Novel nicotine decomposition enzyme mutants

Abstract

Nicotine degrading enzyme variants that exhibit increased nicotine degrading activity and / or decreased immunogenicity compared to wild-type NicA2 and NOX enzymes, compositions comprising the variants, and methods of using them are described.

Description

[Technical field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 454,331, filed February 3, 2017, and U.S. Provisional Patent Application No. 62 / 535,507, filed July 21, 2017, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure generally relates to the field of treating nicotine addiction or nicotine addiction. Nicotine degrading enzyme variants that exhibit increased nicotine degrading activity and / or reduced immunogenicity compared to wild-type NicA2 enzyme, compositions containing them, and methods of using them are described. [Background technology]

[0003] The following discussion is provided to aid the reader in understanding the present disclosure and is not admitted to describe or constitute prior art thereto.

[0004] Smoking is a global health problem. The World Health Organization (WHO) estimates that today there are 1.3 billion smokers worldwide, with approximately 5 million tobacco-related deaths annually. If current smoking patterns continue, smoking will cause approximately 10 million deaths annually by 2020. According to the Centers for Disease Control (CDC), tobacco use is the only leading preventable cause of death in the United States, causing approximately 438,000 deaths annually. In addition, smoking is estimated to result in approximately $157 billion in annual health-related economic costs. The CDC estimates that of the 45 million adult smokers in the United States, 70% want to quit, but less than 5% of those who try remain abstinent after one year.

[0005] One of the reasons why it is difficult to quit smoking is the addictive nature of nicotine from cigarettes and other tobacco products. Nicotine is a small molecule that enters the bloodstream quickly when inhaled into the body and subsequently reaches the brain by crossing the blood-brain barrier. Once in the brain, nicotine binds to nicotinic receptors, resulting in the release of stimulants such as dopamine, activating the reward system and providing the smoker with a positive and pleasurable reinforcement experience, which leads to addiction.

[0006] In addition to the harmful health effects associated with smoking and other tobacco use, nicotine addiction, which results from ingesting or inhaling too much nicotine, is another nicotine-related health problem. 50 The average nicotine concentration is 50 mg / kg in rats and 3 mg / kg in mice. Doses as low as 30-60 mg (0.5-1.0 mg / kg) can be fatal to adult humans, while children can become ill after ingesting one cigarette, and anything higher can make them seriously ill. On the other hand, some evidence suggests that the lethal dose can be as high as 500 mg or more (1.0-7.1 mg / kg) in human adults. In either case, acute nicotine poisoning usually occurs in children who accidentally chew nicotine gum or patches, or who ingest e-liquid from electronic cigarettes. In rare cases, children have also been known to become ill after ingesting tobacco. Hundreds of cases of acute nicotine poisoning are reported every month in the United States alone.

[0007] Typically, initial treatment for nicotine addiction may involve administration of activated charcoal to attempt to reduce gastrointestinal absorption, while further treatments may address symptoms resulting from nicotine addiction.

[0008] The use of wild-type NicA2 enzyme for smoking cessation has been proposed (see, e.g., Xue et al., J. Am. Chem. Soc. 137:10136-39 (2015)). Nevertheless, there remains a need for additional agents, compositions, and methods for treating nicotine addiction, as well as agents, compositions, and methods for treating nicotine addiction. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Xue et al.,J.Am.Chem.Soc.137:10136-39(2015) Summary of the Invention

[0010] Described herein are nicotine degrading enzyme variants that exhibit increased nicotine degrading activity and / or reduced immunogenicity compared to wild-type NicA2 enzyme, compositions comprising them, and methods of using them.

[0011] In some embodiments, the nicotine degrading enzyme variant comprises an amino acid sequence that is a variant of the amino acid sequence of the wild-type NicA2 enzyme set forth in SEQ ID NO:1, wherein the variant sequence has at least one substitution, addition, or deletion to SEQ ID NO:1 that increases the nicotine degrading activity and / or decreases the immunogenicity of the variant compared to the wild-type NicA2 enzyme.

[0012] In some embodiments, the variants exhibit increased nicotine degradation activity compared to the wild-type NicA2 enzyme. In some embodiments, variants of the wild-type NicA2 sequence have the following substitutions at different positions: AF104L, G106S, A107H, A107P, 107R, A107K, A107T, F355C, F355V, W427Q, W427E, W427S, W427M, W427H, W427L, W427R, R91A, R91Q, R91F, R91G, R91T, R91L, R91S, R91N, T250G, T250L, T250R, T250V, T250P, K340P, K340I, K340V ... In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs: 5-56. In some embodiments, the nicotine degrading activity of the variant is at least 200%, at least 300%, or at least 400% of the nicotine degrading activity of the wild-type NicA2 enzyme. In some embodiments, the variant sequence comprises at least one, at least two, or at least three substitution(s) at amino acid positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1. In some embodiments, the variant may have a deletion of 1-52 amino acids at the N-terminus of the peptide. For example, a variant derived from SEQ ID NO:1 may comprise a deletion of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, or 52 amino acids from the N-terminus of the peptide. In some embodiments, the variant may additionally or alternatively have a deletion of one or more amino acids from the C-terminus of the peptide, such as a deletion of the C-terminal residue.

[0013] Additionally or alternatively, in some embodiments, the NicA2 variants exhibit reduced immunogenicity compared to wild-type NicA2 enzyme. In some embodiments, the immunogenicity compared to wild-type NicA2 enzyme is reduced by 75% or more. In some embodiments, the variant sequence comprises at least one substitution, addition, or deletion in an immunogenic T-cell epitope within a region selected from amino acids 10-32, 68-94, 189-225, 248-285, 296-327, 336-391, or 435-459 of SEQ ID NO:1. In some embodiments, the variant sequence comprises at least one substitution, addition, or deletion in an immunogenic T-cell epitope selected from amino acids 16-24, 73-81, 258-266, 302-310, 373-381, or 447-455 of SEQ ID NO:1. In some embodiments, the variant sequence comprises at least one substitution, addition, or deletion at a position selected from (a) amino acid residue 74, 77, 78, or 80 of SEQ ID NO:1, (b) amino acid residue 262, 263, 264, or 266 of SEQ ID NO:1, (c) amino acid residue 303, 304, 306, or 310 of SEQ ID NO:1, (d) amino acid residue 374, 377, 378, 382, ​​or 383 of SEQ ID NO:1, and / or (e) amino acid residue 450, 451, 452, or 457 of SEQ ID NO:1. In some embodiments, the variant sequence comprises at least one substitution or combination of substitutions selected from those listed for Epitope B in Table 3. Additionally or alternatively, in some embodiments, the variant sequence comprises at least one substitution or combination of substitutions selected from those listed for Epitope 1 in Table 3. Additionally or alternatively, in some embodiments, the variant sequence comprises at least one substitution or combination of substitutions selected from those listed for epitope 2 in Table 3. Additionally or alternatively, in some embodiments, the variant sequence comprises at least one substitution or combination of substitutions selected from those listed for epitope 3 in Table 3. Additionally or alternatively, in some embodiments, the variant sequence comprises at least one substitution or combination of substitutions selected from those listed for epitope 4 in Table 3.In some embodiments, the variant comprises at least one substitution or combination of substitutions selected from: (a) I262T, (b) I262S, (c) I262A, (d) I262T and A264L, and / or (e) I262T and N263R.

[0014] Additionally or alternatively, in some embodiments, the variant may further comprise at least one substitution at amino acid position 74, 77, 78, 80, 262-266, 303, 304, 306, 310, 374, 377, 378, 382, ​​383, 450-452, or 457. For example, the variant may comprise a substitution at amino acid position 262 or 263, or the variant may comprise a substitution at amino acid positions 262 and 263. In some embodiments, the substitution may be I262A, I262T, and / or N263R. For example, in some embodiments, the variant sequence comprises the amino acid sequence of SEQ ID NO: 62 or 63.

[0015] In some embodiments, the nicotine degrading enzyme variant comprises an amino acid sequence that is a variant of the amino acid sequence of a wild-type NOX enzyme set forth in SEQ ID NO:57, wherein the variant sequence has at least one substitution, addition, or deletion to SEQ ID NO:57 that increases the nicotine degrading activity and / or decreases the immunogenicity of the variant compared to the wild-type NOX enzyme or wild-type NicA2.

[0016] In some embodiments, the variant of the wild-type NOX sequence comprises a substitution at position 423 of SEQ ID NO:57. For example, the variant sequence may comprise the substitution W423A, W423S, W423E, or W423H. In some embodiments, the variant sequence comprises an amino acid sequence selected from any one of SEQ ID NOs:58-61. In some embodiments, the nicotine degrading activity of the NOX variant is at least 200% of the nicotine degrading activity of the wild-type NicA2 enzyme. In some embodiments, the nicotine degrading activity of the NOX variant is at least 200% of the nicotine degrading activity of the wild-type NOX enzyme.

[0017] Additionally or alternatively, in some embodiments, the NOX variant exhibits reduced immunogenicity compared to wild-type NOX or NicA2 enzymes, in some embodiments, the immunogenicity compared to wild-type NOX or NicA2 enzymes is reduced by 75% or more.

[0018] Additionally or alternatively, in some embodiments, the variants further comprise a deletion of at least amino acids 1-38 of SEQ ID NO:1 or 57, or amino acids 1-50 of SEQ ID NO:1 or 57, or amino acids 1-51 of SEQ ID NO:1 or 57, or amino acids 1-52 of SEQ ID NO:1 or 57. In some embodiments, the variants may further comprise a His tag, such as a His tag comprising the amino acid sequence of SEQ ID NO: 139. Additionally or alternatively, in some embodiments, the variants comprise a deletion of one or more C-terminal amino acids, such as a deletion of a residue corresponding to S482 of the wild-type sequence.

[0019] In any of the embodiments, the variant may be a long-acting variant, such as the variant is conjugated to an albumin-binding peptide, an albumin-binding protein domain, human serum albumin, or an inert polypeptide, such as recombinant PEG (XTEN®), homoamino acid polymer (HAP), proline-alanine serine polymer (PAS), or elastin-like peptide (ELP). In certain embodiments, the long-acting variant is conjugated to polyethylene glycol (i.e., the variant is PEGylated).

[0020] In some embodiments, a pharmaceutical composition is provided that includes the nicotine degrading enzyme variant described herein and a pharma- ceutically acceptable carrier. In some embodiments, the composition is formulated for injection or infusion. In some embodiments, the composition is formulated for oral administration.

[0021] In some embodiments, a method for treating nicotine addiction or promoting smoking cessation is provided, comprising administering a therapeutically effective amount of the nicotine degrading enzyme variant or composition described herein to a mammalian subject in need thereof.In some embodiments, the mammalian subject is a human subject.In some embodiments, the nicotine addiction is associated with the intake of a nicotine product selected from a tobacco product and an e-cigarette.

[0022] In some embodiments, a method for treating nicotine addiction is provided, comprising administering a therapeutically effective amount of the nicotine degrading enzyme variant or composition described herein to a mammalian subject in need thereof.In some embodiments, the mammalian subject is a human subject, or more specifically, a human child.In some embodiments, the nicotine addiction is associated with the consumption of a nicotine product selected from tobacco products and e-cigarettes.

[0023] Also provided are the nicotine degrading enzyme variants and compositions described herein for use in treating nicotine addiction or promoting smoking cessation.

[0024] There is also provided the use of the nicotine degrading enzyme variants and compositions described herein in the manufacture of a medicament for the treatment of nicotine addiction or promoting smoking cessation.

[0025] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention. [Brief description of the drawings]

[0026] [Figure 1] SDS-PAGE analysis of wild-type NicA2 (SEQ ID NO: 1) and His-tagged versions of NicA2Δ50 (SEQ ID NO: 2) are shown. Lane 1: MW marker (Blue Plus2, Invitrogen), lane 2: wild-type NicA2 (SEQ ID NO: 1), and lane 3: NicA2Δ50 (SEQ ID NO: 2). [Diagram 2]1 shows the results of a nicotine degrading activity assay comparing wild-type NicA2 with mutant nicotine degrading enzymes described herein in the epitope. The assay used a final concentration of 80 nM purified protein. [Diagram 3] Residues around the active site in the NicA2 crystal structure are shown (adapted from Tararina et al., Biochem. 55:6595-98 (2016)). Shell 1 is shown in dark grey and shell 2 is shown in light grey. The residues that make up the first and second shells are shown in Table 2. [Figure 4A] The relative activities of specific NicA2 mutants with mutations in epitopes B (Figure 4E), 1 (Figure 4A), 2 (Figure 4B), 3 (Figure 4C), and 4 (Figure 4D) (as listed in Table 3) that were predicted to reduce immunogenic potential compared to wild-type NicA2 are shown. [Figure 4B] The relative activities of specific NicA2 mutants with mutations in epitopes B (Figure 4E), 1 (Figure 4A), 2 (Figure 4B), 3 (Figure 4C), and 4 (Figure 4D) (as listed in Table 3) that were predicted to reduce immunogenic potential compared to wild-type NicA2 are shown. [Figure 4C] The relative activities of specific NicA2 mutants with mutations in epitopes B (Figure 4E), 1 (Figure 4A), 2 (Figure 4B), 3 (Figure 4C), and 4 (Figure 4D) (as listed in Table 3) that were predicted to reduce immunogenic potential compared to wild-type NicA2 are shown. [Figure 4D] The relative activities of specific NicA2 mutants with mutations in epitopes B (Figure 4E), 1 (Figure 4A), 2 (Figure 4B), 3 (Figure 4C), and 4 (Figure 4D) (as listed in Table 3) that were predicted to reduce immunogenic potential compared to wild-type NicA2 are shown. [Figure 4E] The relative activities of specific NicA2 mutants with mutations in epitopes B (Figure 4E), 1 (Figure 4A), 2 (Figure 4B), 3 (Figure 4C), and 4 (Figure 4D) (as listed in Table 3) that were predicted to reduce immunogenic potential compared to wild-type NicA2 are shown. [Diagram 5]1 shows random PEGylation of NicA2 using SDS-PAGE analysis. These results indicate that PEGylation can be increased by a molar excess of the PEGylation reagent and by increasing the PEG chain length. [Figure 6] We show that PEGylation enhances the pharmacokinetic (PK) properties of NicA2 in the serum of animals administered PEGylated NicA2. [Figure 7] This shows that potential immunogenic epitopes on NicA2 can be masked by PEGylation. [Figure 8] We show that PEGylation can reduce NicA2-specific antibody titers in an immunogenic transgenic HLA-DR4 mouse model. PEGylation of NicA2 resulted in a significant (≧10-fold) reduction in the mean NicA2-specific antibody titers in transgenic DR4 mice (4, 2, and 2 animals from the NicA2-PEG1, -PEG2, and -PEG3 groups, respectively, had titers below the limit of detection (LOD)), suggesting the possibility of lower immunogenicity in a clinical setting. [Figure 9] Figure 2 shows that PEGylation attenuates human T cell proliferation responses and release of the cytokine TNFγ mediated by exposure to NicA2. PEGylation of NicA2 resulted in a significant decrease in the mean T cell proliferation stimulation index (left panel) as well as IFNγ secretion levels (right panel). An increase of ≥ 3 (dashed line) is considered a significant increase and a positive response. [Figure 10] We show that PEGylated NicA2 enzyme retains full nicotine degradation activity in serum. PEGylation did not interfere with the ability of NicA2 to degrade nicotine in rat serum. [Figure 11] 1 shows that a NicA2 mutant with an A107R substitution has increased activity in serum at low nicotine concentrations compared to wild-type NicA2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Described herein are nicotine degrading enzyme variants that exhibit increased nicotine degrading activity and / or decreased immunogenicity compared to wild-type NicA2 or NOX enzymes, compositions comprising them, and methods of using them, including methods for treating nicotine addiction and promoting nicotine cessation (e.g., smoking cessation) in a subject in need thereof.

[0028] I. Definition As used herein, the singular forms "a," "an," and "the" are used interchangeably and are intended to include the plural forms and apply to each meaning unless the context clearly indicates otherwise. Also, as used herein, "and / or" refers to and includes any and all possible permutations and combinations of one or more of the listed items.

[0029] As used herein, the term "about" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If there are uses of terms that are not clear to those of ordinary skill in the art, taking into account the context in which it is used, "about" will mean up to plus or minus 10% of the particular term.

[0030] As used herein, the phrases "therapeutically effective amount" and "therapeutic level" refer to the dosage or plasma concentration of nicotine degrading enzyme in a subject that provides a certain pharmacological effect when administered to a subject who needs such treatment, i.e., to degrade nicotine in the subject, and / or to treat nicotine addiction, and / or to promote smoking cessation, and / or to treat nicotine addiction.Even if such dosage is considered to be a therapeutically effective amount by those skilled in the art, it is emphasized that a therapeutically effective amount or therapeutic level of nicotine degrading enzyme is not always effective in treating nicotine addiction or promoting smoking cessation in a given subject.For the sake of convenience, exemplary amounts are provided below.

[0031] Those skilled in the art can adjust such amount according to standard practice as required to treat a particular subject.The therapeutically effective amount can vary based on the condition of the subject, including the route of administration and dosage form, the age and weight of the subject, and / or the degree of nicotine addiction, the amount of nicotine that is generally consumed / ingested by the subject, and / or the nicotine level in the plasma of the subject at the time of treatment, and / or the amount of nicotine that is localized in the brain at the time of treatment.

[0032] As used herein with respect to nicotine addiction or smoking cessation, the term "treatment" or "treating" refers to one or more of reducing, ameliorating, or eliminating one or more symptoms or effects of nicotine withdrawal, reducing the number of cigarettes or amount of nicotine consumed by a subject, and / or reducing plasma nicotine levels in a subject, and / or reducing the amount of nicotine localized in particular tissues of a subject (e.g., the brain / central nervous system, the heart and vascular system, etc.).

[0033] As used herein with respect to nicotine addiction, the term "treatment" or "treating" refers to reducing, ameliorating, or eliminating one or more symptoms or effects of nicotine, and / or reducing plasma nicotine levels in a subject, and / or reducing the amount of nicotine localized in particular tissues of a subject (e.g., the brain / central nervous system, heart and vascular system, etc.).

[0034] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any individual mammalian subject, for example, a cow, dog, cat, horse, or human.

[0035] As used herein, in accordance with FDA guidelines, a "child" refers to a human subject between the ages of 0 and about 19. A child may be a subject who begins a course of treatment before about age 19, even if the subject continues treatment beyond age 19.

[0036] II. Nicotine, Nicotine Addiction, and Nicotine Toxicity Nicotine is a nitrogen-containing chemical made by several plants, including tobacco and other members of the Solanaceae family. When humans, mammals, and most other animal species are exposed to nicotine, it increases heart rate, myocardial oxygen consumption, and stroke volume. Nicotine consumption is also associated with heightened alertness, euphoria, and feelings of relaxation. However, nicotine is highly addictive.

[0037] By binding to nicotinic acetylcholine receptors in the brain, nicotine elicits its psychoactive effects and increases the levels of several neurotransmitters in various brain structures. Nicotine exhibits a higher affinity for nicotinic receptors in the brain than in skeletal muscle, but in toxic amounts it can cause contractions and respiratory paralysis. The selectivity of nicotine is thought to be due to differences in certain amino acids for these receptor subtypes. The structure of nicotine is shown in Formula I below. [ka]

[0038] People who take nicotine regularly and then suddenly stop experience withdrawal symptoms that can include cravings, emptiness, anxiety, depression, irritability, and inattention. The American Heart Association states that nicotine (from smoking tobacco) is one of the most difficult substances to quit, at least as difficult as heroin.

[0039] Nicotine poisoning can occur when an individual ingests tobacco, cigarettes, nicotine gum, patches, or e-cigarette "e-liquid" (e.g., the nicotine-containing liquid used in e-cigarettes and other vaporizer devices) or other products containing tobacco or tobacco extracts, or other products, supplies, or intermediates that contain nicotine. Indeed, a recent study showed that the incidence of nicotine poisoning due to exposure to e-cigarettes increased by 1492.9% between January 2012 and April 2015 (Kamboj et al. PEDIATRICS 137(6):e20160041(2016)). Although exposure can occur through inhalation of tobacco smoke (either directly or indirectly), nicotine poisoning or nicotine overdose occurs more commonly when a subject (typically a child) ingests nicotine, for example, by chewing or ingesting nicotine gum, ingesting cigarettes or other tobacco leaf products, ingesting a nicotine patch, or ingesting an e-liquid. Additionally, nicotine can be absorbed through the skin, and therefore nicotine poisoning can result from direct contact of toxic levels of nicotine with the skin.

[0040] Nicotine poisoning can result in neurological (convulsions, coma, depression, confusion, fainting, headache), cardiovascular (rapid heart rate, high blood pressure), respiratory (difficulty breathing, rapid breathing), gastrointestinal (increased salivation, abdominal cramps, vomiting), and musculoskeletal (muscle cramps, weakness) symptoms, as well as death.

[0041] III. Nicotinase Mutants Described herein are nicotine degrading enzyme variants comprising an amino acid sequence that is a variant of the amino acid sequence of wild-type NicA2 or NOX enzyme as set forth in SEQ ID NO:1 or SEQ ID NO:57, respectively, wherein the variant sequence has at least one substitution, addition, or deletion relative to SEQ ID NO:1 or SEQ ID NO:57 that increases the nicotine degrading activity and / or decreases the immunogenicity of the variant compared to wild-type NicA2 or NOX enzyme, respectively.

[0042] NicA2 (nicotine oxidoreductase, PPS_4081, GenBank accession number: AEJ14620.1) was isolated from Pseudomonas putida strain S16. See, for example, Tang et.al., PLOS GENETICS, 9(10): e1003923 (2013). The activity of NicA2 is the first critical step in nicotine degradation by S16, catalyzing the oxidation of nicotine to N-methylmyosmine. It has been reported to be an essential enzyme in the Pseudomonas putida S16 metabolic cascade responsible for degrading nicotine. The structural analysis of the wild-type NicA2 enzyme has been reported in Tararina et al., Biochem. 55: 6595-98 (2016).

[0043] NOX (nicotine amine oxidase, GenBank accession number: AGH68979.1) was isolated from Pseudomonas sp. HZN6 (see, e.g., Qiu et al., Appl. Environ. Microbiol. 78, 2154-2160 (2012); Qiu et al., Appl. Environ. Microbiol. 79, 2164-2171 (2013)). NOX is closely related to NicA2, with 83% amino acid identity. NOX has been reported to have catalytic activity like NicA2 and to degrade nicotine to N-methylmyosmine.

[0044] The present disclosure provides mutants of wild-type NicA2 and NOX with improved activity and / or reduced immunogenicity. In some embodiments, the disclosed mutants can have about 80, about 85, about 90, about 95, about 96, about 97, about 98, or about 99 percent amino acid identity with wild-type NicA2 or NOX. In some embodiments, the disclosed mutants can share about 80, about 85, about 90, about 95, about 96, about 97, about 98, or about 99 percent homology with wild-type NicA2 or NOX. For example, in some embodiments, the disclosed mutants can include amino acid residues that are conserved between NicA2 and NOX.

[0045] The amino acid sequences of wild-type NicA2, wild-type NOX, and their exemplary mutants are listed in Table 1 below. The disclosed mutants were generated with a linker and His tag (GGGGSGSGHHHHHH, SEQ ID NO: 139) at the C-terminus, which was then removed. The His tag was used to aid in the purification of the mutants, but other purification means or methods that do not require a His tag can also be used. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14]

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

[0046] As mentioned above, nicotine degrading enzyme variants may show increased nicotine degrading activity and / or decreased immunogenicity compared to wild-type NicA2 enzyme. Variants may include one or more mutations to the amino acid sequence of wild-type NicA2, including one or more deletions, additions, or substitutions. Substitution mutations may be "conservative" or "non-conservative". "Conservative" refers to substitutions within the same amino acid family, and "non-conservative" refers to substitutions across the entire amino acid family. Families of amino acids and "conservative" and "non-conservative" substitutions therefor are known in the art. For example, naturally occurring amino acids can be divided into the following four families, and conservative substitutions occur within these families, while non-conservative substitutions occur across different families: 1) Amino acids with basic side chains: lysine, arginine, histidine. 2) Amino acids with acidic side chains: aspartic acid, glutamic acid 3) Amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine. 4) Amino acids with non-polar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.

[0047] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations in the active site of the wild-type NicA2 enzyme associated with its nicotine degrading activity, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions at the positions listed in Table 2 and shown in the structure of FIG. 3, such as amino acid residues 90-93, 95, 102-109, 113, 116, 130, 132, 138, 155, 159, 210, 213-215, 217-220, 234, 245, 246, 248-251, 253, 254, 258, 334, 336, 339-342, 353, 355, 363-367, 378-382, 415-418, 423-429, 459-463, 465, or 466 of SEQ ID NO:1. Shell 1 residues identified in Table 2 constitute the cavity surface, while shell 2 residues contact shell 1 (see FIG. 3). For example, in some embodiments, the disclosed nicotine degrading enzyme variants may contain at least one substitution at amino acid position 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO: 1. In some embodiments, variants may contain one, two, three or more substitutions. [Table 2-1] [Table 2-2]

[0048] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations relative to the wild-type NOX enzyme that are associated with its nicotine degrading activity, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, such as a mutation at amino acid residue 423 of SEQ ID NO: 57. For example, in some embodiments, the variant may comprise the substitutions W423A, W423S, W423E, or W423H.

[0049] In some embodiments, at least one mutation is introduced into the variant that increases the nicotine decomposition activity or increases the catalytic activity of the enzyme, allowing the variant to decompose nicotine more quickly and / or more efficiently. In some embodiments, such a mutation increases the k cat Increase in K M Decrease in k cat / K M Increase in and / or V max Thus, in some embodiments, the mutants may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations in the active site and / or aromatic cage of wild-type NicA2 or wild-type NOX enzymes, and may improve various measures of enzyme performance, including, but not limited to, an increase in k. cat Increase in KM, decrease in k cat / k M Increase in and / or V max These samples show increased nicotine degrading activity as measured by an increase in

[0050] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations in the aromatic cage of the wild-type NicA2 enzyme formed by the tryptophan at position 427 and the asparagine at position 462 of SEQ ID NO:1, such as a mutation at one or more of these positions, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 427 or 462 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 427 or 462 of SEQ ID NO:1.

[0051] In some embodiments, the mutation at position 427 is the substitution W427Q, where the tryptophan (W) at position 427 (of the aromatic cage) of SEQ ID NO:1 is replaced with a glutamine (Q). A variant having the amino acid sequence of SEQ ID NO:5 is an example of this type of variant. This is a non-conservative substitution in which a non-polar aromatic amino acid is replaced with a polar uncharged amino acid. Generally, non-conservative substitutions in the active site of an enzyme are expected to render the enzyme dysfunctional or ineffective, but surprisingly, this variant shows a significant increase in enzymatic degradation activity compared to the wild-type NicA2 enzyme.

[0052] In some embodiments, the mutation at position 427 is a substitution W427E, where the tryptophan (W) at position 427 of SEQ ID NO:1 is replaced with glutamic acid (E). A variant having an amino acid sequence of SEQ ID NO:6 is an example of this type of variant. In some embodiments, the mutation is a substitution W427S, where the tryptophan (W) at position 427 of SEQ ID NO:1 is replaced with serine (S). A variant having an amino acid sequence of SEQ ID NO:7 is an example of this type of variant. In some embodiments, the mutation is a substitution W427M, where the tryptophan (W) at position 427 of SEQ ID NO:1 is replaced with methionine (M). A variant having an amino acid sequence of SEQ ID NO:8 is an example of this type of variant. In some embodiments, the mutation is a substitution W427R, where the tryptophan (W) at position 427 of SEQ ID NO:1 is replaced with arginine (R). A variant having an amino acid sequence of SEQ ID NO:135 is an example of this type of variant. In some embodiments, the mutation is the substitution W427H, where the tryptophan (W) at position 427 of SEQ ID NO:1 is replaced with a histidine (H). A variant having the amino acid sequence of SEQ ID NO:137 is an example of this type of variant. In some embodiments, the mutation is the substitution W427M, where the tryptophan (W) at position 427 of SEQ ID NO:1 is replaced with a leucine (L). A variant having the amino acid sequence of SEQ ID NO:138 is an example of this type of variant.

[0053] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, including one or more conservative substitutions, non-conservative substitutions, additions, or deletions, at arginine (R) at position 91 of SEQ ID NO:1. Thus, in some embodiments, the mutations that increase nicotine degrading activity are at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as conservative substitutions, non-conservative substitutions, additions, or deletions at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0054] In some embodiments, the mutation at position 91 is a substitution R91A, where the arginine (R) at position 91 of SEQ ID NO:1 is replaced with an alanine (A). A variant having an amino acid sequence of SEQ ID NO:9 is an example of this type of variant. In some embodiments, the mutation at position 91 is a substitution R91Q, where the arginine (R) at position 91 of SEQ ID NO:1 is replaced with a glutamine (Q). A variant having an amino acid sequence of SEQ ID NO:10 and 129 is an example of this type of variant. In some embodiments, the mutation at position 91 is a substitution R91F, where the arginine (R) at position 91 of SEQ ID NO:1 is replaced with a phenylalanine (F). A variant having an amino acid sequence of SEQ ID NO:11 is an example of this type of variant. In some embodiments, the mutation at position 91 is a substitution R91G, where the arginine (R) at position 91 of SEQ ID NO:1 is replaced with a glycine (G). A variant having an amino acid sequence of SEQ ID NO:12 is an example of this type of variant. In some embodiments, the mutation at position 91 is a substitution R91T, where the arginine (R) at position 91 of SEQ ID NO:1 is replaced with a threonine (T). A variant having an amino acid sequence of SEQ ID NO:13 is an example of this type of variant. In some embodiments, the mutation at position 91 is a substitution R91L, where the arginine (R) at position 91 of SEQ ID NO:1 is replaced with a leucine (L). A variant having an amino acid sequence of SEQ ID NO:14 is an example of this type of variant. In some embodiments, the mutation at position 91 is a substitution R91S, where the arginine (R) at position 91 of SEQ ID NO:1 is replaced with a serine (S). A variant having an amino acid sequence of SEQ ID NO:15 is an example of this type of variant. In some embodiments, the mutation at position 91 is a substitution R91N, where the arginine (R) at position 91 of SEQ ID NO:1 is replaced with an asparagine (N). A variant having an amino acid sequence of SEQ ID NO:16 is an example of this type of variant.

[0055] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at threonine (T) at position 250 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0056] In some embodiments, the mutation at position 250 is the substitution T250G, where the threonine (T) at position 250 of SEQ ID NO:1 is replaced with a glycine (G). A variant having an amino acid sequence of SEQ ID NO:17 is an example of this type of variant. In some embodiments, the mutation at position 250 is the substitution T250L, where the threonine (T) at position 250 of SEQ ID NO:1 is replaced with a leucine (L). A variant having an amino acid sequence of SEQ ID NO:18 is an example of this type of variant. In some embodiments, the mutation at position 250 is the substitution T250R, where the threonine (T) at position 250 of SEQ ID NO:1 is replaced with an arginine (R). A variant having an amino acid sequence of SEQ ID NO:19 is an example of this type of variant. In some embodiments, the mutation at position 250 is the substitution T250V, where the threonine (T) at position 250 of SEQ ID NO:1 is replaced with a valine (V). A variant having an amino acid sequence of SEQ ID NO:20 is an example of this type of variant. In some embodiments, the mutation at position 250 is the substitution T250P, in which the threonine (T) at position 250 of SEQ ID NO:1 is replaced with a proline (P). A variant having the amino acid sequence of SEQ ID NO:136 is an example of this type of variant.

[0057] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at lysine (K) at position 340 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0058] In some embodiments, the mutation at position 340 is the substitution K340P, where the lysine (K) at position 340 of SEQ ID NO:1 is replaced with a proline (P). A variant having an amino acid sequence of SEQ ID NO:21 is an example of this type of variant. In some embodiments, the mutation at position 340 is the substitution K340I, where the lysine (K) at position 340 of SEQ ID NO:1 is replaced with an isoleucine (I). A variant having an amino acid sequence of SEQ ID NO:22 is an example of this type of variant. In some embodiments, the mutation at position 340 is the substitution K340V, where the lysine (K) at position 340 of SEQ ID NO:1 is replaced with a valine (V). A variant having an amino acid sequence of SEQ ID NO:23 is an example of this type of variant. In some embodiments, the mutation at position 340 is the substitution K340D, where the lysine (K) at position 340 of SEQ ID NO:1 is replaced with an aspartic acid (D). A variant having an amino acid sequence of SEQ ID NO:24 is an example of this type of variant. In some embodiments, the mutation at position 340 is the substitution K340E, in which the lysine (K) at position 340 of SEQ ID NO:1 is replaced with glutamic acid (E). A variant having the amino acid sequence of SEQ ID NO:25 is an example of this type of variant.

[0059] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at glutamine (Q) at position 366 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0060] In some embodiments, the mutation at position 366 is the substitution Q366K, where the glutamine (Q) at position 366 of SEQ ID NO:1 is replaced with a lysine (K). Variants having the amino acid sequences of SEQ ID NO:26 and 130 are examples of this type of variant. In some embodiments, the mutation at position 366 is the substitution Q366E, where the glutamine (Q) at position 366 of SEQ ID NO:1 is replaced with a glutamic acid (E). Variants having the amino acid sequence of SEQ ID NO:27 are examples of this type of variant. In some embodiments, the mutation at position 366 is the substitution Q366V, where the glutamine (Q) at position 366 of SEQ ID NO:1 is replaced with a valine (V). Variants having the amino acid sequence of SEQ ID NO:28 are examples of this type of variant. In some embodiments, the mutation at position 366 is the substitution Q366L, where the glutamine (Q) at position 366 of SEQ ID NO:1 is replaced with a leucine (L). Variants having the amino acid sequence of SEQ ID NO:29 are examples of this type of variant. In some embodiments, the mutation at position 366 is the substitution Q366I, where the glutamine (Q) at position 366 of SEQ ID NO:1 is replaced with an isoleucine (I). A variant having the amino acid sequence of SEQ ID NO:30 is an example of this type of variant. In some embodiments, the mutation at position 366 is the substitution Q366Y, where the glutamine (Q) at position 366 of SEQ ID NO:1 is replaced with a tyrosine (Y). A variant having the amino acid sequence of SEQ ID NO:31 is an example of this type of variant.

[0061] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at threonine (T) at position 381 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0062] In some embodiments, the mutation at position 381 is a substitution T381P, where the threonine (T) at position 381 of SEQ ID NO:1 is replaced with a proline (P). A variant having an amino acid sequence of SEQ ID NO:32 is an example of this type of variant. In some embodiments, the mutation at position 381 is a substitution T381I, where the threonine (T) at position 381 of SEQ ID NO:1 is replaced with an isoleucine (I). A variant having an amino acid sequence of SEQ ID NO:33 is an example of this type of variant. In some embodiments, the mutation at position 381 is a substitution T381V, where the threonine (T) at position 381 of SEQ ID NO:1 is replaced with a valine (V). A variant having an amino acid sequence of SEQ ID NO:34 and 131 is an example of this type of variant. In some embodiments, the mutation at position 381 is a substitution T381Q, where the threonine (T) at position 381 of SEQ ID NO:1 is replaced with a glutamine (Q). A variant having an amino acid sequence of SEQ ID NO:35 is an example of this type of variant. In some embodiments, the mutation at position 381 is a substitution T381N, where the threonine (T) at position 381 of SEQ ID NO:1 is replaced with an asparagine (N). A variant having an amino acid sequence of SEQ ID NO:36 is an example of this type of variant. In some embodiments, the mutation at position 381 is a substitution T381L, where the threonine (T) at position 381 of SEQ ID NO:1 is replaced with a leucine (L). A variant having an amino acid sequence of SEQ ID NO:37 is an example of this type of variant. In some embodiments, the mutation at position 381 is a substitution T381M, where the threonine (T) at position 381 of SEQ ID NO:1 is replaced with a methionine (M). A variant having an amino acid sequence of SEQ ID NO:38 is an example of this type of variant.

[0063] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at asparagine (N) at position 462 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0064] In some embodiments, the mutation at position 462 is the substitution N462L, where the asparagine (N) at position 462 of SEQ ID NO:1 is replaced with a leucine (L). Variants having the amino acid sequences of SEQ ID NO:39 and 132 are examples of this type of variant. In some embodiments, the mutation at position 462 is the substitution N462Y, where the asparagine (N) at position 462 of SEQ ID NO:1 is replaced with a tyrosine (Y). Variants having the amino acid sequences of SEQ ID NO:40 and 133 are examples of this type of variant. In some embodiments, the mutation at position 462 is the substitution N462S, where the asparagine (N) at position 462 of SEQ ID NO:1 is replaced with a serine (S). Variants having the amino acid sequence of SEQ ID NO:41 are examples of this type of variant. In some embodiments, the mutation at position 462 is the substitution N462F, where the asparagine (N) at position 462 of SEQ ID NO:1 is replaced with a phenylalanine (F). Variants having the amino acid sequences of SEQ ID NO: 42 and 137 are examples of this type of variant. In some embodiments, the mutation at position 462 is the substitution N462G, where the asparagine (N) at position 462 of SEQ ID NO: 1 is replaced with glycine (G). Variants having the amino acid sequence of SEQ ID NO: 43 are examples of this type of variant. In some embodiments, the mutation at position 462 is the substitution N462E, where the asparagine (N) at position 462 of SEQ ID NO: 1 is replaced with glutamic acid (E). Variants having the amino acid sequence of SEQ ID NO: 44 are examples of this type of variant. In some embodiments, the mutation at position 462 is the substitution N462A, where the asparagine (N) at position 462 of SEQ ID NO: 1 is replaced with alanine (A). Variants having the amino acid sequence of SEQ ID NO: 45 are examples of this type of variant. In some embodiments, the mutation at position 462 is the substitution N462M, where the asparagine (N) at position 462 of SEQ ID NO: 1 is replaced with methionine (M). The variant having the amino acid sequence of SEQ ID NO: 138 is an example of this type of variant.

[0065] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at isoleucine (I) at position 463 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0066] In some embodiments, the mutation at position 463 is the substitution I463F, where the isoleucine (I) at position 463 of SEQ ID NO:1 is replaced with a phenylalanine (F). A variant having an amino acid sequence of SEQ ID NO:46 is an example of this type of variant. In some embodiments, the mutation at position 463 is the substitution I463Y, where the isoleucine (I) at position 463 of SEQ ID NO:1 is replaced with a tyrosine (Y). A variant having an amino acid sequence of SEQ ID NO:47 is an example of this type of variant. In some embodiments, the mutation at position 463 is the substitution I463A, where the isoleucine (I) at position 463 of SEQ ID NO:1 is replaced with an alanine (A). A variant having an amino acid sequence of SEQ ID NO:48 is an example of this type of variant. In some embodiments, the mutation at position 463 is the substitution I463V, where the isoleucine (I) at position 463 of SEQ ID NO:1 is replaced with a valine (V). A variant having an amino acid sequence of SEQ ID NO:49 is an example of this type of variant. In some embodiments, the mutation at position 463 is the substitution I463L, where the isoleucine (I) at position 463 of SEQ ID NO:1 is replaced with a leucine (L). A variant having the amino acid sequence of SEQ ID NO:50 is an example of this type of variant.

[0067] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at leucine (L) at position 217 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 217, 250, 340, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0068] In some embodiments, the mutation at position 217 is the substitution L217Q, where the leucine (L) at position 217 of SEQ ID NO:1 is replaced with a glutamine (Q). A variant having an amino acid sequence of SEQ ID NO:51 is an example of this type of variant. In some embodiments, the mutation at position 217 is the substitution L217G, where the leucine (L) at position 217 of SEQ ID NO:1 is replaced with a glycine (G). A variant having an amino acid sequence of SEQ ID NO:52 is an example of this type of variant. In some embodiments, the mutation at position 217 is the substitution L217E, where the leucine (L) at position 217 of SEQ ID NO:1 is replaced with a glutamic acid (E). A variant having an amino acid sequence of SEQ ID NO:53 is an example of this type of variant. In some embodiments, the mutation at position 217 is the substitution L217I, where the leucine (L) at position 217 of SEQ ID NO:1 is replaced with an isoleucine (I). A variant having an amino acid sequence of SEQ ID NO:54 is an example of this type of variant. In some embodiments, the mutation at position 217 is the substitution L217C, where the leucine (L) at position 217 of SEQ ID NO:1 is replaced with a cysteine ​​(C). A variant having the amino acid sequence of SEQ ID NO:55 is an example of this type of variant. In some embodiments, the mutation at position 217 is the substitution L217S, where the leucine (L) at position 217 of SEQ ID NO:1 is replaced with a serine (S). A variant having the amino acid sequence of SEQ ID NO:56 is an example of this type of variant.

[0069] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at alanine (A) at position 107 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0070] In some embodiments, the mutation at position 107 is the substitution A107R, where the alanine (A) at position 107 of SEQ ID NO:1 is replaced with arginine (R). A variant having an amino acid sequence of SEQ ID NO:124 or 134 is an example of this type of variant. In some embodiments, the mutation at position 107 is the substitution A107K, where the alanine (A) at position 107 of SEQ ID NO:1 is replaced with lysine (K). A variant having an amino acid sequence of SEQ ID NO:125 is an example of this type of variant. In some embodiments, the mutation at position 107 is the substitution A107T, where the alanine (A) at position 107 of SEQ ID NO:1 is replaced with tyrosine (T). A variant having an amino acid sequence of SEQ ID NO:126 is an example of this type of variant. In some embodiments, the mutation at position 107 is the substitution A107H, where the alanine (A) at position 107 of SEQ ID NO:1 is replaced with histidine (H). A variant having an amino acid sequence of SEQ ID NO:142 is an example of this type of variant. In some embodiments, the mutation at position 107 is the substitution A107P, where an alanine (A) at position 107 of SEQ ID NO:1 is replaced with a proline (P). A variant having the amino acid sequence of SEQ ID NO:143 is an example of this type of variant.

[0071] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at phenylalanine (F) at position 355 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0072] In some embodiments, the mutation at position 355 is the substitution F355C, where the phenylalanine (F) at position 355 of SEQ ID NO:1 is replaced with a cysteine ​​(C). A variant having the amino acid sequence of SEQ ID NO:127 is an example of this type of variant. In some embodiments, the mutation at position 355 is the substitution F355V, where the phenylalanine (F) at position 355 of SEQ ID NO:1 is replaced with a valine (V). A variant having the amino acid sequence of SEQ ID NO:128 is an example of this type of variant.

[0073] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at phenylalanine (F) at position 104 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0074] In some embodiments, the mutation at position 104 is the substitution F104L, where the phenylalanine (F) at position 104 of SEQ ID NO:1 is replaced with leucine (L). A variant having the amino acid sequence of SEQ ID NO:140 is an example of this type of variant.

[0075] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at glycine (G) at position 106 of SEQ ID NO:1. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, or 463 of SEQ ID NO:1.

[0076] In some embodiments, the mutation at position 106 is the substitution G106S, in which a glycine (G) at position 106 of SEQ ID NO:1 is replaced with a serine (S). A variant having the amino acid sequence of SEQ ID NO:141 is an example of this type of variant.

[0077] In some embodiments, the nicotine degrading enzyme variant comprises one, two, or more mutations relative to SEQ ID NO: 1. For example, in some embodiments, the nicotine degrading enzyme may comprise a mutation at tryptophan (W) at position 427, a mutation at isoleucine (I) at position 262, and a mutation at asparagine (N) at position 263 of SEQ ID NO: 1, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions. Thus, in some embodiments, at least one mutation that increases nicotine degrading activity is at one, two, or more of positions 107, 355, 262, 263, 217, 91, 463, 381, 366, 340, 250, 427, or 462 of SEQ ID NO:1, such as a conservative substitution, a non-conservative substitution, an addition, or a deletion at one or more of positions 107, 355, 262, 263, 217, 91, 463, 381, 366, 340, 250, 427, or 462 of SEQ ID NO:1.

[0078] In some aspects, the mutation is a disubstitution of W427Q and I262A, such as when the tryptophan (W) at position 427 of SEQ ID NO:1 is replaced with glutamine (Q) and the isoleucine (I) at position 262 of SEQ ID NO:1 is replaced with alanine (A). A variant having an amino acid sequence of SEQ ID NO:62 is an example of this type of variant. In some embodiments, the mutation is a disubstitution of W427H and N462F, such as when the tryptophan (W) at position 427 of SEQ ID NO:1 is replaced with histidine (H) and the asparagine (N) at position 462 of SEQ ID NO:1 is replaced with phenylalanine (F). A variant having an amino acid sequence of SEQ ID NO:137 is an example of this type of variant. In some embodiments, the mutation is a disubstitution of W427L and N462M, such as when the tryptophan (W) at position 427 of SEQ ID NO:1 is replaced with leucine (L) and the asparagine (N) at position 462 of SEQ ID NO:1 is replaced with methionine (M). A variant having an amino acid sequence of SEQ ID NO: 138 is an example of this type of variant. In some embodiments, the mutation is a tri-substitution of W427Q, I262T, and N263R, such as where tryptophan (W) at position 427 of SEQ ID NO: 1 is substituted with glutamine (Q), isoleucine (I) at position 262 of SEQ ID NO: 1 is substituted with threonine (T), and asparagine (N) at position 263 of SEQ ID NO: 1 is substituted with arginine (R). A variant having an amino acid sequence of SEQ ID NO: 63 is an example of this type of variant.

[0079] In some embodiments, the nicotine degrading enzyme variant comprises one or more mutations, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions, including a mutation at tryptophan (W) at position 423 of SEQ ID NO: 57. Thus, in some embodiments, the mutation that increases the nicotine degrading activity is at one or more of positions including 423 of SEQ ID NO: 57, such as a conservative substitution, non-conservative substitution, addition, or deletion at one or more of positions 423 of SEQ ID NO: 57.

[0080] In some embodiments, the mutation at position 423 is a substitution W423A, where the tryptophan (W) at position 423 of SEQ ID NO:57 is replaced with an alanine (A). A variant having an amino acid sequence of SEQ ID NO:58 is an example of this type of variant. In some embodiments, the mutation at position 423 is a substitution W423S, where the tryptophan (W) at position 423 of SEQ ID NO:57 is replaced with a serine (S). A variant having an amino acid sequence of SEQ ID NO:59 is an example of this type of variant. In some embodiments, the mutation at position 423 is a substitution W423E, where the tryptophan (W) at position 423 of SEQ ID NO:57 is replaced with a glutamic acid (E). A variant having an amino acid sequence of SEQ ID NO:60 is an example of this type of variant. In some embodiments, the mutation at position 423 is a substitution W423H, where the tryptophan (W) at position 423 of SEQ ID NO:57 is replaced with a histidine (H). A variant having an amino acid sequence of SEQ ID NO:61 is an example of this type of variant.

[0081] Additionally or alternatively, in some embodiments the nicotine degrading enzyme variant comprises one or more mutations in an immunogenic T cell epitope, such as one or more mutations in an immunogenic T cell epitope selected from positions 16-24, 73-81, 258-266, 302-310, 373-381, or 447-455 of SEQ ID NO:1, such as one or more conservative substitutions, non-conservative substitutions, additions, or deletions in one or more of these regions. Thus, in some embodiments, the variants may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations in 1, 2, 3, 4, 5, 6, or 7 immunogenic T cell epitopes, in some embodiments, such variants exhibit reduced immunogenicity when administered to a mammalian subject.

[0082] In some embodiments, the nicotinic enzyme variants include a mutation in an immunogenic T cell epitope at one or more positions selected from 74, 77, 78, 80, 262-266, 303, 304, 306, 310, 374, 377, 378, 382, ​​383, 450-452, or 457 of SEQ ID NO:1, including all sequences and combinations thereof. For example, the variants may include any one or more of the mutations described below, including one or more of the exemplary mutations in epitope B, one or more of the exemplary mutations in epitope 1, one or more of the exemplary mutations in epitope 2, one or more of the exemplary mutations in epitope 3, and / or one or more of the exemplary mutations in epitope 4. For example, in some embodiments, the nicotinic enzyme may have an amino acid substitution at positions 262 and / or 263 of SEQ ID NO:1, such as an I262A substitution or an I262T / N263R substitution. [Table 3]

[0083] Additionally or alternatively, in some embodiments the nicotine degrading enzyme variant comprises an N-terminal deletion of 1-52 amino acid residues of SEQ ID NO:1 or SEQ ID NO:57. For example, in some embodiments the variant comprises an N-terminal deletion of amino acid residues 1-16, 1-25, 1-38, 1-50, 1-51, or 1-52 of SEQ ID NO:1 or SEQ ID NO:57. Thus, the disclosed variants can include an N-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 consecutive amino acids.

[0084] In some embodiments, the disclosed variant may additionally or alternatively comprise a deletion at the C-terminus of peptide.For example, the disclosed variant may comprise one or more amino acid deletions at the C-terminus of peptide.For example, the NicA2 variant may delete the amino acid corresponding to S482 of wild-type sequence.

[0085] In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the wild-type NicA2 enzyme (SEQ ID NO:1), or an N-terminal deletion variant thereof having a deletion of up to 52 N-terminal amino acid residues of SEQ ID NO:1.

[0086] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 5. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:5.

[0087] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 6. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 6.

[0088] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 7. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 7.

[0089] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 8. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:8.

[0090] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 9. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:9.

[0091] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 10. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 10.

[0092] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 11. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 11.

[0093] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 12. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 12.

[0094] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 13. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 13.

[0095] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 14. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 14.

[0096] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 15. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 15.

[0097] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 16. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 16.

[0098] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 17. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 17.

[0099] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 18. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 18.

[0100] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 19. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 19.

[0101] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 20. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 20.

[0102] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 21. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 21.

[0103] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 22. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:22.

[0104] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 23. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:23.

[0105] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 24. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:24.

[0106] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 25. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:25.

[0107] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 26. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:26.

[0108] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 27. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 27.

[0109] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 28. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:28.

[0110] In some embodiments, the nicotine degrading enzyme variants described herein are or comprise SEQ ID NO: 29. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 29.

[0111] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 30. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 30.

[0112] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 31. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 31.

[0113] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 32. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 32.

[0114] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 33. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:33.

[0115] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 34. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:34.

[0116] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 35. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:35.

[0117] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 36. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:36.

[0118] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 37. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 37.

[0119] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 38. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:38.

[0120] In some embodiments, the nicotine degrading enzyme variants described herein are or comprise SEQ ID NO: 39. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:39.

[0121] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 40. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 40.

[0122] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 41. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 41.

[0123] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 42. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 42.

[0124] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 43. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 43.

[0125] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 44. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 44.

[0126] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 45. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 45.

[0127] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 46. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 46.

[0128] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 47. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 47.

[0129] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 48. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 48.

[0130] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 49. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 49.

[0131] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 50. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:50.

[0132] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 51. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:51.

[0133] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 52. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:52.

[0134] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 53. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:53.

[0135] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 54. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:54.

[0136] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 55. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:55.

[0137] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 56. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:56.

[0138] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 57. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:57.

[0139] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 58. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:58.

[0140] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 59. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:59.

[0141] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 60. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:60.

[0142] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 61. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 61.

[0143] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 62. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:62.

[0144] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 63. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO:63.

[0145] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 124. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 124.

[0146] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 125. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 125.

[0147] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 126. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 126.

[0148] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 127. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 127.

[0149] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 128. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 128.

[0150] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 129. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 129.

[0151] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 130. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 130.

[0152] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 131. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 131.

[0153] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 132. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 132.

[0154] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 133. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 133.

[0155] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 134. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 134.

[0156] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 135. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 135.

[0157] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 136. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 136.

[0158] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 137. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 137.

[0159] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 138. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 138.

[0160] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 140. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 140.

[0161] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 141. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 141.

[0162] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 142. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 142.

[0163] In some embodiments, the nicotine degrading enzyme variants described herein are or include SEQ ID NO: 143. In some embodiments, the nicotine degrading enzyme variants described herein have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the variant sequence of SEQ ID NO: 143.

[0164] In some embodiments, the variants described herein are assayed using the AMPLEX® Red assay (Thermo Fisher Scientific). and the like, such that the activity is at least about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400% greater than the activity of the wild-type NicA2 or NOX enzyme. ,approx. 410%,approx. 420%,approx. 430%,approx. 440%,approx. 450%,approx. 460%,approx. 470%,approx. 480%,approx. 490%,approx. 500%,approx. 550%,approx. 600%,approx. 650%,approx. 700%,approx. 750%,approx. 800%,approx. 850%,approx. 900%,approx. 950%,approx. 1000%,approx. 1100%,approx. 1200%,approx. 1300% , about 1400%, about 1500%, about 1600%, about 1700%, about 1800%, about 1900%, about 2000%, about 2250%, about 2500%, about 2750%, about 3000%, about 3250%, about 3500%, about 3750%, about 4000%, about 4250%, about 4500%, about 4750%, or about 5000% or more.

[0165] In some embodiments, the variants described herein are incubated with a fixed concentration of enzyme at 37° C. in either buffer or rat serum, and activity is quenched at fixed time points by mixing with MeOH, followed by analysis by gas chromatography (GC; Hieda et al.: Immunization of rats reduces nicotine distribution to brain. Psychopharmacology, 143, 150-157, 1999) to measure residual nicotine concentrations, such that the activity is at least about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360% greater than the activity of wild-type NicA2 or NOX enzyme. ,approximately 370%,approximately 380%,approximately 390%,approximately 400%,approximately 410%,approximately 420%,approximately 430%,approximately 440%,approximately 450%,approximately 460%,approximately 470%,approximately 480%,approximately 490%,approximately 500%,approximately 550%,approximately 600%,approximately 650%,approximately 700%,approximately 750%,approximately 800%,approximately 850%,approximately 900%,approximately 950%,approximately 1000%,approximately 1100%, about 1200%, about 1300%, about 1400%, 1500%, about 1600%, about 1700%, about 1800%, about 1900%, about 2000%, about 2250%, about 2500%, about 2750%, about 3000%, about 3250%, about 3500%, about 3750, about 4000%, about 4250%, about 4500%, about 4750%, or about 5000% or more.

[0166] In some embodiments, the variants described herein exhibit reduced immunogenicity in mammalian subjects compared to wild-type NicA2 or NOX, such that they are at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% less immunogenic than wild-type NicA2 or NOX enzymes. Unless otherwise specified, "reduced immunogenicity" as used herein compared to wild-type NicA2 or NOX enzymes refers to reduced immunogenicity as demonstrated by one or more of in silico approaches, in vitro assays, in vivo studies (e.g., using genetically modified animals), ex vivo studies using human T cells, or clinical studies using human subjects.

[0167] IV. Pharmaceutical Compositions The nicotine degrading enzyme variants disclosed herein can be formulated into pharmaceutical compositions suitable for administration to a target subject (i.e., a human or other mammal) via a predetermined route of administration, as discussed in more detail below.

[0168] A pharmaceutical composition may include one or more of the variants described herein and a pharma- ceutically acceptable carrier or diluent.

[0169] The composition may be formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, oral, nasal, pulmonary, ocular, vaginal, or rectal administration. In some embodiments, the composition is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration, such as a solution, suspension, emulsion, liposomal formulation, etc. The pharmaceutical composition may be formulated to be an immediate release composition, a sustained release composition, a delayed release composition, etc., using techniques known in the art.

[0170] Pharmaceutically acceptable carriers for various dosage forms are known in the art.For example, excipients, lubricants, binders and disintegrants for solid formulations are known, and solvents, solubilizers, suspending agents, isotonicity agents, buffers and soothing agents for liquid formulations are known.In some embodiments, pharmaceutical compositions contain one or more additional components such as one or more preservatives, antioxidants, coloring agents, sweeteners / flavoring agents, adsorbents, wetting agents, etc.

[0171] In some embodiments, the compositions are formulated for administration by injection or infusion, hi some embodiments, the compositions are formulated for oral administration.

[0172] In some embodiments, the nicotinic enzyme variant is a long-acting variant that has been modified to extend its half-life in vivo (after administration). Various techniques for extending the circulating half-life of peptides are known in the art. For example, in some embodiments, the variant is conjugated to polyethylene glycol (PEG) or a similar polymer that extends half-life. As discussed in more detail in Example 3 below, conjugation of PEG to the disclosed nicotinic enzyme variant can improve the pharmacokinetic properties of the variant. In some embodiments, PEGylation has one or more effects selected from masking one or more immunogenic epitopes of the variant, reducing variant-specific antibody titers, and attenuating T cell proliferation and / or cytokine responses. Additionally or alternatively, in some embodiments, conjugation of the variant to PEG does not reduce or significantly reduce or eliminate the enzymatic activity of the nicotinic enzyme variant.

[0173] As illustrated in the Examples below, the length and structure of the PEG chain (i.e., linear vs. branched) can be selected and varied to affect, impart or promote different properties. PEG can be conjugated to the variants by known methods for conjugating PEG to proteins, including those illustrated in the Examples below. Any of the variants described herein can be PEGylated, including variants defined by or including any of SEQ ID NOs: 2-56, 58-63, or 124-134. For purposes of conjugating PEG to the disclosed enzyme variants, the size or length of the PEG polymer can be varied. For example, linear PEG conjugated to the disclosed enzyme variants can range from 1 to 50 kDa, 5 to 40 kDa, or 10 to 20 kDa, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 kDa. In addition, the PEG polymer may be branched, ranging in size from 20 to 80 kDa, such as 20, 40, 60, or 80 kDa.

[0174] In some embodiments, the variant is fused with an albumin-binding peptide, an albumin-binding protein domain, human serum albumin, or an inactive polypeptide. Exemplary inactive polypeptides that have been used to increase the circulating half-life of peptides include, but are not limited to, XTEN® (also known as recombinant PEG or "rPEG"), homoamino acid polymer (HAP, HAP-modified), proline-alanine serine polymer (PAS, PAS-modified), or elastin-like peptide (ELP, ELP-modified). As used herein, "fused to" includes direct or linker-mediated genetic fusion, resulting in a single polypeptide that includes multiple domains, unless otherwise specified.

[0175] V. Methods for Treating Nicotine Addiction or Promoting Smoking Cessation As noted above, the variants described herein are useful in methods of treating nicotine addiction and / or promoting smoking cessation (or cessation of use of other tobacco products) or preventing relapse to smoking (or consumption of other tobacco products) in a mammalian subject in need thereof. (For convenience, in the following discussion, these methods are collectively referred to as treating nicotine addiction or promoting smoking cessation.) In some embodiments, the subject is a human subject who is addicted to nicotine or who desires to quit smoking or maintain abstinence from smoking or consumption of other nicotine products, or to prevent relapse to smoking or consumption of other nicotine products.

[0176] The method generally involves administering to a subject a therapeutically effective amount of the nicotine degrading enzyme variants described herein (or pharmaceutical compositions comprising them). However, in some embodiments, the method involves administering a nucleic acid encoding the nicotine degrading enzyme variant in a construct that expresses the variant in vivo. For example, in such embodiments, the nucleic acid may be provided in a suitable vector, such as an adeno-associated virus (AAV) gene transfer vector. Other exemplary vectors suitable for use in such methods are known in the art. See, for example, Lukashev and Zamyatnin, Biochem., 81(7):700-8 (2016). Exemplary vectors may include one or more enhancers (e.g., cytomegalovirus (CMV) enhancer), promoters (e.g., chicken β-actin promoter), and / or other elements that enhance the properties of the expression cassette. Methods of making suitable vectors and general methods of using expression vectors in vivo are known in the art. See, e.g., (Hicks et al., Sci. Transl. Med., 4(140):140ra87 (2012)).

[0177] In some embodiments, the subject in need of treatment for nicotine addiction or smoking cessation promotion is a human subject who consumes nicotine products, such as smoking cigarettes, chewing tobacco, e-cigarettes, and / or other nicotine delivery devices. Such subjects may or may not be physically addicted to nicotine and / or psychologically addicted to consuming nicotine products. A typical subject in need of smoking cessation treatment smokes or uses tobacco or other nicotine products daily, such as smoking at least one or more cigarettes per day, such as at least about 5, at least about 10, at least about 15, at least about 20 or more cigarettes per day, including less than 10, 10-20, 20-30, 30-40, or 40 or more cigarettes (or equivalent use of other tobacco products or nicotine products).

[0178] In some embodiments, the therapeutically effective amount of the nicotine degrading enzyme variant is an amount effective to reduce plasma nicotine levels, reduce localized nicotine levels in the brain, or both.

[0179] Nicotine exerts many of its important effects after passing through the blood-brain barrier. In some embodiments, the methods and uses described herein reduce or prevent nicotine from passing through the blood-brain barrier. Thus, in some embodiments, administration of the nicotine degrading enzyme variants described herein degrades nicotine circulating in the subject's bloodstream, thereby reducing or preventing nicotine from passing through the blood-brain barrier. Thus, in some embodiments, the methods described herein reduce or prevent the physiological and psychological effects of nicotine from the brain. As the subject experiences a reduction or cessation of these effects, he / she will lose the desire to consume nicotine products. Additionally or alternatively, the nicotine degrading enzyme variants disclosed herein may exert their effects by affecting the ability of nicotine to stimulate the peripheral nervous system.

[0180] The specific amount of nicotinic enzyme administered may depend on one or more of the subject's age and / or weight, the amount of nicotine regularly ingested (e.g., smoked, chewed, or inhaled), and / or the nicotine levels in the subject's brain or plasma at the time of treatment. In some embodiments, the variant is administered at a dose of about 0.01 to about 20 mg / kg, about 0.1 mg / kg to about 18 mg / kg, about 1 mg / kg to about 16 mg / kg, about 2 mg / kg to about 14 mg / kg, or about 5 mg / kg to about 10 mg / kg. In some embodiments, the variant is administered at a concentration of about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about The compound is administered at a dose of about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, about 10 mg / kg, about 10.5 mg / kg, about 12 mg / kg, about 12.5 mg / kg, about 13 mg / kg, about 13.5 mg / kg, about 14 mg / kg, about 14.5 mg / kg, about 15 mg / kg, about 15.5 mg / kg, about 16 mg / kg, about 16.5 mg / kg, about 17 mg / kg, about 17.5 mg / kg, about 18 mg / kg, about 18.5 mg / kg, about 19 mg / kg, about 19.5 mg / kg, or about 20 mg / kg.In some embodiments, the variant may be administered at a concentration of about 0.5 mg, about 1 mg, about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600, about 650 mg, 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 250 mg, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg, about 3000 mg, about 3100 mg, about 3200 mg, about 3300 mg, about 3400 mg, about 350 mg, about 3600 mg, about 3700 mg, about 3800 mg, about 3900 mg, about 4000 mg, about 4000 mg, about 450 mg, about 4000 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, The variant may be administered in a dose of about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, about 2000 mg, about 2050 mg, about 2100, about 2150 mg, about 2200 mg, about 2250 mg, about 2300 mg, about 2350 mg, about 2400 mg, about 2450 mg, or about 2500 mg. If more than one variant is administered, the total amount of variant administered may follow the guidelines set forth above.

[0181] In some embodiments, the method comprises administering a single dose of the nicotine degrading enzyme variant(s) (or a composition comprising same). In some embodiments, the method comprises administering repeated doses, such as for a predetermined period of time until the symptoms or effects of nicotine addiction are reduced, ameliorated, or eliminated, or until the subject stops smoking or otherwise ingesting nicotine. In some embodiments, if the signs / symptoms / effects persist, or if the subject continues to experience or is newly experiencing nicotine cravings, treatment is repeated with additional doses of the variant(s).

[0182] In some embodiments, the method comprises administering the nicotine degrading enzyme variant(s) (or a composition comprising same) three or more times a day, twice a day, or once a day. In some embodiments, the method comprises administering the nicotine degrading enzyme variant(s) (or a composition comprising same) once every other day, three times a week, twice a week, once a week, once every other week, once every three weeks, once a month, or less frequently. In such embodiments, the nicotine degrading enzyme variant may be a long-acting nicotine degrading enzyme variant as described above.

[0183] In some embodiments, treatment may continue for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days or more, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, or 1, 2, or 3 years or more, or until the subject no longer experiences nicotine cravings or other nicotine withdrawal symptoms, or has stopped smoking or using other tobacco products.

[0184] VI. Methods for Treating Nicotine Addiction The disclosed nicotine degrading enzyme variants can be used to treat nicotine addiction, nicotine overdose, or nicotine toxicity. For convenience, these methods are collectively referred to herein as treating nicotine addiction. In some aspects, the methods of treating nicotine addiction described herein include administering the nicotine degrading enzyme variants disclosed herein or pharmaceutical compositions comprising the same to a mammalian subject in need thereof. In some embodiments, the methods include administering the nicotine degrading enzyme variant to a subject who has ingested or consumed a toxic amount of nicotine. In some embodiments, the methods may include administering both the nicotine degrading enzyme variant and another compound useful for treating nicotine addiction, such as activated charcoal or other agents. In such embodiments, the enzyme variant and the second compound (e.g., activated charcoal) can be administered sequentially or simultaneously from the same or different compositions. Thus, the treatment may include administering activated charcoal and / or other supportive therapies to address the symptoms and / or effects of nicotine addiction.

[0185] In some embodiments, the therapeutically effective amount of the nicotine degrading enzyme variant is effective to reduce, ameliorate, or eliminate one or more symptoms or effects of nicotine addiction or overdose. The specific amount administered may depend on one or more of the following: the age and / or weight of the subject, the amount of nicotine believed to have been ingested, and / or the nicotine level in the subject's plasma at the time of treatment, and / or the nicotine level in the subject's brain at the time of treatment. In some embodiments, the subject being treated for nicotine addiction is an adult, and in some embodiments, the subject is a child (i.e., under 19 years of age). In some embodiments, the therapeutically effective amount of the nicotine degrading enzyme variant is an amount effective to reduce the nicotine level in the plasma and / or to reduce the amount of nicotine localized in a particular tissue of the subject (e.g., brain / central nervous system, heart and vascular system, etc.). In certain embodiments, the therapeutically effective amount of the nicotine degrading enzyme variant is an amount effective to reduce the nicotine level in the plasma, to reduce the nicotine level localized in the brain, or both.

[0186] The specific amount of nicotinase administered may depend on one or more of the subject's age and / or weight, the amount of nicotine acutely ingested, and / or the nicotine levels in the subject's brain or plasma at the time of treatment. In some embodiments, the variant is administered at a dose of about 0.01 to about 20 mg / kg, about 0.1 mg / kg to about 18 mg / kg, about 1 mg / kg to about 16 mg / kg, about 2 mg / kg to about 14 mg / kg, or about 5 mg / kg to about 10 mg / kg. In some embodiments, the variant is administered at a concentration of about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about The compound is administered at a dose of about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, about 10 mg / kg, about 10.5 mg / kg, about 12 mg / kg, about 12.5 mg / kg, about 13 mg / kg, about 13.5 mg / kg, about 14 mg / kg, about 14.5 mg / kg, about 15 mg / kg, about 15.5 mg / kg, about 16 mg / kg, about 16.5 mg / kg, about 17 mg / kg, about 17.5 mg / kg, about 18 mg / kg, about 18.5 mg / kg, about 19 mg / kg, about 19.5 mg / kg, or about 20 mg / kg.In some embodiments, the variant may be administered at a concentration of about 0.5 mg, about 1 mg, about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600, about 650 mg, 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 250 mg, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg, about 3000 mg, about 3100 mg, about 3200 mg, about 3300 mg, about 3400 mg, about 350 mg, about 3600 mg, about 3700 mg, about 3800 mg, about 3900 mg, about 4000 mg, about 4000 mg, about 450 mg, about 4000 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, The variant may be administered in a dose of about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, about 2000 mg, about 2050 mg, about 2100, about 2150 mg, about 2200 mg, about 2250 mg, about 2300 mg, about 2350 mg, about 2400 mg, about 2450 mg, or about 2500 mg. If more than one variant is administered, the total amount of variant administered may follow the guidelines set forth above.

[0187] Since nicotine addiction is associated with vomiting, parenteral administration route may be used.In addition, intravenous administration may be more effective than intraperitoneal administration.Therefore, in some embodiments of the method for treating nicotine addiction, the nicotine degrading enzyme variant(s) (or the composition comprising it) is administered intravenously.

[0188] In some embodiments, the method involves administering a single dose of the nicotine degrading enzyme variant(s) (or a composition comprising same). In some embodiments, the method involves administering repeated doses, such as for a predetermined period of time or until symptoms or effects of nicotine addiction or toxicity are reduced, ameliorated, or eliminated, or until the subject has stopped smoking or otherwise ingesting nicotine. In some embodiments, if signs / symptoms / effects persist, treatment is repeated with additional doses of the variant(s).

[0189] In some embodiments, treatment may continue for more than one day after the overdose, such as 1-3 days, or 1-5 days, or 1, 2, 3, 4, or 5 days after the overdose. In some embodiments, treatment may continue until the subject no longer experiences any symptoms of nicotine intoxication or toxicity, or until the nicotine levels in the subject's plasma and / or brain have fallen to a sufficiently safe level. In some embodiments, the nicotinic enzyme variant may be a long-acting nicotinic enzyme variant as described above.

[0190] Those skilled in the art will readily recognize that the present disclosure is well adapted to carry out the objects and obtain the objects and advantages mentioned, as well as those inherent therein. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the present disclosure. The following examples are given to illustrate the present invention. However, it should be understood that the present invention is not limited to the specific conditions or details of these examples. EXAMPLES

[0191] Example 1 - Development and testing of nicotine degrading enzyme variants A synthetic gene (custom DNA synthesis by GeneArt / Invitrogen) codon optimized for E. coli expression of wild-type (wt) NicA2 amino acid sequence (GenBank accession number: AEJ14620.1) with a C-terminal His6-tag was cloned into the NdeI-XhoI sites of pET-22b(+) (Novagen) and the expression plasmid was transformed into E. coli BL21(DE3). The predicted wild-type NicA2 amino acid sequence expressed from this construct is shown in Table 1 (SEQ ID NO:1).

[0192] Size heterogeneity was identified by SDS-PAGE of protein expressed from a construct encoding SEQ ID NO:1 and purified by immobilized metal affinity chromatography (IMAC) using Cobalt TALON™ His-Tag purification resin (Clontech) according to the manufacturer's protocol (Figure 1, major band around the 49 kDa marker and minor band). Since the protein was purified via the His tag, it was speculated that the heterogeneity was at the N-terminus. The online search tool PRED-TAT (compgen.org / tools / PRED-TAT; Pantelis et al., Combined prediction of Tat and Sec signal peptides with Hidden Markov Models, 2010 BIOINFORMATICS) was utilized to identify a putative TAT leader cleavage site with associated cleavage sites following the alanine (A) residue at position 37 of SEQ ID NO:1.

[0193] In an attempt to eliminate non-essential bacterial sequences (including a specific in silico predicted T cell epitope sequence at positions 16-24 of SEQ ID NO:1), to reduce immunogenicity risk, and to eliminate putative N-terminal cleavage sites and associated product heterogeneity, a deletion construct was generated that removes the first 50 N-terminal residues (NicA2Δ50, SEQ ID NO:2).

[0194] It was believed that this region could potentially be deleted without impairing catalytic activity, and as a result, NicA2Δ50 (SEQ ID NO:2) was expressed in E. coli and purified as described above. As can be seen in Figure 1, purified NicA2Δ50 (SEQ ID NO:2) appears homogeneous by SDS-PAGE analysis.

[0195] Analysis of enzyme activity on purified proteins was performed using the Amplex Red assay (Reszka, et al., Effects of peroxidase substrates on the Amplex red / peroxidase assay: Antioxidant properties of anthracyclines, ANALYTICAL BIOCHEMISTRY 342:327-337 (2005)). Briefly, oxidation of nicotine by NicA2 results in the creation of H2O2, which is coupled to the conversion of the colorless Amplex Red reagent to its red fluorescent product, resorufin, by HRP (horseradish peroxidase). The assay was essentially performed as recommended by the supplier of the assay kit (Thermo, Cat#A22188), except that S-nicotine (Sigma) was added to a final assay concentration of 10 μM. The assay was performed in a total volume of 100 μL / well in black half-area flat-bottom 96-well assay plates (Corning Cat#3993). Fluorescence development was detected in a SpectraMax M2 multimode microplate reader (Molecular Devices) using settings Ex at 555 nm; Em at 590 nm, and "plate blank" wells were used to subtract values ​​from no enzyme controls for each time point in the SoftMax Pro data evolution package (Molecular Devices). Activity was expressed as the relative slope of increase in fluorescence plotted as a function of time compared to wild-type NicA2 enzyme run in parallel.

[0196] Amplex Red assays revealed that purified NicA2Δ50 protein exhibited a 23% reduction in activity compared to wild-type NicA2 (FIG. 2 and Table 4). Two shorter deletion constructs (NicA2Δd25 (SEQ ID NO:3) and NicA2Δ38 (SEQ ID NO:4)) were made and tested and similarly exhibited reduced activity compared to the wild-type enzyme. In summary, all three deletion mutants exhibited reduced activity compared to the wild-type (Table 5). [Table 4] [Table 5-1] [Table 5-2] [Table 5-3]

[0197] It was therefore decided to design and identify a NicA2Δ50 mutant that would exhibit nicotine degradation activity at least equivalent to that of the wild type. NOX mutants were also identified for further testing.

[0198] Discovery Studio 4.5 (Dassault Systemes, BIOVIA Corp., San Diego CA) was used to visualize the NicA2 protein and determine the active site residues. Based on inspection of the structure, the location of FAD, and reports of key putative active site residues (Tararina et al., 2016), a putative active site cavity was defined. All residues constituting the exposed surface of this cavity, including both side chains and backbone atoms, were classified as shell 1 residues (Table 2). Based on the defined shell 1 residues, residues in direct contact with shell 1 as well were defined as shell 2 (Table 2). The positions of the residues were confirmed by manual inspection of the structure.

[0199] [ka] This was digested with the restriction enzymes ClaI and BamHI and the fragment was cloned into the corresponding unique sites in an expression plasmid used for expression of NicA2Δ50.

[0200] The resulting library had a NNK randomization of shell 1 codon W427 (Table 2), resulting in a 32-member library (at the DNA / codon level) encoding variants with all 20 possible amino acids at that position. The library was transformed into BL21 Gold (DE3) and single colonies were picked and grown overnight in 96-well plates in LB medium containing carbenicillin (100 μg / ml). The overnight LB culture was diluted into a new 96-deep-well plate containing 475 μl of Autoinduction Magic Medium (Invitrogen) + carbenicillin (100 μg / ml) and grown for 18 hours at room temperature with vigorous shaking. Bacteria were harvested in the plate by centrifugation at 4000 rpm for 15 minutes and the pellet was frozen at -80°C. The pellet was dissolved by dissolving in 100 μl of room temperature Bug Buster HT reagent (Novagen) containing 1 KU r-lysozyme (Novagen) per ml and incubating for 20 minutes at room temperature on a shaking platform. Clarified lysates were prepared by diluting 1:1 (volume:volume) with Bug Buster HT reagent and removing insoluble cell debris by centrifugation at 4400 rpm for 20 min at 4°C. 25 μl of clarified lysates were transferred to a new 96-well plate and diluted 1:1 (volume:volume) in 2% milk in PBS. The diluted lysates were transferred to assay plates (black 96-well half-area high-binding plates (Corning) coated overnight at 4°C with 5 μg / ml anti-His tag antibody (R&D Systems)) in 50 μl per well of PBS, then blocked with 4% milk (in PBS) for 2 h at room temperature and incubated with gentle shaking for 3 h at room temperature to ensure saturation of the immobilized anti-His mAb with a molar excess of the expressed His-tagged enzyme. This step essentially normalizes for any differences in concentration resulting from differences in growth, induction conditions, inherent expression levels, etc., and ensures that a consistent amount of enzyme is assayed for activity in each well in subsequent steps. This also obviates the need for quantification of enzyme in individual wells to accurately measure and compare the activities of mutants.Plates were washed six times with PBST and once with Amplex Red reagent buffer (Thermo) to remove unbound material. The enzyme assay was performed by adding 50 μl of Amplex Red solution (as described above) containing 10 μM S-nicotine to each well and monitoring the development of fluorescence over time.

[0201] From one 96-well assay plate, ten mutants with the highest assay activity (all elevated compared to values ​​from included colonies expressing wild-type NicA2) were isolated from the master plate in overnight LB cultures, and plasmid DNA was prepared and sequenced. Sequencing revealed sequence changes compared to NicA2Δ50 (SEQ ID NO:2) resulting in single mutations from W427 to Q, E, S, and M, resulting in SEQ ID NOs:5, 6, 7, and 8, respectively. In addition, further mutants with mutations at positions 91, 217, 463, 381, 366, 340, 250, 427, or 462 of SEQ ID NO:1, as well as a mutant of NOX with a mutation at position 423 of SEQ ID NO:57, were also designed and screened. A list of the activities of the mutants identified in the Amplex Red screening assay (average for eight individual colonies of each mutant re-assayed as described above) is shown in Table 5.

[0202] Mutant NicA2Δ50W427Q (SEQ ID NO:5) was expressed and purified as described above. Activity assays were performed in parallel with purified wild-type NicA2 and NicA2Δ50 using the Amplex Red assay as described above. As shown in FIG. 2 and Table 4, surprisingly the W427Q mutation was not only "recovered" but actually significantly increased in activity, with the NicA2Δ50W427Q mutant exhibiting approximately 250% activity (a 2.5-fold increase) compared to the wild-type NicA2 enzyme. This data obtained using purified protein is consistent with data generated in the screening assay format (Table 5).

[0203] A protein BLAST homology search using NicA2 as the query sequence yielded NOX, nicotine amine oxidase from Pseudomonas sp. HZN6 (Qiu et al., Appl. Environ. Microbiol. 78, 2154-2160 (2012); Qiu et al., Appl. Environ. Microbiol. 79, 2164-2171 (2013)) as the closest relative with 83% identity. A synthetic gene (custom synthesis by GeneArt / Invitrogen) codon optimized for E. coli expression of the wild-type (wt) NOX amino acid sequence (GenBank accession number: AGH68979.1) with a C-terminal His6-tag was cloned into the NdeI-XhoI sites of pET-22b(+) and the expression plasmid was transformed into E. coli BL21(DE3). The predicted wild-type NOX amino acid sequence expressed from this construct is shown in Table 1 (SEQ ID NO:57). NOX was purified and assayed in an Amplex Red activity assay. The enzyme certainly exhibited activity consistent with nicotine degradation and H2O2 formation, albeit with a 20% reduction in activity compared to wild-type NicA2 (Table 5). Identifying an improved NicA2 variant with a mutation at W427 prompted the creation and screening of an NNK randomized library at the homologous W423 position of NOX. The library was created using the QuikChange site-directed mutagenesis kit (Agilent), a DNA template encoding NOX in the pET22 expression vector described above, and the NNK primer NOX-W423NNK (Table 6, SEQ ID NO:65) according to the kit's instructions. After plating the transformation and incubating overnight at 37°C, eight random colonies were sequenced for library QC, and the remaining colonies were scraped from the agar plate and pooled for DNA minipreps. This DNA was then transformed into the BL21(DE3) expression strain and individual clones were screened as described for the NicA2Δ50W427NNK library above.By screening this library, we identified four mutants with 2-4.5-fold improved activity over wild-type NicA2: NOXW423A, S, E, and H (Table 5). Interestingly, given that all these mutants have increased activity compared to wild-type NicA2, they could potentially be equally good starting points for the development of biotherapeutics, and beneficial mutations directed in NicA2 (e.g., all of the mutations disclosed in Table 5) may also be beneficial at homologous positions in the NOX scaffold.

[0204] Given the successful identification of improved mutants at NicA2 position W427, similar "NNK libraries" in the NicA2Δ50 backbone were created for other active site (shell 1) positions: R91, T250, K340, Q366, T381, N462, I463F, as well as second shell residue L217. These subsequent libraries were created using the QUIKCHANGE™ Site-Directed Mutagenesis Kit (Agilent), a DNA template encoding NicA2Δ50 in a pET22 expression vector, and the NNK primers listed in SEQ ID NOs:66-73 in Table 6, respectively, according to the kit's instructions. As shown in Table 5, screening of these libraries as described above led to the identification of mutants with a range of 1-6.5-fold improvement in activity relative to wild-type NicA2 (SEQ ID NOs:5-56), despite having the Δ50 N-terminal deletion. Interestingly, even though L217 is a second shell residue (Table 3), six substitutions were identified at this position that conferred a 1.7- to 2.8-fold increase in activity over the wild type (Table 5). [Table 6]

[0205] To cover the additional active site positions listed in Table 2, a custom synthetic saturation mutagenesis (CSM) library was provided by Revolve Biotechnologies, Inc. (Firnberg et al., PLos One, 7:e52031 (2012)) exploring all single amino acid (aa) substitutions (one mutation per variant) at the following positions in full-length NicA2: PHE104, GLY105, GLY106, ALA107, TRP108, TYR214, TYR218, GLU249, PHE355, TRP364, TRP417, ALA426, and ALA461.

[0206] Screening of this library in the Amplex Red screening assay as previously described leads to the identification of improved mutants NicA2A107R, NicA2A107K, NicA2A107T, and NicA2F355C (Table 5, SEQ ID NOs:124-127).

[0207] To screen more efficiently through larger library sizes, a fluorescence-activated cell sorting (FACS) sorting protocol was implemented. The library containing the NicA2 variants was cloned into the vector pET22b and transformed into the E. coli BL21(DE3) strain. Cells were incubated in LB + 100 μg / mL ampicillin and grown at 37 °C until the OD600 reached 0.6-0.8. Expression was induced with 1 mM IPTG and the culture was transferred to 18 °C for overnight expression. The next day, cells were washed with 5x PBS + 1 mM EDTA and the cells were permeabilized with nicotine before being resuspended in the same buffer containing 5 μM dihydrorhodamine 123 (DHR123), a redox-sensitive dye that becomes fluorescent upon reaction with hydrogen peroxide released by NicA2, and 5 mM nicotine. The cell suspension was transferred to a clean, sterile flask and incubated at room temperature with shaking. After 2 hours, cells were washed with 5x PBS + 1mM EDTA, sorted by FACS using the FITC channel, and DHR123 fluorescence was quantified. Recovered cells were regrown in LB + 100μg / mL ampicillin and the protocol repeated as necessary.

[0208] Sequencing of clones from the output resulting from one round of selection of the CSM library enriched for putative NicA2 activity yielded mutants NicA2W427R, NicA2T250P, NicA2W427H, N462F, and NicA2W427L, N462M (SEQ ID NOs: 135-138, respectively).

[0209] Based on the results of the improved mutants NicA2Δ50R91Q, NicA2Δ50Q366K, NicA2Δ50T381V, and NicA2Δ50N462Y, the same mutations were introduced into full-length NicA2 (resulting in NicA2R91Q, NicA2Q366K, NicA2T381V, and NicA2N462Y with SEQ ID NOs: 129, 130, 131, and 133, respectively), and activity was evaluated in the Amplex Red assay as previously described. As shown in Table 5, all of these exemplary mutations resulted in activity enhancement in the full-length enzyme. Correspondingly, the high activity of the NicA2A107R mutant (SEQ ID NO: 124) was retained in the NicA2Δ50A107R mutant (SEQ ID NO: 134). As a result, all of the mutations listed in Table 5 are expected to improve activity in the context of both full-length NicA2 and deletion mutants such as NicA2Δ50, NicA2Δ25, NicA2Δ38, or any similar deletion including at least the first 50 N-terminal residues of NicA2, or any N- or C-terminal deletion mutant provided that it has at least 20% of the enzymatic activity of full-length wild-type NicA2.

[0210] Specific variants with mutations at multiple residues selected from Table 5 can be generated by site-directed mutagenesis, and libraries consisting of multiple mutations at multiple positions selected from Table 5 can be generated and screened as described above. These efforts can allow the identification of variants with mutations at several positions in the same molecule that have higher enzymatic activity than any of the individual single mutations listed in Table 5.

[0211] Using mutants having SEQ ID NOs: 2-4, we have shown that it is possible to delete the first epitope with only a moderate effect on activity, and that this reduction in activity can be alleviated or overcome by the mutations listed in Table 5.

[0212] To determine immunogenic epitopes, the structural coordinates of the NicA2 protein were examined on Discovery Studio 4.5 (Dassault Systèmes, BIOVIA Corp., San Diego CA). Regions of the NicA2 sequence that were flagged as potential epitopes via T cell epitope scanning (Immune Epitope Database and Analysis Resource, IEDB, iedb.org) against the MHC allele DRB1*0401 were evaluated and mutational energy was assessed using a predicted site saturation mutagenesis protocol implemented in Discovery Studio. Favorable scoring mutations in each previously defined region were cross-referenced with a new round of IEDB T cell epitope predictions against the MHC allele DRB1*0401 to find changes that would reduce predicted immunogenicity. The final selection of potential mutations was confirmed by visualization of predicted structural changes in Discovery Studio and manual evaluation. Table 3 lists exemplary mutations in immunogenic T cell epitopes that are predicted to reduce immunogenicity while substantially retaining nicotine degrading activity and stability (based on in silico scoring as described above).

[0213] Ten mutants based on mutations for epitope 1 were generated by cloning synthetic double-stranded DNA fragments (SEQ ID NOs: 74-83, Table 7) digested with restriction enzymes EcoRI and SacII into corresponding unique sites in an expression plasmid used for expression of wild-type NicA2 (SEQ ID NO: 1). As discussed in more detail below, the nucleic acid sequences of other exemplary epitope mutants are shown in SEQ ID NOs: 84-113. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8]

[0214] Ligations of individual constructs were transformed in parallel into BL21 Gold (DE3), and eight single colonies from each transformation were picked and grown overnight in 96-well plates in LB medium containing carbenicillin (100 μg / ml). For screening of the W427NNK library, screening of eight individual clones of each mutant was performed as described above, and assay values ​​were reported as the average after eliminating potential rare outliers, as seen in Figure 4A-E (only one out of eight total clones, typically arising from clones without insertions or mutations within the cloned synthetic DNA). Five mutants were identified with assay values ​​≥ 90% of the activity of the wild-type NicA2 enzyme, and these five mutants (I262T, I262S, I262A, I262T&A264L, and I262T&N263R, see Figure 4) have similar enzyme activity to the wild-type and are predicted to be less likely to be immunogenic based on in silico predictions.

[0215] Mutants based on mutations in the four other epitopes listed in Table 3 were prepared and evaluated as described above. Mutants were created by cloning synthetic double-stranded DNA fragments digested with restriction enzymes NdeI and EcoRI (SEQ ID NOs: 84-93, Table 7) for epitope B, DNA fragments digested with SacII-ClaI (SEQ ID NOs: 94-103, Table 7) for epitope 2, DNA fragments cut with SacII-ClaI (SEQ ID NOs: 102-113, Table 7) for epitope 3, and DNA fragments cut with ClaI-XhoI (SEQ ID NOs: 114-123, Table 7) for epitope 4 into the corresponding unique sites in the expression plasmid used for expression of wild-type NicA2 (SEQ ID NO: 1). Eight random colonies from each ligation and transformation were tested as described above. As seen in Figure 4A-E, 2, 4, and 7 mutants were identified with assay values ​​of ≥ 90% of the activity of the wild-type NicA2 enzyme from epitopes B, 2, and 3, respectively, and these mutants (L74N&Y77R, R78Q, V304A&M306Q, V394A, V304T&M306I, M306I&L310R, L374Q&I377S, L374A&I377A, L374N&I382Q, I377A&I382T, I377T and I382T, I377T, and L374N&A383Q, see Figure 4) are predicted to have similar enzyme activity as the wild-type and lower immunogenic potential based on in silico predictions. Interestingly, none of the proposed mutants for epitope 4 showed more than 40% activity compared to wild-type NicA2 (see FIG. 4D). The mutations identified in this example (one mutation from each epitope) can be introduced into the d50W427Q backbone or combined with any of the other identified single mutation mutants listed in Table 5, or any number of mutations resulting from combinations of mutations listed in Table 5, to generate a single deimmunized enzyme mutant with enzymatic activity equal to or better than wild-type NicA2.

[0216] Two mutants were created combining deletion of the first epitope within the TAT leader sequence, mutating epitope 1 using the mutants identified in Figure 4, and containing the activity enhancing mutation W427Q: NicA2Δ50W427Q, I262A (SEQ ID NO: 62) and NicA2Δ50W427Q, I262T, N263R (SEQ ID NO: 63). These mutants were purified and assessed for enzymatic activity in the Amplex Red assay as described above. As can be seen in Table 4, both mutants have increased activity over wild type, despite having changes that may reduce immunogenicity.

[0217] Example 2 - Identification of NicA2 MHCII epitopes An in silico search for NicA2 MHCII epitopes was performed based on the eight most common HLA-DR alleles (Cantoret et al., PNAS 108:1272-1277 (2011); DRB1*0101, DRB1*1501, DRB1*1301, DRB1*1101, DRB1*0801, DRB1*0701, DRB1*0401, and DRB1*0301). Searches were performed using the immune epitope database (Vita et al., Nucleic acids research, 43:D405-D412 (2015)) and the percentile consensus rank method to assess the predicted immunogenic potential of NicA2. Percentile consensus scores for each overlapping 15-mer NicA2 peptide were averaged across all eight HLA-DR alleles. We next determined the immunogenic potential of NicA2 by selecting all NicA2 sequences that were >1 standard deviation in more closely predicted binding to MHCII compared to the overall averaged binding score. This method revealed eight contiguous sequences across 45% of the NicA2 sequence reflecting residues 10-32, 68-94, 189-225, 248-285, 296-327, 336-391, and 435-459 of SEQ ID NO:1 that are predicted to be highly immunogenic (highlighted in grey in Table 1).

[0218] A narrower search using only the DRB1*0401 allele was performed in silico, which yielded the six highest ranked immunogenic T cell epitopes, underlined in Table 1, SEQ ID NO:1.

[0219] Example 3 - PEGylation of nicotinic enzyme Protocols for conjugating polyethylene glycol (PEG) to NicA2 and other nicotine degrading enzymes, such as NOX and the NOX variants disclosed herein, were developed and experiments were performed to determine the effect of PEGylation on activity and half-life.

[0220] SDS-PAGE analysis showed that the degree of PEGylation increased with increasing molar excess of PEGylation reagent and PEG chain length. Random PEGylation of wild-type (wt) NicA2 was performed using a 7- to 20-fold molar excess of 10 or 20 kDa NHS-PEG reagent (Sunbright® ME-100TS or ME-200TS, NOF America Corporation) in 100 mM Na3PO4, pH 7.6 on ice for ≥2 h at a protein concentration of 5 mg / mL. Exclusion of unconjugated PEG reagent was performed using Amicon Ultra-15 centrifugal filter units with a 50 kDa cutoff. Samples corresponding to 2 μg of protein were loaded onto SDS-PAGE gels run in MOPS running buffer and stained using SimplyBlue SafeStain (Invitrogen). As seen in Figure 5, the degree of PEGylation could be controlled by controlling the excess molar ratio of the PEGylation reagent. Preparations NicA2-PEG1, -PEG2, and -PEG3, in which no residual unconjugated protein was detected by SDS-PAGE, were selected for further analysis.

[0221] To determine whether PEGylation could enhance the pharmacokinetic (PK) properties of NicA2, serum concentrations were determined as a function of time following intravenous (iv) administration to rats (5 mg / mL, N=4, 2M+2F). Data from these experiments are shown in Figure 6. Briefly, MaxiSorp ELISA plates (Nunc) were coated overnight with anti-His tag antibody (R&D Systems), which was able to bind to the C-terminal His tag on NicA2 and PEGylated NicA2 proteins. Plates were blocked with 1% nonfat dry milk (NFDM) in phosphate buffered saline (PBS) for approximately 1 hour. Dilutions of NicA2 and NicA2-PEG1-3 standards and serum samples in 1% NFDM in PBS+0.1% Tween-20 were added to the plates and incubated at room temperature for 2 hours. After washing away unbound material (all washing steps were performed in PBS + 0.1% Tween-20), rabbit anti-NicA2 polyclonal primary detection antibody was added to the wells for 1 hour of incubation. Following a washing step, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Fc) (KPL International) was added. The plates were washed and remaining bound complexes were detected with TMB substrate (3,3',5,5'-tetramethylbenzidine, KPL International). Once acid-quenched, the plates were read in a spectrophotometer at 450 nm and data were analyzed with SoftMax® Pro, version 5.4 (Molecular Devices).

[0222] Additional experiments were performed to determine whether PEGylation masks epitopes on NicA2. Serial dilutions in PBS of preparations of unPEGylated or PEGylated NicA2 were tested in the same sandwich ELISA assay used to measure serum concentrations in the PK experiments described above (Figure 6), and the signal (A450) was plotted as a function of concentration. With increasing degrees of PEGylation (approximately 1000-fold higher concentrations of NicA2-PEG2 and -PEG3 required to obtain an A450 of 1.0 compared to the unPEGylated molecules), the assay sensitivity decreased dramatically, indicating that the epitopes recognized by the detection antibody reagents are less accessible in the PEGylated molecules. These results are shown in Figure 7.

[0223] PEGylation was shown to reduce the titer of NicA2-specific antibodies in an immunogenic genetically engineered HLA-DR4 mouse model. Specifically, we investigated the reduction in NicA2-specific antibody titers 10 days after subcutaneous (sc) injection in Freund's incomplete adjuvant in human DR4 genetically engineered mice (N=6, 3M+3F, Taconic Biosciences). This mouse model carries hybrid MHC class II molecules with the antigen-binding domains of human HLA-DRA and HLA-DRB*0401 (representative of the DR4 supertype) and does not express endogenous mouse MHC class II molecules. Titers were defined as the serum dilution to achieve OD450=0.5 in an ELISA using NicA2-coated plates and detected by goat α-mouse IgG-γ-HRP. The lowest serum dilution tested was 50-fold (limit of detection (LOD), shown by the dashed line in Figure 8). PEGylation of NicA2 resulted in a significant ≧10-fold decrease in the mean NicA2-specific antibody titers in transgenic DR4 mice ( FIG. 8 ; 4, 2, and 2 animals from the NicA2-PEG1, -PEG2, and -PEG3 groups, respectively, had titers below the LOD), indicating that the PEGylated variant may be less immunogenic in a clinical setting.

[0224] As shown in Figure 9, PEGylation was also observed to attenuate human T cell proliferation responses and cytokine TNFγ release mediated by exposure to NicA2. PEGylation of NicA2 resulted in a significant decrease in the mean T cell proliferation stimulation index (Figure 9, left panel) as well as a decrease in IFNγ secretion levels (Figure 9, right panel) (independent experiments from five healthy volunteers). Positive control: phytohemagglutinin (PHA). Test results for five independent experiments are shown for the T cell proliferation response stimulation index (SI, measured by 3H-thymidine incorporation) (left) and the fold increase in IFNγ secretion levels (measured by flow cytometry and Cytometric Bead Array kit (BD Biosciences) (right)) for NicA2 and NicA2-PEG2 at three test concentrations compared to the baseline response (PBMC). An increase of 3 or more (dashed line) is considered a significant increase and a positive response.

[0225] As shown in Figure 10, the PEGylated enzyme was also shown to retain full nicotine degradation activity in serum. Wild-type NicA2 or NicA2-PEG2 were added to a final concentration of 0.075 mg / mL in rat serum containing 40 ng / mL S-nicotine (250 nM, equivalent to the plasma level observed in a typical smoker) pre-incubated at 37°C. Samples were collected at various time points and the enzyme activity was immediately quenched by the addition of methanol and rapid mixing. Residual nicotine levels were determined by gas chromatography (GC). The LOD of the GC assay was 2 ng / mL, as indicated by the dashed line. PEGylation did not appear to interfere with the ability of NicA2 to degrade nicotine.

[0226] Example 4 - Nicotine degrading activity in serum To confirm improved activity not only at the assay buffer and high (10 μM) nicotine concentrations of the Amplex Red assay (discussed in Example 1, see, e.g., Tables 4 and 5), but also at serum concentrations more relevant to those encountered by a typical smoker, an exemplary representative variant from the Amplex Red assay (NicA2A107R) was evaluated in an assay that utilizes serum rather than buffer (the same assay used to generate the data shown in FIG. 10).

[0227] Briefly, NicA2A107R was added to a final concentration of 0.075 mg / ml in rat serum containing 40 ng / ml S-(-)-nicotine (250 nM). As can be seen in Figure 11, mutant NicA2A107R has increased nicotine degrading activity under these conditions compared to wild-type NicA2 at serum concentrations that would be found in smokers.

[0228] Example 5 - Treatment of Nicotine Addiction The sublethal dose of nicotine in BALB / c mice was determined to be 2mg / kg intraperitoneally. The effects of nicotine were dose-dependent and included sedation, tail lift, trembling, tachypnea, hunched posture, rapid leg movements, lurching, loss of righting response, and clonic / tonic seizures. In this experiment, wild-type NicA2 was used as a proof of concept to show that the disclosed enzyme mutants can treat nicotine addiction or toxicity.

[0229] 7-8 week old BALB / c mice (N=5 for each group) were pre-treated with 775 mg / kg wild type NicA2 or negative control (both iv) 15 min prior to administration of nicotine at a sublethal dose of 2 mg / kg. Mice were monitored for 5 min for seizures and phenotypic signs of nicotine intoxication or toxicity and scored according to the criteria in Table 8, where a score of less than 3 indicates no seizures. Susceptibility to seizures was calculated by determining the percentage of animals with a score of 4 or 5. [Table 8]

[0230] As shown in Table 9 below, all untreated mice exhibited severe seizures, whereas none of the mice pretreated with NicA2 exhibited seizures. Thus, these results indicate that the disclosed nicotinic enzyme variants can be used to treat nicotine addiction. [Table 9]

[0231] Similar experiments in a rat seizure model using lower doses of wild-type NicA2 (70 mg / kg and 140 mg / kg) (using an intraperitoneal challenge dose of 4 mg / kg nicotine because rats are more tolerant to nicotine than mice) were ineffective. Thus, the dose of the enzyme may be important to provide an effective treatment.

[0232] The dose of 775mg / kg that has been successfully used in mouse experiments is not a practical dose for humans.However, as shown above, the nicotine decomposition enzyme variants described herein have higher nicotine decomposition activity than wild-type enzyme.Therefore, it is believed that the enzyme variants disclosed herein are effective against nicotine addiction at doses suitable for use in humans.

[0233] Example 6 - Treating Nicotine Addiction and / or Promoting Smoking Cessation This example illustrates methods of treating nicotine addiction and / or promoting smoking cessation in human adults using the variants described herein.

[0234] A therapeutically effective amount of a pharmaceutical composition comprising a nicotine degrading enzyme variant (e.g., NicA2Δ50W427Q, SEQ ID NO:5, or its long-acting form) is administered orally or by intravenous or subcutaneous injection to an adult human subject who regularly smokes cigarettes but wants to quit. The subject is evaluated for the nicotine levels circulating in the plasma, and the presence and / or severity of signs and symptoms associated with nicotine withdrawal, such as headache, irritability, anxiety, and insomnia, and the number of cigarettes smoked on a given day. The subject is treated with repeated administrations until the nicotine levels circulating in the plasma reach a target (reduced) level, and / or until one or more signs / symptoms of nicotine withdrawal are reduced, improved, or eliminated, and / or until the subject reduces the level of consumption of nicotine products (e.g., smokes fewer cigarettes per day), and / or until the subject stops consuming nicotine products (e.g., quits smoking).

[0235] Example 7 - Treatment of pediatric patients with nicotinic enzyme variants This example illustrates the use of nicotine degrading enzyme variants in the treatment of nicotine addiction in pediatric patients.

[0236] A therapeutically effective amount of a pharmaceutical composition comprising a nicotine degrading enzyme variant is administered by intravenous, intramuscular, or subcutaneous injection to a child known to have or suspected of having ingested nicotine. The child is evaluated for the presence and / or severity of signs and symptoms associated with nicotine addiction, including but not limited to seizures, coma, shortness of breath, and elevated heart rate, and the child is treated until one or more signs / symptoms are reduced, improved, or eliminated. Optionally, if the signs / symptoms persist and / or nicotine plasma / brain levels remain elevated, another dose of the pharmaceutical composition is administered. * * * * *

Claims

1. A nicotine degrading enzyme mutant comprising an amino acid sequence which is a mutant of the amino acid sequence of the wild-type NicA2 enzyme set forth in SEQ ID NO: 1, said mutant sequence comprising: (i) SEQ ID NO:1, comprising at least one substitution at an amino acid position selected from 91, 104, 106, 107, 217, 250, 340, 366, 381, 427, 462, and 463 of SEQ ID NO:1, which increases nicotine degrading activity; and (ii) SEQ ID NO: 1 with an N-terminal truncation of up to 52 amino acids, comprising at least one substitution at an amino acid position selected from 91, 104, 106, 107, 217, 250, 340, 366, 381, 427, 462, and 463 of SEQ ID NO: 1 that increases nicotine degrading activity. having at least 90% sequence identity to a sequence selected from The at least one substitution is i. R91A, R91Q, R91F, R91G, R91T, R91L, R91S, and R91N; ii. F104L; iii. G106S; iv. A107H, A107P, A107R, A107K, and A107T; v. L217Q, L217G, L217E, L217I, L217C, and L217S; vi. T250G, T250L, T250R, and T250V; vii. K340P, K340I, K340V, K340D, and K340E; viii. Q366K, Q366E, Q366V, Q366L, Q366I, and Q366Y; ix. T381P, T381I, T381V, T381Q, T381N, T381L, and T381M; x. W427Q, W427E, W427S, and W427M; xi. N462L, N462Y, N462S, N462F, N462G, N462E, and N462A; and xii. I463F, I463Y, I463A, I463V, and I463L A nicotine degrading enzyme variant selected from:

2. The variant sequence is SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:129, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:130, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, 131, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:134, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:137, SEQ ID NO:140, and SEQ ID NO:

141.

3. 3. The variant of claim 1 or 2, having at least 97% sequence identity to a sequence selected from (i) SEQ ID NO: 1, and (ii) SEQ ID NO: 1 with an N-terminal truncation of up to 52 amino acids.

4. 2. The variant of claim 1, having at least about 95%, or at least 97%, sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 5-56, 62, 63, 124-127, 129-134, 137 and 140-143.

5. 5. The variant of claim 4, wherein the variant sequence comprises at least one substitution selected from A107R and A107T.

6. The mutant according to any one of claims 1 to 5, wherein the mutant is a long-acting mutant.

7. The variant of claim 6 , wherein the long-acting variant is fused to polyethylene glycol (PEG).

8. The mutant sequence is a sequence similar to SEQ ID NO:1 (a) i. R91A, R91Q, R91F, R91G, R91T, R91L, R91S, and R91N; ii. F104L; iii. G106S; iv. A107H, A107P, A107R, A107K, and A107T; v. L217Q, L217G, L217E, L217I, L217C, and L217S; vi. T250G, T250L, T250R, and T250V; vii. K340P, K340I, K340V, K340D and K340E; viii. Q366K, Q366E, Q366V, Q366L, Q366I, and Q366Y; ix. T381P, T381I, T381V, T381Q, T381N, T381L, and T381M; x. W427H, W427Q, W427E, W427S, and W427M; xi. N462L, N462Y, N462S, N462F, N462G, N462E, and N462A; and xii. I463F, I463Y, I463A, I463V, and I463L; At least one substitution selected from (b) a deletion of amino acids 1 to 38 of SEQ ID NO:1, or a deletion of amino acids 1 to 50 of SEQ ID NO:1, and (c) optionally comprising at least one substitution, addition, or deletion in an immunogenic T cell epitope selected from amino acids 74, 77, 78, 80, 262-266, 303, 304, 306, 310, 374, 377, 378, 382, ​​383, 450-452, and 457 of SEQ ID NO:1; The variant of claim 1, comprising the modification:

9. The mutant sequence is a sequence similar to SEQ ID NO:1 (a) i. R91A, R91Q, R91F, R91G, R91T, R91L, R91S, and R91N; ii. F104L; iii. G106S; iv. A107H, A107P, A107R, A107K, and A107T; v. L217Q, L217G, L217E, L217I, L217C, and L217S; vi. T250G, T250L, T250R, and T250V; vii. K340P, K340I, K340V, K340D and K340E; viii. Q366K, Q366E, Q366V, Q366L, Q366I, and Q366Y; ix. T381P, T381I, T381V, T381Q, T381N, T381L, and T381M; x. W427H, W427Q, W427E, W427S, and W427M; xi. N462L, N462Y, N462S, N462F, N462G, N462E, and N462A; and xii. I463F, I463Y, I463A, I463V, and I463L; At least one substitution selected from (b) a deletion of amino acids 1 to 38 of SEQ ID NO:1, or a deletion of amino acids 1 to 50 of SEQ ID NO:

1. The variant of claim 1, comprising the modification:

10. The variant of claim 8 or 9, which is fused to PEG.

11. 3. The variant of claim 1 or 2, wherein the variant sequence comprises: (a) an amino acid substitution at amino acid position 104 or 107 that increases nicotine degrading activity; and (b) a deletion of amino acids 1 to 50 of SEQ ID NO:

1.

12. 12. The variant of claim 11, consisting of the amino acid sequence of SEQ ID NO: 134, optionally fused to PEG.

13. 12. The mutant of claim 11, which consists of the amino acid sequence of SEQ ID NO: 126, having a deletion of amino acids 1 to 50 corresponding to SEQ ID NO:

1.

14. A pharmaceutical composition comprising the nicotine degrading enzyme variant according to any one of claims 1 to 13 and a pharma- ceutically acceptable carrier.

15. 15. A pharmaceutical composition according to claim 14 for treating nicotine addiction or promoting smoking cessation, or for treating nicotine addiction or nicotine toxicity.

16. 16. The pharmaceutical composition of claim 15, wherein the nicotine addiction is associated with consumption of a nicotine product selected from tobacco products and e-cigarettes.

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