical administration). Exemplary embodiments for Such are suitable subcombination or as suitable in any other described provided in U.S. Pat. Application No. 20040109853. embodiment of the invention. Certain features described in Surface decontamination may be further assisted by con the context of various embodiments are not to be considered tacting the Surface with a caustic agent; a decontaminating essential features of those embodiments, unless the embodi foam, a combination of baking condition heat and carbon 35 ment is inoperative without those elements. dioxide, or a combination thereof. Sensitive surfaces and Various embodiments and aspects of the present invention equipments may require non corrosive decontaminants such as delineated hereinabove and as claimed in the claims sec as neutral aqueous solutions with active ingredient (e.g., tion below find experimental support in the following paraOXonases). examples. In addition to the above described coating compositions, 40 OP contamination may be prevented or detoxified using an EXAMPLES article of manufacture which comprise the PON1 immobi lized to a solid Support in the form of a sponge (as described Reference is now made to the following examples, which above), a wipe, a fabric and a filter (for the decontamination together with the above descriptions illustrate some embodi of airborne particles). Chemistries for immobilization are 45 ments of the invention in a non limiting fashion. provided in U.S. Pat. Application 2004.0005681, which is Generally, the nomenclature used herein and the laboratory hereby incorporated in its entirety. procedures utilized in the present invention include molecu The terms “comprises”, “comprising”, “includes”, lar, biochemical, microbiological and recombinant DNA “including”, “having and their conjugates mean “including techniques. Such techniques are thoroughly explained in the but not limited to’. 50 literature. See, for example, “Molecular Cloning: A labora The term “consisting of means “including and limited to. tory Manual” Sambrook et al., (1989): “Current Protocols in The term “consisting essentially of means that the com Molecular Biology Volumes I-III Ausubel, R. M., ed. position, method or structure may include additional ingre (1994); Ausubel et al., “Current Protocols in Molecular Biol dients, steps and/or parts, but only if the additional ingredi ogy”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, ents, steps and/or parts do not materially alter the basic and 55 “A Practical Guide to Molecular Cloning”, John Wiley & novel characteristics of the claimed composition, method or Sons, New York (1988); Watson et al., “Recombinant DNA, Structure. Scientific American Books, New York; Birren et al. (eds) As used herein, the singular form “a”, “an and “the "Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, include plural references unless the context clearly dictates Cold Spring Harbor Laboratory Press, New York (1998); otherwise. For example, the term “a compound' or “at least 60 methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683, one compound may include a plurality of compounds, 202; 4,801,531; 5,192,659 and 5.272,057; “Cell Biology: A including mixtures thereof. Laboratory Handbook'', Volumes I-III Cellis, J. E., ed. Throughout this application, various embodiments of this (1994): “Current Protocols in Immunology” Volumes I-III invention may be presented in a range format. It should be Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clini understood that the description in range format is merely for 65 cal Immunology” (8th Edition), Appleton & Lange, Norwalk, convenience and brevity and should not be construed as an Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in inflexible limitation on the scope of the invention. Accord Cellular Immunology”. W.H. Freeman and Co., New York US 8,735,124 B2 25 26 (1980); available immunoassays are extensively described in used as a template in a standard 50 lul PCR reaction. The the patent and scientific literature, see, for example, U.S. Pat. purified PCR product was digested with Ncol and Not1, and Nos. 3,791,932; 3,839,1533,850,752; 3,850,578; 3,853.987; cloned into the pET32 vector with a C-terminal 6-His tag 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; (FIG. 1). 3,996.345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and Constructing PON1 Gene Libraries by Using Designed 5,281.521; “Oligonucleotide Synthesis' Gait, M. J., ed. Oligonucleotides at Targeted Positions. (1984): “Nucleic Acid Hybridization’ Hames, B. D., and The PON1 gene having H115W mutation was used as a Higgins S. J., eds. (1985): “Transcription and Translation' template to construct a library using synthetic oligos by ISOR Hames, B. D., and Higgins S. J. Eds. (1984); "Animal Cell protocol. Briefly, H115W mutant gene was digested with Culture' Freshney, R.I., ed. (1986): “Immobilized Cells and 10 DNasel. Approximately 5 lug of purified DNA in 50 ul reac Enzymes' IRL Press, (1986): “A Practical Guide to Molecu lar Cloning Perbal, B., (1984) and “Methods in Enzymol tions was digested with 0.01 UDNase (Takara) at 37°C. for ogy” Vol. 1-317, Academic Press: “PCR Protocols: A Guide 2, 4, and 6 min. The reactions were terminated with 15ul of To Methods And Applications”. Academic Press, San Diego, 0.5 M EDTA, and heating at 90° C. for 10 min, and were run Calif. (1990); Marshak et al., “Strategies for Protein Purifi 15 on a 2% agarose gel. Fragments of 50-150 bps size were cation and Characterization—A Laboratory Course Manual excised and purified using a gel extraction kit (Qiagen). The CSHL Press (1996); all of which are incorporated by refer PON1 gene was reassembled using 100 ng of purified DNA ence as if fully set forth herein. Other general references are fragments with oligonucleotides encoded one mutation and provided throughout this document. The procedures therein 20 flanking nucleotides matching the PON1 gene (Table 9, are believed to be well known in the art and are provided for below). Assembly PCR was performed in a 50 ul reaction the convenience of the reader. All the information contained mixture that contained 2.5 U Pfu Ultra (Stratagene). The therein is incorporated herein by reference. cycling included: one denaturation step at 96° C. for 3 min, then 35 cycles composed of: (i) a denaturation step at 94° C. Example 1 (30s); (ii) nine successive hybridization steps separated by 3° 25 C. each, from 65° C. to 41° C., for 1.5 min each (total 13.5 Materials and Methods min), and (iii) an elongation step of 1.5 min at 72°C. Finally, a 10 minelongation step at 72°C. was performed. The assem Constructing PON1 Gene Libraries by Random Mutagen bly product was amplified by a nested PCR reaction with CS1S. primers pET-Nes 1-Bc and plT-Nes0-Fo. In this step, 1 ul of Recombinant PON1 variant G3C9 (Gene bank entry: 30 the assembly reaction was used as a template in a standard 50 AY499193) was used as a template to create H1 15W and ul PCR reaction. The purified PCR product was digested with V346A amino acid mutations by primer designing, pFT NcoI and Notl, and cloned into the pET32 vector with a Nes2-Bc and pPT-Nes 1-Fo primer (Table 9, below) was used C-terminal 6-His tag (FIG. 1). to amplify 10ng of template with a mutator Taq polymerase Double Emulsion and Sorting by FACS. (mutazyme, Genemorph) in 25ul of reaction mixture for 15 35 Substitution libraries were sorted by compartmentalization cycles. On average 1.7+0.65 amino acid mutations/gene with of single E. coli cells, each expressing an individual library ~60% transition and ~40% transversion were found. The variant in double emulsion droplets, and sorting these drop PCR product was treated with DpnI (to destroy the template lets by fluorescent activated cell sorter (FACS), essentially as plasmid), purified, and served as a template (10 ng) for described (references are provided hereinbelow under the another 15 cycles of nested PCR performed with Taq poly 40 “Reference Section”). BL21 (DE3) cells possessing GFPuv merase. The PCR products were digested with NcoI and NotI gene in the genome were used for expression of the PON 1 and cloned to pET32 vector with a C-terminal 6-His tag (FIG. under the T7 promoter. Plasmid DNA was transformed and 1). grown while shaking at 250 RPM in 5 ml 2xYT media con Constructing PON1 Gene Libraries by Gene Shuffling. taining 100 ug/ml amplicillin and 1 mM CaCl, for 12 hrs at The improved PON1 variants were separately amplified 45 30° C., followed by another 24 hrs at 20° C. The cells were from their respective plasmids using Taq polymerase and centrifuged at 3000 g for 10 min at 4°C., resuspended in primers pET-Nes2-Bc and pET-Nes 1-Fo. To facilitate the 2xYT, and kept for 1 hr at room temperature. They were then removal of non-beneficial mutations, the PCR amplified rinsed twice in 0.1 M Tris-HCl, 1 mM CaCl, 0.1 MNaCl, pH wild-type PON1 gene was added at a 1:3 ratio to a mixture of 8.0, resuspended in the same buffer, and passed through a 5 PCR products from all the improved variants. Approximately 50 um filter (Sartorius). Filtered cells were compartmentalized 5ug of purified DNA mixture in 50 ul reactions was digested in the first emulsion (water-in-oil), and 100 mM solutions of with 0.01 UDNasel (Takara) at 37° C. for 2, 4, and 6 min. The CMP-MeCyC racemate or DEPCyC was added to the oil reactions were terminated with 15 ul of 0.5 M EDTA, and phase (0.8 ul, to a final concentration of 50 LM). The produc heating at 90° C. for 10 min, and were run on a 2% agarose tion of the second emulsion (water-in-oil-in-water) and sort gel. Fragments of 50-150 bps size were excised and purified 55 ing were performed as described. More than 10° events, at using a gel extraction kit (Qiagen). The PON 1 gene was 2000 events/sec. were stored using FACSAria (Becton-Dick reassembled using 100 ng of purified DNA fragments and inson) (FIG. 2). Events corresponding to single E. coli cells thermocycling in a 50l reaction mixture that contained 2.5 U were gated by GFP emission (at 530 nm, using blue laser for Pfu Ultra (Stratagene). The cycling included: one denatur excitation). Approximately 5000 events were sorted to ation step at 96° C. for 3 min, then 35 cycles composed of: (i) 60 96-well plates containing 200 ul of 2xYT media (1000 events a denaturation step at 94° C. (30s); (ii) nine successive per well). The plates were immediately moved to 37° C. hybridization steps separated by 3° C. each, from 65° C. to incubated for 1 hr while shaking at 250 rpm, plated on LB 41°C., for 1.5 min each (total 13.5 min), and (iii) an elonga agar plates containing 100 ug/ml amplicillin and 20 mM glu tion step of 1.5 min at 72°C. Finally, a 10 minelongation step cose, and grown overnight at 30° C. Recovery of the sorted at 72°C. was performed. The assembly product was amplified 65 cells was determined by comparing the number of colonies on by a nested PCR reaction with primers pET-Nes1-Bc and the LB plates to the number of events sorted by the FACS, and pET-Nes0-Fo. In this step, 1 ul of the assembly reaction was was found to be 20-40%. US 8,735,124 B2 27 28 Screens in 96-Well Plates. marin leaving group, using 100-fold dilution in 50 mM Tris Randomly picked colonies were individually grown with buffer pH 8 and measuring the absorption at 400 nm (the shaking in 96-deep-wells plates, using 0.5 ml 2xYT medium molar absorption of free coumarin was 3.7x10" M' cm). containing 100 g/ml amplicillin and 1 mM CaCl, for 8 hrs at The observed release of the coumarin was >95% of the cal 30°C., followed by another 16hrs at 20° C. Several repeats of 5 culated value. "P-NMR also indicated the complete and full wild-type PON1 were grown as controls. Following growth conversion of CMP-coumarin into CMP-F. This was apparent (ODocs4), the plates were centrifuged at 3000 g for 15 from the disappearance of the 'P signal attributed to CMP mins at 4°C., and pellets were kept at -70° C. for few hours. coumarin (6, 29.8 ppm) and the concomitant rise of a doublet centered at 6, 32.2 ppm with a J. value of 1045 Hz, which is The pellets were resuspended in 200 ul of lysis buffer (0.1M typical to O-alkyl methylphsphonofluoridates. To determine Tris-HCl pH 8.0, 1 mM CaCl2, 10 ug/ml lysozyme (Sigma), 10 the concentration of the GF stock solution produced in 0.2 M 0.2% TritonX-100, and 5 units/mlbenzonase (Novagen)), and NaF, a -10-fold dilution into a known concentration (~5 nM) lysed by shaking at 1300 rpm for 30 min at 37°C. The pellet of Torpedo Californica AChE (TcAChE) in 50 mM phosphate was removed by centrifugation at 4000 rpm for 20 min at 4 buffer pH 8.0, permitted to titrate the content of the toxic S C., and the Supernatant was transferred to a new set of plates isomer of GF, which was found to be 0.43-0.48 mM (ca. half and stored at 4°C. 15 of the racemic GF generated from 1 mM racemic CMP Apparent enzymatic rates (vo) for different substrates were Coumarin). The conversion of the IMP-, and PNP-Coumarin measured in a plate reader (Synergy-HT BioTek) using an analogs to the respective fluoridates, and the determination of appropriate volume of clarified lysates (0.1-10 ul depending the kinetic parameters, was similarly performed and moni on the substrate). 25uM of IMP-MeCyC and CMP-MeCyC tored. were used and release of coumarin was measured at 405 nm. The formation of GF, and the irreversibility of the conver In order to get S isomer, 10-20 nM of 3B3 purified enzyme sion process, were further validated by three experiments. were used to cleave R isomer from the 25 M of racemic Firstly, the bimolecular rate constant of the toxic isomer of the mixture. All rates were determined at the linear range of CMP-coumarin analog when inhibiting TcAChE was 3.3x product release, and background rates (lysates containing no 10 M'min'. In contrast, the in-situ generated GF exhibited PON1) were subtracted to give the observed initial rate (vo). 25 a bimolecular rate constant of 1.3x10 M'min'). The Enzymes Purification and Kinetics. 40-fold increase in the inhibition potency of TcAChE is con Variants exhibiting the highest rates with the target sub sistent with what would be expected from the inhibition rate strate were grown in 50 ml cultures; cells were harvested by constant of AChE by authentic GF. Secondly, the half-life of centrifugation, resuspended, and disrupted by Sonication. the in situ generated GF in activity buffer (50 mM Tris, 1 mM Ammonium sulfate was added to the lysate to 55% (wt/vol). 30 CaCl, 0.1%Tergitol, pH 8.0) is 85 to 130 min (based on loss The precipitate was dissolved and dialyzed against activity of anti-AChE activity; see below), while t of S-CMP buffer (Tris-HCl 50 mM pH=8, CaCl, 1 mM, NaCl 50 mM, coumarin under the same conditions is -530 min. (The in situ Tergitol 0.1%.) and purified on Ni-NTA (Novagen). Fractions generated GF solution were therefore freshly made before were analyzed for paraoxonase activity and purity (by SDS screening and kept in an ice bath until used). Thirdly, since the PAGE), pooled, dialyzed against activity buffer supple 35 released coumarin was present in the reaction mixture, we mented with 0.02% sodium azide, and stored at 4°C. Protein aimed to exclude the possibility that the conversion is revers purity was typically 70-80% by SDS-PAGE gel. Variants 4E9 ible, and that we were actually monitoring PON1-mediated and rePON1 (G3C9) were further purified by FPLC purifica hydrolysis of CMP-Coumarin and a subsequent shift in equi tion using a mono-Q column (HiPrep 16/10 QFF, GE health librium. Therefore the effect of an excess of free coumarin on care) eluted by activity buffer with 250 mM NaCl, concen 40 the hydrolysis of in-situ generated GF was tested (FIG. 4). trated (vivaspin 20 MWCO 20 KDa), loaded on a gel filtration The above described assay was repeated with 4 additional column (HiLoad 26/60 Superdex 75, GE healthcare) and equivalents of coumarin (one equivalent was generated by the dialyzed against activity buffer supplemented with 0.02% fluoride exchange). The results indicated a small reduction in Sodium azide for long term storage at 4°C. Protein purity was catalytic activity (1.15x107 to 0.96x107M min') of 4E9 on assessed to be >97% by SDS-PAGE gel. A range of enzyme 45 GF. Because reversibility is expected to result in higher rates concentrations (0.01-4 uM) and Substrate concentrations was in the presence of excess of coumarin, this result indicates that applied (from 0.3xKup to 2-3xKm). Product formation was the reverse conversion of GF into CMP-coumarin does not monitored spectrophotometerically in 96-well plates with occur under the assay conditions. 200-ul reaction volumes. For each purified variants, at least Determination of k/K Values with In-Situ Generated three independent repeats were done for kinetic parameters 50 GF. and values were determined by fitting the data directly to the The GF Stock was diluted 1000-fold in cold distilled water Michaelis-Menten using KaleidaGraph (FIG. 3). The cata and then further diluted 20-fold into the 0.005 to 0.2 MPON lytic activity of evolved variants with R-CMP-coumarin was variant in activity buffer (Tris-HCl 50 mM pH=8, CaCl. 1 obtained by measuring kinetics of the 2" phase observed mM, NaCl 50 mM, Tergitol 0.1%.). The nominal racemic GF with racemic CMP-coumarin after consumption of the S 55 concentration was set to 40-50 nM. At various time intervals, CMP-coumarin by one of the Sevolved variants (e.g. variant the reaction mixture is diluted 10-fold into 2.5 nMTcAChE in OC9 at 30 nM). By measuring initial rates using several 50 mM phosphate buffer pH 8.0, 25°C. The phosphate buffer Substrate concentrations, we could estimate the apparent k/ that chelates calcium, and the dilution, quenched the PON1 K for this isomer. activity with GF. Residual TcAChE activity was measured Conversion of CMP-Coumarin to GF. 60 after 10 and after 20 min (to ascertain completion of inhibi Caution: Although the total amount of the in situ generated tion) by aliquating 10 ul into 1 ml Ellman assay solution cyclosarin (GF) in aqueous Solution is non-hazardous, the containing 1 mMacetylthiocholine as substrate. The %-inhi reader should be aware of its high potency as inhibitor of bition of TcAChE by the same GF solution without PON was AChE. CMP-coumarin (0.5 ml of 1 mM) was incubated at considered as 100% anti-AChEpotency attributed to the toxic room temperature in 0.2 MNaF. pH 5.0. The quantitative and 65 isomer of GF. This%-inhibition decreased over time of incu completion of the conversion of CMP-coumarinto GF (about bation with PON1, and k was calculated by fitting the 30 min) was verified by monitoring the release of the cou %-loss of anti-TcAChE potency versus time to a mono-expo US 8,735,124 B2 29 30 nential equation. The concentration of PON1 was set so that mild, moderate or severe reactions. Mild reactions were char degradation of D-50% of GF (i.e., gain of 50% AChE activity) acterized by Straub tail and ataxia. Moderate reactions con occurred within less than 10 mins (although this was impos sisted in addition decreased motor activity and tremors while sible with the poorly active variants such as wild-type-like animals with severe reactions exhibited in addition ventral rePON1-G3C9). position, fasciculation and dyspnea as well. The overall reac AChE Protection Assays. The assays were performed by pre-incubation of the PON1 tions observed following Sp-CMP-coumarin intoxication variant and the OP (as exemplified in the below protocol for were scored using semi-quantitative grading of five grades CMP-Coumarin), or by direct competition of the PON1 vari (0-4), taking into consideration the severity of the reactions ant and AChE, as exemplified in the second protocol with 10 (0=No Reactions, 1 =Mild Reactions, 2=Moderate Reactions, CMP-F. Briefly, randomly picked colonies of library variants 3=Severe Reactions, 4=Mortality). were grown and lysed as above. Clarified cell lysates were diluted 1:4 in activity buffer, and 500 diluted lysate were mixed with 10ul of 6 uMCMP-coumarin. The reactions were incubated (15 mins), and an equal volume of AChE solution 15 Example 2 (0.25 nM AChE, in PBS, 0.1% BSA) was added. Following 15 min’s incubation, samples (20 ul) were mixed with Ell Directed Evolution of PON1 for S-CMP Hydrolysis man's reagent and the AChE substrate (180 ul, 0.85 mM DTNB, 0.55 mMacetylthiocholine, in PBS), and initial rates Several variants of rePON1 with an enhanced activity were measured at 412 nm. Residual AChE activity was deter towards a racemic mixture of CMP-Coumarin were previ mined by comparing initial rates those without OP. The ously isolated by screening neutral drift libraries of rePON1 screen with CMP-F was performed with the following modi (e.g. 1G3, 2G9). The most active variant was found to be 3B3 fications: undiluted cell lysates were mixed with an equal with ~250-fold higher catalytic efficiency (k/K 20x10 volume of AChE solution (0.5 nM), and freshly made CMP-F M'min') compared to the wild-type-like rePON1 (k/K was added to 1 LM final concentration. Reactions were incu 25 0.08x10 M'min': Table 1b: Table 2 below). Although the bated for 15 mins, and residual AChE activity was determined hydrolysis by rePON1-3B3 was also restricted to the R iso as above. The catalytic specificity (k/K) of purified vari mer (FIG. 6a), the high catalytic efficiency and R-stereose ants was measured by mixing the in situ prepared OP-fluori lectivity could be used to isolate the S isomers of CMP dates (40 nM) with purified PON1 variants (0.1-0.01 uM) in coumarin and IMP-coumarin from the corresponding activity buffer. Samples of this reaction mix were taken at 30 racemates, and apply them for the Subsequent screens. Low different times, diluted (1:10) with the AChE solution (4 nM activity of rePON1 mutants H115W and V346A rePON1 AChE, 0.1% BSA, 1 mM EDTA, in PBS), incubated for 15 towards S-CMP-coumarin was also identified. However, mins, and residual AChE activity was determined as above. their activity with S-CMP-coumarin was too low for detec The apparent k/K values were derived from the slope of tion under library screening conditions. Therefore the first the resulting single exponential curve. 35 rounds IMP-coumarin, a less bulky G-agent analogue whose Prophylactic Activity of 4E9 in a Mouse Model. S isomer is more reactive with PON1 were used (Table 2, Eight weeks old male mice of strain C57BL/6J strain, were below). Supplied under germ-free conditions by the Animal Breeding Random mutagenesis of rePON1-H115W-V346A and Center of The Weizmann Institute of Science (Rehovot, screening of the resulting library in 96-well plates with S Israel). The mice were housed in a light- and temperature 40 IMP-Coumarin yielded several improved variants that typi controlled room. All animals were handled according to the cally carried one mutation in addition to H1 15W and V346A regulations formulated by the Institutional Animal Care and (Table 3, below). A second round of mutagenesis and screen ing with S-IMP-Coumarin led to the isolation of variants in Use Committee (application number 04590909-2). Prior to which V346A was removed and the H115W and F222S muta treatments, blood samples were taken (50-75 ul, retero-or 45 tions dominated (Table 3, below). As the evolving variants bital) into heparin (10 ul. 1:10). Mice were then weighted became more reactive with the (average weight 24.5 (gr)+2.2) and PON1 variant 4E9 or Seisomer, the3" generation library could be screened with rePON1 (210-260 ug/ml, >97% pure in isotonic activity both S-IMP- and S-CMP-Coumarin. Indeed, this round buffer: Tris 50 mM pH=8, CaCl. 1 mM, NaCl 100 mM, resulted in several variants with improved activities towards tergitol 0.02%) were injected i.v to the tail vein at different 50 S-CMP-Coumarin (e.g. 3A7, 8C8: Table 1b: Table 4, doses (1.1, 2.1 or 2.2 mg/kg). After 55' or 5 hi55' blood below). However, since the 4" round of mutagenesis and samples were obtained as described and mice were screening yielded no further improvements, a structure-based reweighed. After 1 or 6 hours, intoxication was induced by a targeted library was designed and Subjected it to high single i.v. administration of Sp-CMP-coumarin (26.5 ug/ 55 throughput screening (>10 variants per run) by FACS sort ml. PBS) at a dose of 290 g/Kg. All animals were observed ing, as described below. closely for clinical signs following CMP-coumarin intoxica tion during the first 24 hours and were kept for at least 14 days before sacrifice. Control mice were injected i.v. to the tail vein Example 3 with either: isotonic activity buffer (200 ul) or Atropine sul 60 fate 20 mg/kg and 2-PAM 25 mg/kg in PBS just 5 min Highly Proficient Variants by FACS Screening of prior to intoxication as indicated. The toxicity of PON1 vari Double Emulsion Droplets ant 4E9 or the isotonic activity buffer were assayed by inject ing them to mice without an OP challenge, as described, and 65 The targeted substitutions library was based on PON1s monitoring for at least 14 days. All clinical signs noted fol active-site structure. In particular, a recently obtained crystal lowing Sp-CMP-coumarin intoxication were categorized to Structure of the re-G3C9-H115W indicated movements of US 8,735,124 B2 31 32 several side-chains in response to this mutation, including k. However, to be able to Screen at concentrations that correspond to the very low toxic concentrations of cyclosarin those of residues 69, 134 which are in direct contact with in vivo (~1 uM)', and for variants that efficiently degrade W115, and of the more remote residues 346, 347 and 348. cyclosarin itself a screen was developed based on monitoring therefore a library was generated by randomizing these posi the rescue of AChE, the OP's physiological target. AChE was tions and those of residues 115 and 222 that were found to be added to crude bacterial lysates expressing the library PON1 mutated in all active variants of the 3" round (Table 4, below). variants. The OP was added, and the residual activity of AChE An oligo spiking strategy was used that incorporated the was Subsequently measured using a chromogenic assay to indicate the level of OP degradation by the tested variant. The randomizing oligos onto re-PON1-H1 15W in a combinato FACS sorted 6" round library was re-screened in 96-well rial manner so that each library variant carried on average 4 10 plates using the AChE assay and 1 uM of racemic CMP mutated positions. Due to the intense level of mutagenesis, coumarin. 730 randomly-picked colonies were screened and and the targeting of the active-site, the libraries comprised 13 variants were isolated with improved activity by 2-12 fold mostly inactive variants. To purge inactive library clones, we relative to 3D8 (Table 7, below). Although at this stage variants that exhibited sufficiently employed a high-throughput FACS screen using a fluoro 15 high catalytic proficiency were identified, these were selected genic phosphotriester dubbed DEPCyC that was found to and tested with coumarin Surrogates. In fact, the fluoride correlate well with the activity with S-CMP-Coumarin'. E. leaving group of the actual threat agents Substantially differs coli cells transformed with the plasmid library were compart from coumarin (FIG. 5) fluoride is more reactive and is a mentalized in water-in-oil emulsion droplets. The fluorogenic much smaller leaving group. However, the toxicity of nerve agents prevents their use in ordinary labs. Therefore a non Substrate was added, and the primary emulsion droplets were hazardous screening protocol was developed based on converted to double-emulsion droplets that were sorted by CMP-F generated in situ, in dilute aqueous solutions, by FACS. Cells in isolated droplets were plated, picked and replacing the coumarin leaving group of CMP-coumarin with assayed in 96-well plates for Sp-CMP-coumarin activity. fluoride. This exchange was spectroscopically monitored by The most active variants isolated from this 5" round (Table 25 following the release of the coumarin. The inhibition of 5, below) carried mutations L69G/A and H134R, in addition TcAChE by in situ generated CMP-F proceeded 40-fold to H1 15W and F222S that appeared in the previous rounds. faster than with S-CMP-Coumarin, with the expected k, of These variants were shuffled, together with random mutagen 1.3x10 M'min'. Using the in situ generated CMP-F, we esis at low rates (~1.7 amino acid exchanges per gene), and measured the ki/K values of the evolved variants under the resulting 6" round library was sorted by FACS, and then 30 pseudo-first-order conditions (CMP-Fs.50 nM, well under a screened in 96-well plates. The most improved variants car likely K). Encouragingly, the activities with the coumarin and fluoridate S isomers were comparable, and at least three ried five key mutations: L69G, H 115W, H134R, F222S and variants (0C9, 2D8, 1A4) exhibited ki/K values of >107 T332S (Table 6, below). The best variant, 3D8 exhibited a M'min' with CMP-F (Table 8, below). The AChE protec k/K value of 1.2x107 M'min' with S-CMP-coumarin tion assays therefore confirmed the ability of the evolved (Table 1 below: FIG. 6a). Further, 3D8 and other variants 35 variants to protect AChE from cyclosarin in vitro, and vali from the 6" round exhibited similar rates with both the S. and dated the coumarin analogues as faithful Surrogates of the R isomers as indicated by the complete hydrolysis of the actual G-agents. The assay also confirmed that the starting racemic CMP-coumarin with monophasic kinetics (FIG. 6a). point, rePON1-G3C9 is much more active with CMP-F than FIG. 6b shows how the toxic isomer (S) of a Cyclosarin CMP-Cuomarin (Table 1b), as is human PON1, although the coumarin analogue called CMP is hydrolyzed by different 40 k/K (~10 M'min') is >100-fold too low for in vivo variants and the wild-type like rePON1 (G3C9) with time. We detoxification using reasonable amounts of enzyme. added the purified variants and the substrate at the indicated The evolved variants were sufficiently active to enable amounts and followed the hydrolysis by reading the increase library Screens using the in situ generated agent. This in absorption of the coumarin leaving group with time. As can approach is highly attractive, since the assay of AChE pro be seen, both rePON1 and 3B3 can hardly hydrolyze the 45 tection against the actual threat agent mimics the in vivo substrate while 4E9 and 3D8 do so readily. FIG. 6c show how protection challenge whereby the catalytic scavenger must be these variants hydrolyze the in-situ generated agent (Cyclosa sufficiently active to intercept the threat agent before the latter rin) termed here CMP-Fasitis the fluoride derivative of CMP. reacts with AChE. Therefore, the 13 most improved variants Purified variants and the substrate were added at the indicated from the last round were re-screened using CMP-F at the amounts and followed the hydrolysis by sampling the reac 50 expected plasma concentration for 1xLDso exposure (1 uM). tionat different time points and adding the samples to purified Nine variants exhibited improved activities relative to 3D8 AChE. The residual AChE activity was used as a measure of (Table 7, below). Of these, 3 variants (4E9, 5F3 and 6A3) the amount of substrate hydrolyzed sine they are correlated. exhibited the highest specific activity upon examination of As can be seen, variant 3B3 is the worst while 4E9 is the best the amount of soluble expressed protein. hydrolyzer. By fitting the curve to a first-order equation we 55 Following sequencing and protein purification, 4E9 was can derive the apparent rate constant for the hydrolysis of GF identified as the most active variant with both S-CMP-cou by these variants. marin and S-CMP-F (k/K 2.23x10'M'min', and 1.7x107 M'min', respectively: Table 1b below).

60 Example 4 Example 5 Acetylcholinesterase Protection Assay Prophylactic Protection Assays Along the selection for variants with higher rates, the con 65 centration of the CMP-coumarin substrate was decreased to To validate that hydrolysis of the toxic isomer by a variant enable the isolation of variants with improved K as well as with ki/K values of >10 M'min' should protect against US 8,735,124 B2 33 34 lethal OP exposure at a low protein dose, rePON1-4E9 was TABLE 2 tested as a prophylactic in a mouse model. Due to safety issues, the CMP-Cuomarin surrogate was applied, but the Activity of PON1 mutants with both isomers challenge was upgraded by using the toxic Somer only (S- of CMP-coumarin and with Sp-IMP-coumarin CMP-coumarin) and by administrating it directly by i.v. injection. The results indicated a survival rate of 45% for mice R-CMP S-CMP S-IMP coumarinx pretreated with 1.1 mg/kg 4E9 one hour prior to the OP coumarin coumarin 10 Apparent Non exposure (Table 10 below). Increasing the 4E9 dose to 2.2 (k.ca/K) (k.ca/K.) (k.K.) Synonymous mg/kg increased the percent of Surviving animals to 75%, Variants M'min M'min M'min' mutations Supporting the predicted correlation between ki/K and in 10 vivo and protection level. Twenty-four hours after exposure, Wild- <200 n.d. O.O8 OOO34 - the survival rate was 75% for mice receiving 4E9 either one, type-like or six hours before the OP challenge. A similar survival ratio rePON1- (1) (1) G3C9 (63-75%) was observed 14 days later. As expected, the wild H115W 331 - 39 1983 - 30 O45 H115W 15 type-like rePON1-G3C9 (estimated ki/K for S-CMP (>1.6) (1) (6) coumarins2x10 M'min'), which served as a starting point V346A 88576 3633 - 126 O.2 V346A for the directed evolution of 4E9, conferred no protection. (>4.4) (1.8) (2.5) Notably, treatment of mice with atropine and 2-PAM, even 5 H11SW- 813 - 79 1014O7 0.4 H115W, minutes prior to challenge, gave very poor protection against V346A (>4.1) (5) (5) V346A the cyclosarin coumarin Surrogate, as is the case with cyclosa For each variant, enzymatic activities (ke K.) were measured with purified proteins and rin itself: the 24 h survival was only 22%, and there was no denoted are the average+standard deviation values obtained from the 3 independent repeats, survival 96 h post challenge. Further, whereas 4E9 protected The values withoutstandard deviations hads.d. a 20% of their values. Values in parentheses denoted the fold increase and decrease as compare to wt like rePON1 for either isomers of mice exhibited only mild intoxication symptoms 2-12 h after CMP and as compared to H115W for Sp-DMP. n.d. denotes non detectable activity, the challenge, all atropine plus 2-PAM treated mice displayed The catalytic efficiency was estimated as described in the “Reference Section” severe intoxication symptoms with no improvement until 25 Denoted in bold are mutations in active-site residues, death. TABLE 1b. Representative variants along the directed evolution process Re-CMP- SP Coumarin CMP-F Sp-CMP-Coumarin Apparent Apparent kca, KM kcal/KM kcal/KM k.ca/KM Variant Mutations (min) (IM) (IM'min') (IM'min') (M'min') rePON1 (Wild-type-like) ind ind <0.0002 O.O8 OOO34 0.13 - O.O3 G3C9 (1) a (1) (1) 3B3 N41D, S11OP, ind ind <0.0002 2O 1.7 --OOOO1 L240S, H243R, (1) a (250) F264L, N324D, T332A H115 W. H.115W, V346A ind ind O.OOO8 0.4 O.O2 OOO3 V346A (>4) (5) 3A7 V97A, H115W, ind ind O.OO27 O16 O.OO8 O.OO3S P135A, F222S, (>13.5) (2) M289 8C8 L69S, V97A, 11.60.18 12455.8 O.093 O.OO3 O.OO3S O.2 H115W, (>465) (23) (1.5) P135A, F222S 2D8 L69G, H 115W, 268 - 1.6 76.3 3.2 3.52 0.13 O.465 14.3 H134R, F222S, (>17600) (5.8) (110) T332S 3D8 L69G, H 115W, 295 1.62 25.4 O.S 11.6 0.23 ind 3.3 H134R, M196V (>58000) (25) F222S, T332S 4E9 L69G, S111T, 513 23 22.3 indf 16.8 H115W, (>111500) (129) H134R, F222S, T332S

Annotation of variants: The first digit relates to the plate number, and the following letter-digit to its location within this plate. For example, variant 3A7 = plate #3, well A7; n.d., not detectable. Denoted in bold are mutations in active-site residues. Enzymatic parameters were measured with purified proteins and comprise the average obtained from the 3 independent repeats. Error ranges represent the standard deviations observed between measurements. A more complete set of parameters including separatek andKf values when available, are provided in the Tables below, Values in parentheses describe the fold-change compare to the starting point, rePON-G3C9. The kinetic parameters for SP-CMP-coumarin were spectrophotometerically measured with pure substrate samples'. Parameters for RP-CMP coumarin were determined with the racemate, and for CMP-F with the in situ prepared substrate and an AChE inhibition assay (see Methods for details). The catalytic efficiency was estimated in the reference section Variants exhibited a single-phase kinetics of product release when reacted with racemic CMP-coumarin, suggesting that the rates of hydrolysis for RP-and SP-CMP-coumarin are similar, US 8,735,124 B2 35 36 TABLE 3 TABLE 3-continued Improved 1 and 2" round variants from libraries Improved 1 and 2" round variants from libraries derived from rePON1-FH115W-V346A. derived from rePON1-FH115W-V346A.

Fold Fold improvement improvement with S-IMP with S-IMP Variants Round coumarin Non-Synonymous mutations Variants Round coumarin Non-Synonymous mutations 1G3 1 2x H115W, P135A, V346A 3A7 2 22x V97A, H115W., P135A, 2A10 1 2.8x I109T, H115W, S139P, 10 V346A 3G6 1 4.8x F17S, H115W, V346A 3F11 1 3.7x H11SWV346A 4B8 1 5.6x H115W F347I The annotation or the variants: The first letter relates to the plate number, and the letter-digit SF2 1 3.3x H11SW F222L V346A to the location of the clone within this plate. For example, variant 1G3 = plate #1, well G3. Round of mutagenesis and screening 5F11 1 2.5x H115W, M289I, V346A 15 6G1 1 Sx H11SW F222L V346A Shown are all variants that exhibited higher S-IMP-coumarinactivity in crude cell lysates relative to the H115W +V346APON1 mutant. For each variant, enzymatic activities were 6E3 1 3.1x H115W, S139P, V167M, measured in crude lysate and denoted are the average values offold improvement obtained V346A from 3 independent repeats. The values hads.d. a 20% of their value. 6D10 1 4.3x H115W, V346Af Non-synonymous mutations observed in each variant, Mutations inactive site residues are noted by underlined. 7F6 1 74x H115W, F222S, D309N, These two variants had the same amino acid exchanges. The small differences in activity V346A may relate to differences in the composition and number of synonymous mutations,

TABLE 4 Improved 3 round variants from libraries derived from rePON1-H11SW-V346A. Fold improvement Fold improvement

Variants

The annotation of the variants: The first letter relates to the plate number, and the letter-digit to the location of the clone ywithin this plate. For example, variant 4D2 = plate #4, well D2, Shown are all variants that exhibited higher S-IMP-coumarin and S-CMP-coumarin activities in crude lysates relative to the H115W+V346APON1 mutant. For each variant, enzymatic activities were measured in crude lysate and denoted are the average values offold improvement obtained from 3 independent repeats. The values hads, d. a 20% of their value. Non-synonymous mutations observed in each variant, Mutations in active site residues are noted by underline.

45 TABLE 5 Improved variants from the targeted substitutions library 5 round).

50

55

60

The annotation of the variants: The first letter relates to the plate number, and the letter-digit to the location of the clone within this plate. For example, variant 2F8 = plate # 2, well F8. Shown are all variants that exhibited higher S-CMP-coumarin activities in crude lysates relative to the 8C8 mutant. For each variant, enzymatic activities were measured in crude lysate and denoted are the average values offold improvement obtained from 3 independent 65 repeats. The values had s.d. a 20% of their value. Non-synonymous mutations observed in each variant, Mutations in active site residues are underlined, US 8,735,124 B2 37 38 TABLE 6 Improved variants from shuffling of the targeted Substitutions library (6' round). CMPb CMPb IMP. EMP. EMP. CMP(Sp)" (R) (racemic) (S) (S) (S) kcar K. kcal/Kn kcal/Kn Non-synonymous Variant (min) M uM'min' uM'min' mutations 8C8 11.60.18 12455.8 O.093 O.OO3 O.OO3S O.O3 O.O22 O.08 0.2 L69S, V97A, H115W, (1) (1) (1) (1) (1) (1) P135A, F222S 2C3 1495 - 0.87 212.7 S.S O.7 OO1 O.O88 O.62 O.O79 O.88 0.16 L69G, H 115W, H134R, (7.5) (25) (21) (3.6) (11) (1) F222S, K233E 5H5 126 - 2 1023.8 1.25 O.OS O.2088 2.43 O.76 3.8 0.8 L10S, F28Y, L69G, (13.4) (60) (81) (35) (48) (4) H115W, H134R, F222S, T332S OC9 1852.5 653.6 2.85 O1 O.296 3.56 1.04 6.67 2.6 L14M, L69G, S111T, (31) (85) (119) (47) (83) (13) H115W, H134R, F222S, T332S 2D8 2681.6 76.3 3.2 3.52. O.13 O465 3.04 O.85 6.8 0.98 L69G, H 115W, H134R, (38) (133) (101) (39) (85) (5) F222S, T332S 1A4 1851.5 51 1.7 3.63 - 0.1 O.39 3.11 O.85 7.15 2.2 A6E, L69G, H 115W, (39) (111) (104) (39) (89) (11) H134R, F222S, K233E, T332S, T326S 3D8 295 1.62 25.4 OS 11.6 O.23 ind 4.73 4.6 12 6.2 L69G, H 115W, H134R, (125) (158) (209) (150) (31) M196V, F222S, T332S The annotation of variants: The first letter relates to the plate number, and the letter-digit to the location of the clone within this plate. For example, variant 3B3 = plate #3, well B3. For each variant, enzymatic activities (k.K.) were measured with purified proteins and denoted are the average + standard deviation values obtained from the 3 independent repeats. The individual values exhibiteds.d. a 20%. Values in parentheses denoted the fold increase and decrease as compare to 8C8, the best variant of the previous round. Tenoted in bold are mutations in active-site residues, Variant exhibited a single-phase kinetics of product release when reacted with racemic CMP-coumarin, suggesting that the rates of hydrolysis for Rp-and SP-CMP-coumarin are similar,

TABLE 7 30 TABLE 8-continued PON1 variants selected using the AChE inhibition assay. Activities of selected rePON1 variants on Sp-CMP-coumarin and its fluoridated product CMP-F (cyclosarin).” CMP-coumarin CMP-F hydrolysis hydrolysis (S)CMP- CMP- fluoridate? Variant activity activity mutant coumarin fluoridate coumarin l8le fold over 3D8) fold over 3D8) 35 The kK values for the fluoridates were determined by monitoring the rate of loss of 1 VH10 11.6x 4.3x anti-AChEpotency of the in situ-generated compound, assuming K->P-F. Calculations 2 VE2 11.0x 6x are based on a single enzyme concentration selected to fit the dynamic range for determi 3 V-F3 s.s 6 nation of the apparent k of loss of anti-AChEpotency, SX X The coumarin leaving group was replaced by fluoride in racemic CMP-coumarinto yield 4 VC11 8.3x 6x the racemic fluoridates of CMP (CMP-F). Note that the data for the hydrolysis of CMP-F can 5 V-D6 7.2x 3.4x 40 be attributed mostly to the toxic (Sp) isomer of CMP-F. 6 V-A6 6.2x 2.1X 7 VIII- 2.3x 3.3x A12 TABLE 9 8 VIII-D1 2.1X S.SX 9 VI-G8 2.0x 6x List of the oligonucleotides and primers 10 VI-H12 1.7x 2x 45 SE 11 IV-HS 1.6x 6x Q 12 IV-E9 1.6x 1.6x Orienta- ID 13 VI-A3 1.6x 4.5x Designation tion Primer sequence NO : pET-Nes2-Bc Forward 5' - GATGGCGCCCAACAGTCC-3' O9 The variants were tested using AChE (0.5 nM) with racemic CMP-coumarin (1 uM), and ranked relative to 3D8, isolated in Round 6 (Table 6). The variants were ranked relative to 3D8 using AChE (0.5 nM) and in-situ generated 50 pET-Nes1-Fo Backward 5'-GCGCGTCCCATTCGC-3' O racemic CMP-F (1 uM) pET-Neso-Fo Backward 5'-TGATCTAGTGCGGCCGCCAGCTCA 1. CAGTAAAGAGCTTTGTGAAACAC-3' TABLE 8 pET-Nes1-Bc Forward 5' - GTCCGGCGTAGAGGATCG-3' 2 Activities of selected rePON1 variants on Sp-CMP-coumarin 55 and its fluoridated product CMP-F (cyclosarin).” L69NNS 5 - GGCTTTCATCAGCTCCGGANNSAA 3 GTATCCTGGAATAATGAGC-3' (S)CMP- CMP- fluoridate? mutant coumarin fluoridate coumarin H115NNS Forward 5 - CTTCATTTAACCCTNNSGGGATTAG 114 CACATTC-3' 8C8 O.09 O.2 2.2 60 3D8 11.6 3.3 O.3 H134NNS Forward 5 - CTACTGGTGGTAAACNNSCCAGAC 5 OC9 2.8 11.1 3.9 TCC TCGTCC-3 2D8 3.5 14.3 4.1 1A4 3.6 11.3 3.1 F222NNS Forward 5 - GTTGATTCCGTTAGCSNNATCAAAT 6 2C3 0.7 O.47 0.7 CCTTCTGC-3 The figures shown are values ofk, Kix 10° (M'min') 65 v346NNS Backward 5 - GAGCTTTGTGAAASNNTGTGCCAA 7 Data for OP-coumarin are based on release of the chromophore monitored at 400 mm. TCAGCAG-3 US 8,735,124 B2 39 TABLE 9- continued TABLE 12

List of the oligonucleotides and primers Key residues in the 2" round variants listed in Table 4 Amino wt- II- MG2- IV. VII- VI SEQ Acid PON1 A1 IA4 D11 D11 D2 VB3 Orienta- ID Designation tion Primer sequence NO : 64 F F F F F F L 69 L V V V V V V 115 H L A. A. A. V V 134 H R R R R R R F247NNS Backward 5 - GTAAAGAGCTTTGTGSNNCACTGT 118 10 196 M M M L M M M GCCAATCAG-3 222 F M M M M V M 309 D D D D G D D H348NNS Backwards" - CAGTAAAGAGCTTTSNNAAACACT 119 332 T S S S S S S GTGCCAATC-3 kK for Round 3 best mutants, Gagents were at 0.5 L.Mand the enzymes at concentrations well below the OP, thus fulfilling the catalytic conditions for the hydrolysis of the nerve 15 agents, Data shown (LM'min). meant SD, n = 3.

TABLE 10 TABLE 13 Prophylactic protection in mice GD Time prior to Enzyme OP challenge dose % Survival recorded at: Variant GB Fast Slow GF 2-II-D12 180 O.28 2.69 O.14 2.69 O.14 9.6 1.5 hours Treatment group mg/kg) 12 h 24h 96 h 14 day 1-I-D10 3.11 - O.63 8.73 O.76 8.73 O.76 21.1.3.3 Untreated (12) O% 0% O% O% I-IV-H9 3.13 - 0.16 42.15.O 42.1 S.O. 23.8 - 1.5 1 Buffer (3) O% 0% O% O% 25 1-I-F11 3.89 O.38 44.3 6.2 44.3 6.2 45.87.2 5 min Atropine plus 2- 66%. 22%. 22% O% Average of G3 3.0 24.5 24.5 25.1 PAM (9) Average of G2 O.9 18.2 11.1 9.1 1 rePON1 (18) 2.2 O% 0% O% O% Average of Gl O.17 6.7 1.3 4.7 1 4E9 (11) 1.1 45%. 45%. 45%. 45% 1. A significant systematic improve across all G agents, when compared to libraries G1 and 1 4E9 (16) 2.2 75% 75%. 63%. 63% G2 6.3 4E9 (4) 2.1 75% 75% 75% 75% 30 2 SMP-coumarin was injected i.v, at 290 ugkg to male mice weighing on average 24.5+ 2.2 gr. Treatment given prior to OP challenge. All figures in parentheses relate to the number of mice in each group, Buffer content: Tris 50 mM pH8, CaCl, 1 mM, NaCl 100 mM, Tergitol 0.02%, d Atropine plus 2-PAM: Atropine sulfate 20 mg/kg) plus 2-PAM25 mg/kg in PBS, Example 7 Purified rePON1-G3C9 or variant4E9 (see Methods) were injected i.v. at the indicated dose prior to OP challenge. Round 4 PON1 Variants that have High Catalytic Example 6 Efficiency for Detoxification of Organophosphates 40 Materials and Methods TABLE 11 Gene libraries. Recombinant PON1 variants cloned into a Activities of Round 2 and Round 3 directly evolved PON1 variants pET vector with a C-terminal 6-His tag (Gupta, et al., 2011) Selected for the hydrolysis of G-type nerve agents were used as the template for library construction using Syn 45 thetic oligonucleotides and the ISOR protocol (Herman and Tawfik, 2007). Briefly, the genes of PON1 variants were PCR amplified, treated with DpnI (NEB) and purified. Purified DNA (20 ug in 150 ul) was digested with 0.3 U DNasel Variant round Fast Slow M'min' M'min' 50 (Takara) at 37°C. for 0.5, 1, 1.5 and 2 min. The reactions were VII-D11 2 29 29 10.7 1.2 terminated with 16 ul of 0.5M EDTA, inactivated at 80°C. for VB3 2 26 6.8 12 O.S 15 min, and run on 2% agarose gel. Fragments of 50-150 bps II-A1 2 25 7.3 5.9 0.7 IV-D11 2 25 7.3 10.6 1.3 size were excised and purified by a gel extraction (Qiagen). MG2-I-A4 2 19.5 13 12.8 1.3 The intact gene was reassembled by PCR from the DNA VI-D2 2 14 3.3 2.8 O.2 55 fragments (100 ng) with the addition of synthetic oligonucle Average R2 23.1 11.1 9.1 O.9 PG11 1 14 3.2 2.12 O.2 otides (1-10 nM, as described in previous examples). The SH8 1 5.7 O.64 2.44 O.1 assembly product was amplified by nested PCR using primers Average R1 9.9 1.9 2.3 O.2 pET-Nes 1-Bc and pET-Nes0-Fo (Gupta, et al., 2011), puri 4E9 O 7.4 O.S6 16.8 O.3 2D8 O 4.11 O.15 14.3 O.23 fied, digested. (NcoI, NotI) and recloned into the pET32 1A4 O 4.1 O.33 11.3 O.21 60 Vector. OC9 O 3.4 O.26 11.1 O.32 Screening. 8C8 O O.O28 OO14 O.21 O.O3 Average RO 3.8 O.3 10.7 O.2 Plasmid transformed BL21/DE3 cells expressing PON1 rePON1-G3C9 O.043 O.O1 O.13 O.08 variants were plated on LB-agar plates (plus 100 mg/lampi The figures shown are values ofkK, LM'min 65 cillin and 1% glucose) and grown overnight at 37° C. Ran Fast and slow hydrolysis of the two equally toxic isomers of GD domly picked colonies were individually grown in 96-deep wells plates (500 ul 2YT per plate, plus amplicillin and CaCl US 8,735,124 B2 41 42 1 mM) for 24hrs at room temperature with shaking. The cells equation using KaleidaGraph. Substrate concentrations were were pelleted and frozen (-80° C.). Cell pellets were verified by monitoring their complete hydrolysis using NaF defrosted, resuspended in lysis buffer (200ul, 0.1M Tris-HCl (100 mM). pH 8.0, 1 mM CaCl, 10 g/ml lysozyme, 0.2% Triton X-100, Kinetic Assay with PMP-Coumarin and 5 units/ml benzonase (Novagen)), lysed (1300 rpm, 45 5 Purified variants (0.5uM) were mixed with racemic PMP min, 37° C.) and centrifuged (4000 rpm, 20 min, 4° C.). coumarin (10 uM) and activity buffer as above. The rate of Clarified cell lysates (400) were mixed with an AChE solution PMP-coumarin hydrolysis was monitored by measuring the (40 ul, 1 nM reAChE (Gupta, et al., 2011), in PBS, 0.1% absorbance of released coumarin at 405 nm for up to 200 min. BSA) in 96 well plates using an automated liquid-handling 10 Alternatively, purified R-specific variant 3B3 (Ashani, et al., system (Precision 2000-BioTEk). In-situ generated G-agents 2010) (0.5 M) was mixed with racemic PMP-coumarin (10 (Gupta, et al., 2011) (20 ul, 0.1-1.5uM) were added. Follow ing a 30 min incubation, reaction samples (200) were mixed uM) and activity buffer and product release was monitored for with the AChE substrate and DTNB (180 ul, 0.85 mMDTNB, 50 min. Then, the newly evolved variants were added (0.5 0.55 mM acetylthiocholine, in PBS) and initial rates were 15 uM) and product release was monitored for additional 150 measured at 412 nm. The percentage of residual AChE activ 1. ity was determined by comparing initial rates in the presence Kinetics of GA Hydrolysis by P NMR. of the screened PON1 variants with to controls, without A Varian 300 MHz was used to monitor the changes in the enzyme (full inactivation), no OP (no inactivation), and a 'P NMR signal of reaction mixtures. In an NMR tube, 0.15 reference variant from the previous round of evolution. Vari ml DO were added to 0.3 ml reactivity buffer containing 1 ants exhibiting residual AChE activity >2-fold greater than mg O.O-diisopropyl methylphosphonate and the Solution the reference variant were isolated, re-plated, and >3 indi was spiked with 50 ul of a GA stock solution in aceton to vidual clones were grown for Verification assays and sequenc provide a final concentration of 1 mg/ml. After recording the ing. 25 baseline spectrum, the reaction was initiated by the addition Enzyme Purification. of the corresponding PON1 variant. Twenty pulses were col Cultures of BL21/DE3 plasmid transformed cells were lected for each scan, data acquisition was repeated every 2-3 grown (ODoo-0.5, 37° C.), induced (IPTG 1 mM, 4 h), min. harvested, resuspended, and disrupted by Sonication. Ammo 30 Ex-Vivo Assays. nium sulfate was added (55% w/v). The precipitate was dis Protection of ChE’s in human whole blood samples was solved and dialyzed against activity buffer (Tris-HCl 50 mM measured as previously described. Briefly, purified PON1 pH 8, CaCl. 1 mM, NaCl 50 mM, Tergitol 0.1%) and purified variants (0.3–2.5 LM) were added to pre-heated (37° C.) on Ni-NTA (Novagen). Fractions were analyzed for purity human whole blood samples (obtained from the Israeli blood (by SDS-PAGE), pooled, dialyzed against activity buffer 35 bank), spiked with CaCl (to 2 mM) and adjusted to pH-7.4 supplemented with 0.02% sodium azide, and stored at 4°C. with Tris base (1 M). At selected time intervals, freshlygen Protein purity was typically 70-85% by SDS-PAGE gel. erated GF was added (to 0.1 uM) and the residual activity of Determination of k/K by AChE Inhibition. blood ChEs was assayed as previously described (Ashani, et In situ generated agents (40 nM) (Gupta, et al., 2011) were al., 2011). The ChE activity was compared to the residual mixed with purified PON1 variants (10-0.01 uM) in activity 40 ChEs activity obtained after 5 min incubation in blood. The buffer. Samples of the reaction mixture were taken at different hydrolytic activity of PON1 variants (0.3-2.5uM) following incubation in whole blood (pH 7.4, CaCl2 mM) was evalu times, diluted (1:10) into an AChE solution (4 nM TcAChE, ated by dilution of PON1 containing blood samples (1000 0.1% BSA, 1 mM EDTA, in PBS), incubated 15 min), and fold) at different time intervals into activity buffer, addition of their residual AChE activity was determined as described 45 racemic CMP-coumarin (0.3 mM), and measurement of the above. The %-inhibition of AChE by the G-agent without rate of hydrolysis as above. These rates were compared to the preincubation with a PON variant was considered as 100% rates obtained after 5 min incubation. inhibition attributed to the toxic isomers of the G-agents. Results Normalized '%-inhibition values were derived for each time 50 2D8 was chosen as the starting point for further evolution, point, and the apparent k/K was derived from the slope of the mutations of which are described in Table 15, herein the resulting single exponential curve. below. Determination of ki/K Values with S/R-CMP-Cou Library Design and Screening a1. The present inventors generated gene libraries derived S-CMP-coumarin was obtained by digestion of racemic 55 from 2D8 by structure-guided targeted mutagenesis. This CMP-coumarin with variant 3B3 that is R-CMP-coumarin enabled them to generate smaller and more focused libraries specific (Ashani, et al., 2010), followed by organic extraction that could be screened by a medium-throughput 96-well plate screen. Several strategies were applied, as detailed below. of the intact. S-CMP. R.-CMP-coumarin was obtained in a Strategy 1: Targeted Diversification of Active-Site Resi similar manner using variant VIID2 that is S-CMP-coumarin 60 dues. specific. Purified variants (0.01-0.6 uM) were mixed with Examining the structures of the unbound PON1 (PDB code either S, or R-CMP-coumarin (5-1000 uM) in activity buffer 1V04 (Harel, et al., 2004)), of PON1 in complex with the in 96-well ELISA plates (200 ul per reaction). Product for inhibitor 2-hydroxyquinone (PDB code 3SRG (Ben-David, mation was monitored for 5 min at 405 nm and initial veloci et al., 2011)), and of a docking model with the OP pesticide ties were derived. Values of at least three independent repeats 65 paraoxon (Ben-David, et al., 2011), indicated several active were averaged for determining the kinetic parameters of each site positions that could affect OP binding and catalysis, variant by fitting the data directly to a Michaelis-Menten including residues 70, 71, 196,240 and 292 (Table 14). US 8,735,124 B2 43 44 TABLE 1.4 Fold improvement Mutagenesis Spiked Screening measured in crude Round strategy mutations Shuffling Substrate cell lysates 1 1 Gly69Leu/Val/Ile/Ser 2OOO 21 improved Lys7OAla Seri Gln/ASn clones variants'. s.4- Tyr71 Phe/Cyc/Ala Leu/Ile Screened old with Trp115Leu/Cyc/Gly/Ala? Val with GB and s5 Arg134His/Gln/ASn GB, GD old with GD Met196Leuf Ile? Phe relative to the Leu24OIle, Wal starting point Phe92Serf Val Leu (2D8). Best variant: PG11 2 1 Ser222Leu/Val/Ile/Met/Cys 10 best 1OOO 25 improved clones clones variants. s.4 from Screened old with round 1 with GB, GB, s2 fold GD, GF with GD and sa. old with GF relative to PG11. Best variant: VIID11. 3 1 Library 3.1 7 best 400 clones 18 improved ASn50Gly/Ala clones from variants/. s.4 Met196Phef Ile from library 3.1 fold with His 197Lys/Ser/Arg/Gln/ASn/Thr round 2 screened GB, s1.6 fold le291 Phe/Trp/Leu with GB with GD Phe292Leu/Ile Trp and GD relative to Tyr294Phe/Gln/Asn VIID11 Val346Leu/Ile/Phe/Trp Best variant: Phes47Gly/Ala? Ile/Leu/Val/Thr/ 1-I-F11 Ser/Trp His348Gly/Ala? Ile/Leu/Val/Thr/ Ser/Trp 3 4 Library 3.1: Leu5SIle? MetWall le74Leu Asp136His/Gln Pro189Gly/Ser 3 3 Library 3.2: 7 best 400 clones Gly69Met/Ala clones from Lys70Seri Gln/Thr/Glu/Asp, Arg from library 3.2 Tyr71 Phe/Trp/Met/Cyc round 2 screened Pro72Gly/Ser with GB Gly73Pro/Ser and GD le74Trp/Phe/Pro/Ser/Gly Met75Leu/Trp/Phe/Pro Phe77Gly/Ala Ile/Leu/Val/Thr/ Ser/Trp/Ile/Leu/Met Asp78/ASn/Gln/Ser/Ala Val/ Tyr/Gly/Ser/Pro 4 2 - No mutations spiked - 18 best 700 clones 18 improved variants screened variants". s2 from with GB fold with round 38 and GD GB, s2 fold libraries with GD 3.1 and relative to 3.2 1-I-F11 Best variant: IG1 The average frequency of random mutations in unselected library clones was 0.5 + 0.3. See FIG.8. See FIG. 9. See FIG, 10.

The present inventors also explored mutations in active revealed that the unselected library contained on average 4+1 site residues that were substituted in the earlier rounds of mutations per clone, with each clone exhibiting a different directed evolution towards GF hydrolysis, including posi 60 mutational composition. This Round 1 library was then tions 69,115, and 134. The library of variants was constructed screened with in-situ generated GD and GB, using the AChE by spiking mutations in these 8 active-site positions into the inhibition assay (see materials and methods). This assay mea 2D8 gene in a combinatorial manner using the ISOR method sures the ability of PON1 variants to prevent loss of AChE (Herman and Tawfik, 2007). In general, residues were activity by rapidly hydrolyzing the toxic isomer of the added mutated to amino acids with similar physico-chemical prop 65 OP. erties although more drastic changes were also included (e.g. By the end of Round 1, all improved variants had acquired Gly69Leu/Val/Ile). Sequencing of randomly selected clones mutations at two positions, 69 and 115. Upon selection to a US 8,735,124 B2 45 46 broader range of G-agents, these residues changed again. The mutations. For the Round 4 library, however, shuffling was improved variants carried additional mutations, primarily at applied with no spiking of targeted mutations (Table 14). positions 70, 71, 196, 240 or 292, but these varied from one Strategy 3: Targeted Mutagenesis of the Flexible Active variant to another (FIG. 8). Site Loop The changes in residues 69 and 115 led the present inven PON1's longest active site loop (residues 70-78) is highly tors to explore the re-optimization of another key active-site flexible and is disordered in the crystal structures of unbound PON1 (Harel, et al., 2004). Upon binding of the lactaminhibi residue, 222, the mutagenesis of which to Ser had led to tor 2-hydroxyquinoline (2HO), the loop became structured increased GF hydrolysis (see previous examples). Residue and thereby completes the active-site (Ben-David, et al., Ser222 was therefore targeted for mutagenesis in the 2" 2011). In particular, Tyr71 and Ile74 comprise part of the round library that was screened for neutralization of GB and 10 active-site wall and contact the inhibitor 2HQ. The loop con GD, as well as GF (Table 14). Mutations to hydrophobic figuration, and the position of Tyr71 in particular, seem to residues (Leu, Val, Ile, Met, or Cys) were explored. Indeed, vary between different substrates (Ben-David, et al., 2011). 96% (2/25) of the improved variant from Round 2 were Indeed, mutations in positions 70 (Lys to Asn or Gln) and 71 mutated in position 222, mostly to Met (1724). (Tyr to Phe or Ile) appeared in many improved clones from Positions 69, 115 and 222 that were substituted in the first 15 Round 1 (FIG. 8). Therefore, in addition to the library two rounds reside at the bottom of PON1's active-site and described above, the present inventors designed a second close to the catalytic calcium. However, in the 3" round Round 3 library that explored active-site loop mutations. library, the present inventors targeted residues whose side Given the very small size of the fluoride leaving-group of chains are involved in structuring the top of PON1’s active G-agents, the present inventors explored loop mutations that site cleft. These included positions 50, 197, 291, 294,346, introduced large side-chains such as Trp, Phe and Tyr that 347 and 348. In addition, positions 196 and 292 that had been could reduce the active site's volume and improve substrate explored during the generation of the first round library but binding. The present inventors also attempted to modulate the acquired no mutations were retargeted (Table 14). On aver loop's configuration by exploring mutations to Gly and Pro age, variants in the unselected 3" round library contained 2+1 (Table 14). Since mutations were introduced with oligonucle Substitutions at the targeted positions. Following screening 25 otides (21-29 bp) that flank the mutated codon, substitutions were mostly mutually exclusive and each clone carried, on with GB and GD, the present inventors obtained 18 improved average, only one loop mutation. clones in which positions 197 and 291 were primarily Strategy 4: Ancestral Mutations. mutated, although not in all variants (in 3/1s and /8 of the Ancestral mutations, i.e., Substitutions into residues that improved variants, respectively). Some positions did not appeared along the evolutionary history of a given protein, accept any mutations (196, 294, 346, 347, 348), and others 30 were mutated in single clones (50, 292). have been shown to be powerful modulators of a protein's Strategy 2: Shuffling of Improved Variants stability and function (Alcolombri, et al., 2011; Bridgham, et Shuffling of selected variants can combine beneficial muta al., 2006: Field and Matz, 2010). A library based on active tions that were acquired in separate variants and/or eliminate site Substitutions from the predicted ancestors of mammalian deleterious mutations from individual variants. In particular, 35 PONs has been described (Alcolombri, et al., 2011). Screen since the mutational diversity in the present libraries was ing this library with a coumarin analogue of GF (CMP-cou large compared to the number of clones screened (screening marin) led to the identification of several ancestral mutations covered between 0.04 to 7% of the theoretical library diver that increase this activity. The present inventors included sity), the improved variants generally exhibited different these mutations (Leu55Ile, Ile74Leu, Asp136His, mutational compositions (e.g. Round 3 in which no conver 40 Pro189Gly) together with similar ones (55 Met/Val, 136Gln, gence was seen). In effect, shuffling of improved variants was 189Ser) in the Round 3 substitution library at an average applied in every round as part of the ISOR protocol (Herman frequency of 0.5 mutations per gene in the unselected library and Tawfik, 2007) that was used to introduce the library (Table 14, herein above). TABLE 1.5 Mutations Catalytic efficiency (k.u.K.) x 10 M'min relative to GD

Variant rePON1 Round Fast Slow GF GB GA rePON1 O.OOSS O.OO2 0.0015 + 0.0006 0.013 + 0.003 0.008 + 0.001 0.043 + 0.007 2D8 Leu69Gly, O O.4 0.1 OO15 O.OO3 0.46 + 0.01 O.O25 O.OO2 O.O82, O.OOS His 115Trp, His134Arg, Phe222Ser, Thr332Ser Fold (73) (10) (35) (3) (2) improvement relative to (rePON1) PG11 Leu69Val, 1 1.43 - 0.3 O32- 0.04 O.2 0.03 O.O2 O.OO5 O.OO6 OOO1 His 115Ala, His134Arg, Phe222Ser, Thr332Ser Fold 4 (260) 21 (213) 0.4 (15) 0.8 (3) 0.07 (0.1) improvement R1 relative to variant 2D8 (rePON1) US 8,735,124 B2 47 48 TABLE 15-continued Mutations Catalytic efficiency' (ka/Kit) x 10 M'min relative to GD

Variant rePON1 Round Fast Slow GF GB GA VII-D11 Leu69Val, 2 2.90.9 2.90.1 1.07 - O.O8 O.12 OO1 O.O25 OOO3 His115Ala, His134Arg, Phe222Met, Asp309Gly, Thr332Ser Fold 7 (527) 193 (1933) 2 (82) 5 (15) 0.3 (0.6) improvement R2 relative to variant2D8 (rePON1) 1-I-F11 Leu55Met, 3 4.4 + 0.6 4.4 + 0.6 1520.1 O39 O.04 O.17 O.04 Leu69Val, His115Ala, His134Arg, Phe222Met, Ile291 Leu, Thr322Ser Fold 11 (800) 293 (2933) 3 (117) 16 (49) 2 (4) improvement R3 relative to variant2D8 (rePON1) IIG1 Leu55Ile, 4 5.1 - 0.6 5.1 O.6 3.4 O.3 O32-, O.O1 O.23 O.OO)4 Leu69Val, His115Ala, His134Arg, Asp136Gln, Phe222Met, Ile291 Leu, Thr332Ser Fold 13 (927) 340 (3400) 7 (262) 13 (40) 3 (5) improvement R4 relative to variant2D8 (rePON1)

Directed Evolution of rePON1 for G-Agent Hydrolysis starting variant 2D8 was found (Table 15). However, PG 11's Table 15, herein above summarizes the results of four 40 activity with GF was reduced 2.3-fold and its GB activity was rounds of directed evolution starting from variant 2D8 and the same as 2D8’s. The two best variants shared the same 4 screening for GB and GD hydrolysis. After screening ~2000 active site mutations: Leu69Val. His134Arg, Phe222Ser and variants in Round 1, 21 clones were identified that in crude Thr332Ser, but differed in position 115: variant 5H8 acquired cell lysates were improved up to 4-fold with GB and up to a Val and variant PG 11 an Ala. While mutations Phe222Ser 5-fold with GD relative to the starting point 2D8 (FIG.8). The and Thr332Ser were already present in the parental variant two most active variants with GD: 5H8, and PG11 were 45 2D8, the mutations in positions 69,115 and 134 were selected purified and characterized (Table 15 and 17). An improve from the substitution library of Round 1. ment of 21- and 4-fold in the catalytic activity of the best Table 16 herein below summarizes the catalytic activities variant PG 11 with the two toxic isomers of GD relative to the of improved variants from each round of evolution. TABLE 16 Mutations Catalytic efficiency' (k./Kit) x 10'M'min

relative to GD

Variant rePON1 Round Fast Slow GF GB

rePON1 O.OOSS O.OO17 O.OO15 O.OOO6 O.O13 O.OO3 O.008 O.OO1 8C8 Leu69Ser, O 0.0028 + 0.0006 (0.5) 0.0014 + 0.0004 (0.9) 0.015 + 0.005 (1.2) 0.0034 + 0.001 (0.4) Val97 Ala, His115Trp, Pro135Ala, PheSer OC9 Leu14Met, O 0.34 + 0.037 (62) 0.026 + 0.0006 (17) 1.11 + 0.3 (85) 0.028 + 0.009 (4) Leu69Gly, Ser111Thr, His115Trp, His134Arg, Phe222 Ser, Thr332Ser

US 8,735,124 B2 51 52 TABLE 16-continued Mutations Catalytic efficiency' (k./Kit) x 10M min relative to GD

Variant rePON1 Round Fast Slow GF GB His134Arg, His197Arg, Phe222Met, Ile291 Leu, Thr332Ser Mutations relative to the wild-type like rePON1 variant G3C9(Harel, et al., 2004). Mutations that were newly introduced in a given round are denoted in bold. Fold improvement relative to wt like rePO 1. Errors of values were derived from at least two independent measurements. The maximal deviation between different enzyme preparations was 22 fold (measured for VI D2). Fast and slow hydrolysis of the two equal y toxic isomers of GD (SSP and RCSP), Kinetic parameters were determined with the in situ generated G-agent and by assaying residual AChE activity. They therefore relate to the toxic S, isomer, Values from (Gupta, et al., 2011) Standar CO,

For the 2nd round, a substitution library was constructed catalytic efficiencies were improved up to 11- and 293-fold that explored various substitutions at position 222 whilst with the two toxic isomers of GD, and up to 17-fold with GB shuffling the 10 most active clones found in Round 1 (FIG.9). (Table 15, Table 16). Although 2D8 was evolved for GF, a The resulting library was screened for GD and GB, as well as further s3-fold improvement with GF was identified in GF. The latter was included to eliminate variants that exhibit Round 3 variants. The catalytic efficiency of the most reduced activity with GF, as observed with variants isolated improved variant with GB (1-I-F11, 3.9x10 M'min') was from the 1st round. Of the ~1000 clones screened, 39 variants 25 improved >3-fold relative to the best Round 2 variant. were isolated that were improved relative to the best variants In Round 4 (Table 14, FIG. 13), 700 clones were screened of Round 1. Sequencing revealed 25 singular variants (FIG. with GB and GD, and 22 improved variants were isolated of 10). Following more detailed screens (at different agent con which 18 were singular (FIG. 14). Of these: centrations and incubation times), the 6 most active variants 66% (12/1s) were improved mostly with GB, and 33% (9/s) were identified, purified and characterized. The purified vari 30 were improved also with GD. In addition to the mutations ants exhibited up to 7- and 193-fold higher specific activities fixed in earlier rounds, His115Ala was fixed, and most vari with the toxic isomers of GD, and up to 5-fold higher activi ants also carried Leu55Ile (55%, 10/1s) and/or Ile291 Leu ties with GB, relative to the starting variant 2D8 (Table 15, (83%, 15/18) (FIG. 14). 9 variants were purified and three Table 16). Their activities with GF were improved relative to variants identified that had improved relative to the starting variants from Round 1 and thereby became similar to that of 35 variant 2D8 by s13- and s340-fold with the toxic isomers of 2D8. All Round 2 variants contained three mutations: GD, s7-fold with GB, and s9-fold with GF (Table 15, Table Leu69Val, His 134Arg and Thr332Ser, and all but one con 16). As shown, their catalytic efficiencies with GF and GD tained also the Phe222Met mutation (Table 14, Table 16). The were well over 107 M'min' (Table 15, Table 16, FIG. 15). greatest variability was in position 115 in which either Ala, Neutralization of GA Leu or Val were observed. While no attempt to introduce 40 The in-vivo toxicity of GA is >2-fold lower than that of all random mutations during library construction was made, the other G-agents (Benschop and Dejong, 1988), thus ranking it PCR methodologies applied for library making, produced as the least threatening of the G-agents, and of lower priority random mutations such as Phe64Leu or Asp309Gly that were for detoxification. In addition, its structure and leaving group retained in certain improved variants. By Round 2, the most are significantly different than that of all other G-agents (FIG. improved variants reached the targeted catalytic efficiency of 45 7A), suggesting that variants that are improved for the three >107M min' with GD and GF, but were still 10-fold lower other G-agents might exhibit lower rates with GA, and vise in activity with GB. The best Round 2 variant, VII-D11, versa. Thus, the libraries were not screened for GA neutral hydrolyzed the two toxic isomers of GD with equally high ization, but the present inventors did examine the activity with rates (2.9x107 M'min': Table 15). GA of wild-type like rePON1, and of the most improved In the 3" round, two libraries were constructed and 400 50 variants from the 4 rounds of evolution described here. Using variants from each library were screened with GD and GB the AChE inhibition assay, it was found that rePON1 is sq.0- (Table 13, FIG. 11). 18 improved clones were identified alto fold more efficient at hydrolyzing the toxic isomer of GAthan gether. All these clones were improved with GB, but only the toxic isomers of all other G-agents (Table 15). The most 67% (2/1s) were also improved with GD (FIG. 12). Most of improved variants from Rounds 3 and 4 became 4 to 5-fold the improved variants originated from Library #3.1 (73%, 55 more efficient than rePON1 at hydrolyzing the toxic isomer of 13/18), and their improvements with GB (1.6-4-fold) were GA (1-I-F11 and IIG1: Table 15). Thus, although GA neu greater than with GD (1.1-1.6 fold). Sequencing indicated tralization was not screened for, the catalytic efficiency of the that 55% (9/1s) of these improved variants contained at least present evolved variants with GA had increased and became one ancestral mutation (Leu55Ile, or Asp136His) and 28% similar to that with GB (-3x10 M'min'). (5/18) contained two ancestral mutations. In addition, the 60 Stereospecificity of the Evolved Variants active-site mutation Ile291 Leu became abundant (39%, 71s). The hydrolysis of G-agents was monitored using the AChE Certain mutations introduced in earlier rounds. His134Arg assay that only detects the hydrolysis of the toxic isomers (S, and Phe222Met, were fixed (i.e., appeared in all improved for GB, GD and GF, and Rp for GA). To examine the hydroly variants) and others—Leu69Val and His 115Ala, were nearly sis of both stereoisomers, the present inventors assayed the fixed (90%, 164s; FIG. 12). 65 most improved variants from each round of directed evolution The four most improved variants of Round 3 were purified with the purified R, and S isomers of the coumarin analogue and characterized. Relative to 2D8, the starting variant, their of GF (CMP-coumarin: FIG.7B). The results indicate that the US 8,735,124 B2 53 54 evolution of higher detoxification rates of the three target mers of GA with equal efficiency, while the starting point G-agents (GB, GD and GF) was accompanied by a complete variant2D8 displayed a sharp bi-phasic time-course behavior reversion in rePON1s stereoselectivity, as summarized in Stemming from a strong stereo-preference for one isomer Table 17, herein below. over the other (FIG. 18B). Since the catalytic efficiency (k/ TABLE 17 Sp-CMP-Coumarin RP-CMP-Coumarin Round kcar KM kcal/KM kca, KM kcal/KM E # Variant (min) (IM) (uM'min') (min) (IM) (uM'min') (S/R) - rePON1 n.d. n.d.

The catalytic efficiency (k/K) of the wild-type-like K) value of 2D8 with the toxic R-GA isomer was compa rePON1 with R-CMP-coumain is >1600-fold higher than rable to the hydrolysis rate of the slow rather than the fast GA with S-CMP-coumarin. In contrast, thek/K values of the 25 component in the 'P-NMR data, it was assumed that 2D8 is best variants from Rounds 3 and 4 with S-CMP-coumarin is more efficient at hydrolyzing the non-toxic S, isomer of GA. >2500-fold higher than with R-CMP-coumarin (Table 17, Evolved Round 4 variant (VIID2) displayed an almost equal FIGS. 16A-B). reactivity towards both isomers of GA with an apparent rate The increase in S/R stereoselectivity with CMP-cou constant ratio of less than 4 (FIG. 18B). To summarize, the marin was attributed to changes in both Substrate binding and 30 AChE assay indicated that rePON1 and 2D8 hydrolyze the catalysis. In each round of evolution, a decrease in the K toxic R isomer of tabun with similar efficiencies, 4.3x10 value for S-CMP-coumarin and a concomitant increase in and 8.2x10 M'min', respectively, while variant VIID2 had k value were observed. Parallel, increases in K and improved at R isomer hydrolysis (1 .83x10°M'min'). decreases in k values were observed with R-CMP-cou marin (Table 17). However, the change in the enantiomeric 35 Activity and Stability in Human Blood Samples ratio (E; k/K(S) ki/K(R)) differed between rounds, The use of evolved PON1 variants as prophylactic drugs with the greatest change occurring over the course of evolu depends on their ability to maintain activity in human blood tion leading from wild-type like rePON1 to the starting point over long periods of time. Variants PG 11, VIID1 1, 1-I-F11 variant 2D8. Here, the greatest change occurred in Round 3 and VIID2 (from Rounds 1-4 respectively) were added to variants. Interestingly, as far as indicated by the data for 40 human blood samples and incubated at 37°C. In situ gener CMP-coumarin, this change was driven by a mild increase ated GF was added at different time intervals and the residual (-2-3-fold) in rate of hydrolysis of the S isomer (that was ChE activity was measured (FIG. 19A). Due to its low cata selected for), and a far larger decrease in hydrolysis rate of the lytic efficiency, Round 1 variant PG 11 could not provide R isomer (s.52-fold). measurable ChE protection at the concentration range used A similar trend was observed with the coumarin analogue 45 of GD, PMP-coumarin (FIGS. 17A-B). As with other (0.5-2.1M). However, variants VIID1 1, 1-I-F11 and VIID2 G-agents, the toxicity of GD is determined by the phosphorus showed measurable ChE protection (41-45%). Further, these chirality, and GD's two S isomers (S.R., S.S.; FIG. 7A) are variants maintained 80-95% of their initial protection activity >1000-fold stronger inhibitors of AChE than its two R, iso in human blood after 24 h of incubation at 37° C. (FIG. 19A). mers (Benschop, et al., 1984). While the best Round 1 variant 50 Assaying the rate of hydrolysis of the chromogenic ana hydrolyzed all four diasteroisomers of PMP-coumarin at logue of GF, CMP-coumarin, at different time intervals fol measurable rates, variants from Rounds 2 to 4 hydrolyzed lowing incubation in whole blood (FIG. 19B) or buffer (FIG. only 50% of the racemic mixture (FIG. 17A). To confirm that 20) gave very similar results. This assay also indicated that the the hydrolyzed fraction corresponds to the S isomer pair, the activity of all variants was mildly increased after 1 h incuba present inventors applied the evolved rePON1 variant 3B3 55 tion (4-12% relative to their activity after 5 min). This effect that hydrolyzes almost exclusively the R isomers of the cou is in most likelihood the result of binding of the rePON1 marin G-agent analogues (Ashani, et al., 2010). Indeed, fol variants to the HDL (high-density lipoprotein) particles in lowing the action of 3B3 that hydrolyzed only 50% of a blood (Gaidukov and Tawfik, 2005). Taken together, these racemic mixture of PMP-coumarin, the remaining 50% was results suggest that the interaction of the evolved PON1 vari readily hydrolyzed by evolved variants from Rounds 2-4 60 (FIG. 17B). ants with human blood constituents did not affect their pro In order to examine the stereopreference of a variant ficiency ex vivo within 24 hours. towards GA, the present inventors followed its hydrolysis Neutralization of VX both by the AChE assay, which detects only the hydrolysis of Chemical warfare nerve agents are generally divided into the toxic R, isomer (Table 15) and by 'P-NMR (FIGS. 18A 65 G-agents and V-agents (Romano, et al., 2008). While most B), which provides data on the hydrolysis of both isomers. G-agents have a small and highly reactive fluoride leaving The NMR data indicated that rePON1 hydrolyzes both iso group (pK 3.1), V-type agents have a bulky and far less US 8,735,124 B2 55 56 reactive N.N-dialkylaminoethyl-thiol leaving group 10. Broomfield, C. A. A purified recombinant organophos (pKa=7.9) (Bracha and O’Brien, 1968) (FIG. 7A). V-agents phorus acid anhydrase protects mice against Soman. Chem pose a greater threat due to their increased toxicity and as Such Biol Interact 87,279-84 (1993). are prime targets for detoxification. However, the hydrolysis 11. Li, W. F. Furlong, C. E. & Costa, L. G. Paraoxonase of the toxic S, isomer of VX by wild-type-like rePON1, and protects against chlorpyrifos toxicity in mice. Toxicol Lett by human PON1 (unpublished results), is below the detection 76,219-26 (1995). limits <2 M'min'. Upon longer incubations with high 12. Kassa, J., Karasova, J. Z. Caisberger, F. & Bajgar, J. The enzyme concentrations, Stoichiometric neutralization of VX influence of combinations of oximes on the reactivating may occur, possibly by reacting with Cys284 and thereby and therapeutic efficacy of antidotal treatment of Soman inactivating the enzyme (Sorenson, et al., 1995; Tavori, et al., 10 poisoning in rats and mice. Toxicol Mech Methods 19, 2011). The catalytic efficiencies of variants VIID2 and VH3 547-51 (2009). with S-VX are 132 and 286 M'min', respectively. Thus, 13. Harvey, S. P. et al. Stereospecificity in the enzymatic although these variants were evolved for broad range G-agent hydrolysis of cyclosarin (GF). Enzyme and Microbial hydrolysis, they also exhibit >100-fold higher rates than wild Technology 37, 547-555 (2005). type PON1 for VX neutralization. 15 14. Li, W. S. Lum, K. T., Chen-Goodspeed, M., Sogorb, M. Although the invention has been described in conjunction A. & Raushel, F. M. Stereoselective detoxification of chiral with specific embodiments thereof, it is evident that many Sarin and Soman analogues by phosphotriesterase. Bioorg alternatives, modifications and variations will be apparent to Med Chem 9, 2083-91 (2001). those skilled in the art. Accordingly, it is intended to embrace 15. Amitai, G. et al. Enhanced stereoselective hydrolysis of all such alternatives, modifications and variations that fall toxic organophosphates by directly evolved variants of within the spirit and broad scope of the appended claims. mammalian serum paraoxonase. Febs J 273, 1906-19 All publications, patents and patent applications men (2006). tioned in this specification are herein incorporated in their 16. Aharoni, A. et al. Directed evolution of mammalian entirety by reference into the specification, to the same extent paraoxonases PON1 and PON3 for bacterial expression as if each individual publication, patent or patent application 25 and catalytic specialization. Proc Natl AcadSci USA 101, was specifically and individually indicated to be incorporated 482-7 (2004). herein by reference. In addition, citation or identification of 17. Gaidukov, L. et al. In vivo administration of BL-3050: any reference in this application shall not be construed as an highly stable engineered PON1-HDL complexes. BMC admission that such reference is available as prior art to the Clin Pharmacol 9, 18 (2009). present invention. To the extent that section headings are 30 18. Ashani, Y. et al. Stereo-specific synthesis of analogs of used, they should not be construed as necessarily limiting. nerve agents and their utilization for selection and charac terization of paraoxonase (PON1) catalytic scavengers. REFERENCES FOR EXAMPLES 1-6 Chem Biol Interact Epub ahead of print (2010). 19. Gupta, R. D. & Tawfik. D. S. Directed enzyme evolution (Other References are Cited Throughout the Application) 35 via small and effective neutral drift libraries. Nat Methods 1. Doctor, B. 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SEQUENCE LISTING

<16 Os NUMBER OF SEQ ID NOS: 142

SEO ID NO 1 LENGTH: 355 TYPE PRT ORGANISM: Artificial sequence FEATURE; OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs SEQUENCE: 1 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asin Val His Arg 2O 25 3 O US 8,735,124 B2 61 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Lieu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Thr Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu. 355

<210s, SEQ ID NO 2 &211s LENGTH: 366 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 2 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 63 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Gly Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Arg Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu Ala Ala Ala Lieu. Glu. His His His His His His 355 360 365

<210s, SEQ ID NO 3 &211s LENGTH: 366 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 3 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 65 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Val Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Leu Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Arg Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Leu Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu Ala Ala Ala Lieu. Glu. His His His His His His 355 360 365

<210s, SEQ ID NO 4 &211s LENGTH: 366 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 4 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 67 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Gly Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Thr Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Arg Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu Pro Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu Ala Ala Ala Lieu. Glu. His His His His His His 355 360 365

<210s, SEQ ID NO 5 &211s LENGTH: 366 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 5 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 69 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Val Llys Phe Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Leu Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Arg Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Phe His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu Ala Ala Ala Lieu. Glu. His His His His His His 355 360 365

<210s, SEQ ID NO 6 &211s LENGTH: 366 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 6 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 71 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Val Llys Phe Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Leu Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Arg Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu Ala Ala Ala Lieu. Glu. His His His His His His 355 360 365

<210s, SEQ ID NO 7 &211s LENGTH: 366 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OO > SEQUENCE: 7 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 73 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Val Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Val Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Arg Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu Ala Ala Ala Lieu. Glu. His His His His His His 355 360 365

<210s, SEQ ID NO 8 &211s LENGTH: 366 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 8 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Ser Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 75 - Continued

Glu Val Ser Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Gly Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Arg Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Arg Thr Lieu. 245 250 255 Thr Pro Met Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu Ala Ala Ala Lieu. Glu. His His His His His His 355 360 365

<210s, SEQ ID NO 9 &211s LENGTH: 366 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 9 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 77 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Val Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Arg Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu Ala Ala Ala Lieu. Glu. His His His His His His 355 360 365

<210s, SEQ ID NO 10 &211s LENGTH: 366 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 10 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 79 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Val Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Leu Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Asn Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Lieu. His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu Ala Ala Ala Lieu. Glu. His His His His His His 355 360 365

<210s, SEQ ID NO 11 &211s LENGTH: 366 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 11 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 81 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Lieu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Thr Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu Ala Ala Ala Lieu. Glu. His His His His His His 355 360 365

<210s, SEQ ID NO 12 &211s LENGTH: 363 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 12 Met Ala Lys Lieu. Thr Glu Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 83 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Gly Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Arg Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Glu Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Ser Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu. His His His His His His His His 355 360

<210s, SEQ ID NO 13 &211s LENGTH: 363 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 13 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 85 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Gly Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Arg Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Cys Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Thr Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu. His His His His His His His His 355 360

<210s, SEQ ID NO 14 &211s LENGTH: 363 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 14 Met Ala Lys Pro Thr Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 87 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Lieu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Ala Lieu. Glu Lieu. Gly Ile Thr Gly Asn. Thir Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn His Ala Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Val His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Thr Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu. His His His His His His His His 355 360

<210s, SEQ ID NO 15 &211s LENGTH: 363 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 15 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 89 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Gly Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn Thr Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Lieu Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Thr Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu. His His His His His His His His 355 360

<210s, SEQ ID NO 16 &211s LENGTH: 363 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 16 Met Ala Lys Lieu. Thir Thr Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 91 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Lieu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Ala Lieu. Glu Lieu. Gly Ile Thr Gly Asn. Thir Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn His Ala Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn. Asn Val Arg Val Val Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Ile Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Thr Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu. His His His His His His His His 355 360

<210s, SEQ ID NO 17 &211s LENGTH: 363 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 17 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 93 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Lieu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn His Ala Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Thr Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Ala Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu. His His His His His His His His 355 360

<210s, SEQ ID NO 18 &211s LENGTH: 363 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 18 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 95 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Lieu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Ile Ser Ser Phe 1OO 105 11 O Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Ser 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Ala Thr Gly Asp Lieu. Trp Val Gly Cys Arg Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Ser Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu. His His His His His His His His 355 360

<210s, SEQ ID NO 19 &211s LENGTH: 363 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 19 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 97 - Continued

Glu Val Thr Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Val 35 4 O 45 Asp Asn Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Lieu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 7s 8O Llys Ser Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Val 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Asn Thr Lieu. Asp Thr Ser Ser Phe 1OO 105 11 O Asn Pro Trp Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 12 O 125 Lieu. Leu Val Val Asn His Pro Asp Ser Ser Pro Thr Val Glu Val Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Lieu Lys 18O 185 19 O Ser Trp Glu Met His Leu Gly Lieu Ala Trp Ser Phe Val Thr Tyr Tyr 195 2OO 2O5 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255 Thr Pro Lieu Lys Ser Lieu. Asp Phe Asp Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asp Ile Lieu. Ser Glu Glu Pro Llys Val Thr Val Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Thr Val Ala Ala Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Ala Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu. His His His His His His His His 355 360

<210s, SEQ ID NO 2 O &211s LENGTH: 363 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 2O Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 2O 25 3O US 8,735,124 B2 99 100 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Gly Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn Arg Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Glu Tyr Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 21 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 21 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 101 102 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro His Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Ser 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly Arg Pro Asn Gly 27s 285

Met Arg Ile Phe Ser Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 22 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 22 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 103 104 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asp ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro His Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Ser 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly Arg Pro Asn Gly 27s 285

Met Arg Ile Phe Ser Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Ser Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 23 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 23 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 105 106 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro His Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Thir Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly Arg Pro Asn Gly 27s 285

Met Arg Ile Phe Ser Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Ser Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 24 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 24 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Ser Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 107 108 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Gly Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Ala Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEO ID NO 25 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 25 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Ser Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25

US 8,735,124 B2 111 112 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Ala Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Ala Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Ile Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEO ID NO 27 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 27 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 113 114 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asp ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Pro Ser Phe 105 11 O

Asn Pro His Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Ser 225 23 O 235 24 O

Lell Ala Arg Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Luell Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asp Gly Thir Wall Luell Glin Gly Ser Ala Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 28 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 28 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 115 116 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Gly Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn Arg Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Wall His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Ser Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 29 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 29 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Ser Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 117 118 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Ala Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 3 O LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 3 O Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 119 120 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Gly Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Luell Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 31 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 31 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 121 122 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Ser Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Ala Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 32 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 32 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 123 124 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Ala Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 33 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 33 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 125 126 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Ile His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 34 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 34 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 127 128 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Luell Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Ala Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEO ID NO 35 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 35 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 129 130 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Ile Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Ala Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 36 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 36 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 131 132 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Luell Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Ala Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEO ID NO 37 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 37 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 133 134 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Pro Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Met Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Ala Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 38 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 38 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 135 136 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Ala Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEO ID NO 39 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 39 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 137 138 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Gly Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 4 O LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 4 O Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Ser Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Tyr Asn Val His Arg 25 US 8,735,124 B2 139 140 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Gly Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn Arg Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Ser Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Ser Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 41 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 41 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25 US 8,735,124 B2 141 142 - Continued

Glu Wall Thir Pro Wall Glu Lell Pro Asn ASn Lieu Val Lys Gly Wall 35 4 O 45

Asp Asn Gly Ser Glu Asp Lell Glu Ile Luell Pro Asn Gly Luell Ala Phe SO 55 6 O

Ile Ser Ser Gly Lell Lys Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70

Ser Gly Ile Lell Lell Met Asp Luell ASn Glu Glu Asp Pro Wall 85 90 95

Wall Lel Glu Luell Gly Ile Thir Gly Asn Thir Luell Asp Ile Ser Ser Phe 105 11 O

Asn Pro Trp Gly Ile Ser Thir Phe Thir Asp Glu Asp Asn Thir Wall Tyr 115 12 O 125

Lell Lel Wall Wall Asn His Pro Asp Ser Ser Ser Thir Wall Glu Wall Phe 13 O 135 14 O

Lys Phe Glin Glu Glu Glu Ser Luell Luell His Lell Thir Ile Arg 145 150 155 160

His Luell Luell Pro Ser Wall Asn Asp Ile Wall Ala Wall Gly Pro Glu 1.65 17O 17s

His Phe Ala Thir Asn Asp His Tyr Phe Ala Asp Pro Tyr Luell 18O 185 19 O

Ser Trp Glu Met His Lell Gly Luell Ala Trp Ser Phe Wall Thir Tyr 195

Ser Pro Asn Asp Wall Arg Wall Wall Ala Glu Gly Phe Asp Wall Ala Asn 21 O 215 22O

Gly Ile Asn Ile Ser Pro Asp Gly Wall Ile Ala Glu Luell 225 23 O 235 24 O

Lell Ala His Ile His Wall Glu Lys His Ala Asn Trp Thir Luell 245 250 255

Thir Pro Luell Lys Ser Lell Asp Phe Asp Thir Luell Wall Asp Asn Ile Ser 26 O 265 27 O

Wall Asp Pro Wall Thir Gly Asp Luell Trp Wall Gly His Pro Asn Gly 27s 285

Met Arg Ile Phe Tyr Asp Pro Asn Pro Pro Gly Ser Glu Wall 29 O 295 3 OO

Lell Arg Ile Glin Asp Ile Lell Ser Glu Glu Pro Wall Thir Wall Wall 3. OS 310 315

Ala Glu Asn Gly Thir Wall Luell Glin Gly Ser Thir Wall Ala Ala Wall 3.25 330 335

Gly Lys Lell Lell Ile Gly Thir Wall Phe His Ala Luell Tyr 34 O 345 35. O

Glu Luell His His His His His His His His 355 360

SEQ ID NO 42 LENGTH: 363 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Serum paraoxonase (PON1) Variant amino acid sequence

<4 OOs, SEQUENCE: 42 Met Ala Lys Lieu. Thir Ala Lieu. Thir Lieu. Lieu. Gly Lieu. Gly Lieu Ala Lieu. 1. 5 15 Phe Asp Gly Gln Lys Ser Ser Phe Glin Thr Arg Phe Asn Val His Arg 25