Novel nicotine degrading enzyme variants

Abstract

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

1 - 80 . (canceled) 
     
     
         81 . A method of treating nicotine addiction, facilitating smoking cessation, treating nicotine poisoning or treating nicotine toxicity comprising administering to a mammalian subject in need thereof a therapeutically effective amount of a nicotine-degrading enzyme variant, wherein the variant comprises an amino acid sequence having at least 90% sequence identity to a sequence selected from (i) SEQ ID NO: 1 and (ii) SEQ ID NO: 1 having an N-terminal deletion of up to 52 amino acids, and wherein the variant comprises at least one substitution at an amino acid position selected from positions 91, 104, 106, 107, 217, 250, 340, 355, 366, 381, 427, 462, and 463 of SEQ ID NO:1, and has nicotine-degrading activity, provided that if the variant is F335V then the variant has an N-terminal deletion of up to 52 amino acids. 
     
     
         82 . The method of  claim 81 , wherein the mammalian subject is a human subject. 
     
     
         83 . The method of  claim 82 , wherein the variant sequence has at least 95% sequence identity to a sequence selected from (i) SEQ ID NO: 1 and (ii) SEQ ID NO: 1 having an N-terminal deletion of up to 52 amino acids, and wherein the variant comprises at least one substitution at an amino acid position selected from positions 91, 104, 106, 107, 217, 250, 340, 366, 381, 427, 462, and 463 of SEQ ID NO:1 
     
     
         84 . The method of  claim 83 , wherein the variant comprises at least one substitution selected from:
 a. R91A, R91Q, R91F, R91G, R91T, R91L, R91S, and R91N;   b. F104L;   c. G106S;   d. A107H, A107P, A107R, A107K, and A107T;   e. L217Q, L217G, L217E, L217I, L217C, and L217S;   f. T250P, T250G, T250L, T250R, and T250V;   g. K340P, K340I, K340V, K340D and K340E;   h. Q366K, Q366E, Q366V, Q366L, Q366I, and Q366Y;   i. T381P, T381I, T381V, T381Q, T381N, T381L, and T381M;   j. W427R, W427H, W427L, W427Q, W427E, W427S, or W427M   k. N462M, N462L, N462Y, N462S, N462F, N462G, N462E, and N462A; and   l. I463F, I463Y, I463A, I463V, and I463L.   
     
     
         85 . The method of  claim 82 , wherein the variant comprises an F355C substitution. 
     
     
         86 . The method of  claim 83 , wherein the variant comprises at least one substitution selected from A107R and A107T. 
     
     
         87 . The method of  claim 82 , wherein the variant further comprises at least one substitution, addition, or deletion in an immunogenic T-cell epitope at an amino acid position selected from positions 74, 77, 78, 80, 262-266, 303, 304, 306, 310, 374, 377, 378, 382, 383, 450-452, and 457 of SEQ ID NO:1. 
     
     
         88 . The method of  claim 82 , wherein the variant further comprises at least one substitution, addition, or deletion in an immunogenic T-cell epitope at an amino acid position selected from positions 16-24, 73-81, 258-266, 302-310, 373-381, and 447-455 of SEQ ID NO: 1. 
     
     
         89 . The method of  claim 82 , wherein the variant further comprises at least one substitution, addition, or deletion at a position selected from:
 (a) amino acid positions 74, 77, 78, or 80 of SEQ ID NO: 1;   (b) amino acid positions 262, 263, 264, or 266 of SEQ ID NO: 1;   (c) amino acid positions 303, 304, 306, or 310 of SEQ ID NO: 1;   (d) amino acid positions 374, 377, 378, 382, or 383 of SEQ ID NO: 1; and/or   (e) amino acid positions 450, 451, 452, or 457 of SEQ ID NO: 1.   
     
     
         90 . The method of  claim 83 , wherein the variant further comprises at least one substitution or substitution combination selected from:
 (a) L74N and Y77R;   (b) L74N and Y77K;   (c) L74Q and Y77R;   (d) L74Q and Y77N;   (e) L74N and Y77Q;   (f) L74N and Y77H;   (g) L74N and L80H;   (h) L80F;   (i) Y77R;   (j) R78Q;   (k) I262A and A264Q;   (l) I262K and L266D;   (m) I262T;   (n) I262S;   (o) I262D and L266K;   (p) I262A   (q) I262T and A264L;   (r) I262T and N263R;   (s) M265H;   (t) I262A and A264N;   (u) V303T, V304N, and M306I;   (v) V304A and M306Q;   (w) V304A, M306N;   (x) V304A;   (y) V304A and M306H;   (z) V304N and M306H;   (aa) V304Q and M306H;   (bb) V304N, M306I;   (cc) V304T and M306I;   (dd) L374Q and I377S;   (ee) L374A and I377A;   (ff) L374Q and I377A;   (gg) L374N and I377A;   (hh) L374N and I382Q;   (ii) I377A and I382T;   (jj) I377A and L378N;   (kk) I377T and I382T;   (ll) I377T; and   (mm) L374N and A383Q.   (nn) I448Q and F450S;   (oo) I448E and F450N;   (pp) I448A and F450N;   (qq) I448Q and F450Q;   (rr) I448T and F450Q;   (ss) I448E and F450L;   (tt) T455K;   (uu) L449H and F450A;   (vv) F450A;   (ww) I448A and F450Y.   (xx) I262T;   (yy) I262S;   (zz) I262A;   (aaa) I262T and A264L;   (bbb) I262T and N263R; and   (ccc) M306I and L310R.   
     
     
         91 . The method of  claim 82 , comprising a deletion of at least amino acids 1-38 of SEQ ID NO:1 or a deletion of amino acids 1-50 of SEQ ID NO:1. 
     
     
         92 . The method of  claim 86 , comprising a deletion of at least amino acids 1-38 of SEQ ID NO:1 or a deletion of amino acids 1-50 of SEQ ID NO:1. 
     
     
         93 . The method of  claim 86 , wherein the variant is fused to a compound selected from the group consisting of an albumin-binding peptide, an albumin-binding protein domain, human serum albumin, an inert polypeptide, recombinant PEG (XTEN), a homo-amino acid polymer (HAP), a proline-alanine serine polymer (PAS), and an elastin-like peptide (ELP), and polyethylene glycol (PEG). 
     
     
         94 . The method of  claim 86 , wherein the variant is PEGylated. 
     
     
         95 . The method of  claim 82 , wherein the variant comprises an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 135, 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: 136, 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, SEQ ID NO: 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: 127, SEQ ID NO: 128, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 140, and SEQ ID NO: 141. 
     
     
         96 . The method of  claim 83 , wherein the variant comprises the amino sequence of any of SEQ ID NOs: 124, 126, or 134. 
     
     
         97 . The method of  claim 81 , wherein the variant consists of the amino sequence of any of SEQ ID NOs: 124, 126, or 134 optionally fused to a compound selected from the group consisting of an albumin-binding peptide, an albumin-binding protein domain, human serum albumin, an inert polypeptide, recombinant PEG (XTEN), a homo-amino acid polymer (HAP), a proline-alanine serine polymer (PAS), and an elastin-like peptide (ELP), and polyethylene glycol (PEG). 
     
     
         98 . The method of  claim 86 , wherein the method treats nicotine addiction or facilitates smoking cessation in a patient in need thereof. 
     
     
         99 . The method of  claim 86 , wherein the method treats nicotine poisoning or nicotine toxicity in a patient in need thereof. 
     
     
         100 . The method of  claim 97 , wherein the method treats nicotine addiction or facilitates smoking cessation in a patient in need thereof.