Protoxin-ii variants and methods of use

Abstract

The present invention relates to Protoxin-II variants, polynucleotides encoding them, and methods of making and using the foregoing.

We claim: 
     
         1 . An isolated Protoxin-II variant, comprising: a modified SEQ ID NO: 1, wherein amino acid numbering corresponds to amino acid numbering in SEQ ID NO: 1, comprising a W7Q substitution, a W30L substitution and a conserved M19 wherein the Protoxin-II variant is 30 or 32 amino acid residues and wherein, when the Protoxin-II variant is 32 amino acid residues, residues GP are at the amino-terminus of the Protoxin-II variant; and
 wherein the variant possesses a potency comprising an IC 50  value of 1×10 −7  M or less, wherein the IC 50  value is measured using a veratridine-induced depolarization inhibition assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6  M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7.   
     
     
         2 . The isolated Protoxin-II variant of  claim 1 , wherein the modified SEQ ID NO: 1 further comprises at least one of an N-terminal GP and a C-terminal extension. 
     
     
         3 . The isolated Protoxin-II variant of  claim 1 , wherein the isolated Protoxin-II variant further comprises an N-terminal extension, wherein the N-terminal extension comprises at least one of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384 and 385. 
     
     
         4 . The isolated Protoxin-II variant of  claim 2 , wherein the modified SEQ ID NO: 1 further comprises a C-terminal extension, wherein the C-terminal extension is selected from the group consisting of: (i) at least one of SEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396 and 397; and (ii) a C-terminal extension that is a sequence selected from the group consisting of SEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, and 397 with at least one conservative amino acid substitution therein. 
     
     
         5 . The isolated Protoxin-II variant of  claim 1 , further comprising:
 an N-terminal extension; and   a linker conjugating the N-terminal to the modified SEQ ID NO: 1.   
     
     
         6 . The isolated Protoxin-II variant of  claim 5 , wherein the linker comprises at least one of SEQ ID NOs: 383, 392, 398, 399, 400, 401 and 402. 
     
     
         7 . The isolated Protoxin-II variant of  claim 1 , wherein the isolated Protoxin-II variant comprises the amino acid sequence of SEQ ID NOs: 56, 78, 111, 114, 117, 118, 119, 122, 123, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 145, 146, 147, 149, 150, 151, 152, 153, 154, 156, 158, 159, 165, 172, 173, 175, 177, 178, 183, 184, 185, 186, 189, 190, 193, 197, 199, 207, 210, 211, 216, 217, 224, 266, 273, 282, or 335. 
     
     
         8 . The isolated Protoxin-II variant of  claim 1  comprising the amino acid sequence GPQCX 1 X 2 WX 3 QX 4 CX 5 X 6 X 7 X 8 X 9 CCX 10 X 11 FX 12 CX 13 LWCX 14 KKLL (SEQ ID NO: 433), wherein
 X 1  is Q, R, K, A or S; 
 X 2  is K, S, Q or R; 
 X 3  is M or F; 
 X 4  is T, S, R, K or Q; 
 X 5  is D or T; 
 X 6  is S, A or R; 
 X 7  is E, R, N, K, T or Q; 
 X 8  is R or K; 
 X 9  is K, Q, S or A; 
 X 10  is E, Q or D; 
 X 11  is G or Q; 
 X 12  is V or S; 
 X 13  is R or T; and 
 X 14  is K or R. 
 
     
     
         9 . The isolated Protoxin-II variant of  claim 1 , further comprising at least one of a free C-terminal carboxylic acid, a free C-terminal amide, a free C-Terminal methylamide and a free C-terminal butylamide group. 
     
     
         10 . An isolated fusion protein comprising the isolated Protoxin-II variant of  claim 1  and a moiety selected from the group consisting of a half-life extending moiety selected from the group consisting of human serum albumin (HSA), an albumin binding domain (ABD), and an Fc fragment. 
     
     
         11 . An isolated conjugated polypeptide comprising the isolated Protoxin-II variant of  claim 1  and a half-life extending moiety wherein the half-life extending moiety is polyethylene glycol (PEG). 
     
     
         12 . An isolated polynucleotide encoding a Protoxin-II variant or fusion protein selected from the group consisting of:
 (a) the isolated Protoxin-II variant of  claim 1 ;   (b) an isolated fusion protein comprising the Protoxin-II variant of  claim 10 ;   (c) an isolated Protoxin-II variant, comprising: a modified SEQ ID NO: 1, wherein amino acid numbering corresponds to amino acid numbering in SEQ ID NO: 1, comprising a W7Q substitution, a W30L substitution and a conserved M19 wherein the Protoxin-II variant is 30 or 32 amino acid residues and wherein, when the Protoxin-II variant is 32 amino acid residues, residues GP are at the amino-terminus of the Protoxin-II variant; and   wherein the variant possesses a potency comprising an IC 50  value of 3×10 −8  M or less, wherein the IC 50  value is measured using a veratridine-induced depolarization inhibition assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6  M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7; and   (c) an isolated Protoxin-II variant, comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO: 422 (GPYCQKVVMQTCDSERKCCEGMVCRLWCKKKLL-COOH) wherein the amino sequence comprises:   a Q or W at position 7, when residue numbering is according to SEQ ID NO: 1;   a L, F, Y, or W at position 30, when residue numbering is according to SEQ ID NO: 1, wherein the variant comprises 30 or 32 amino acids; and   a potency comprising an ICs value of about 30×10 −9  M or less, wherein the ICs value is measured using a veratridine-induced depolarization inhibition assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6  M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7; and wherein the conservative amino acid substitutions are selected from the group consisting of: (1) a substitution of one acidic amino acid for another acidic amino acid, wherein the acidic amino acids are aspartic acid and glutamic acid; (2) a substitution of one basic amino acid for another basic amino acid, wherein the basic amino acids are lysine, arginine, and histidine; (3) a substitution of one nonpolar amino acid for another nonpolar amino acid, wherein the nonpolar amino acids are alanine, valine, leucine, isoleucine, phenylalanine, methionine, and tryptophan; and (4) a substitution of one uncharged polar amino acid for another uncharged polar amino acid, wherein the uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine.   
     
     
         13 . An isolated vector comprising the polynucleotide of  claim 10 . 
     
     
         14 . An isolated host cell comprising the vector of  claim 13 . 
     
     
         15 . A method of producing the isolated Protoxin-II variant, comprising culturing the host cell of  claim 14  and recovering the Protoxin-II variant produced by the host cell. 
     
     
         16 . A pharmaceutical composition comprising:
 (a) a Protoxin-II variant, fusion protein, or conjugated polypeptide selected from the group consisting of:
 (i) a Protoxin-II variant selected from the group consisting of:
 (A) the isolated Protoxin-II variant of  claim 1 ; 
 (B) an isolated Protoxin-II variant, comprising: a modified SEQ ID NO: 1, wherein amino acid numbering corresponds to amino acid numbering in SEQ ID NO: 1, comprising a W7Q substitution, a W30L substitution and a conserved M19 wherein the Protoxin-II variant is 30 or 32 amino acid residues and wherein, when the Protoxin-II variant is 32 amino acid residues, residues GP are at the amino-terminus of the Protoxin-II variant; and 
 
 wherein the variant possesses a potency comprising an IC 50  value of 3×10 −8  M or less, wherein the IC 50  value is measured using a veratridine-induced depolarization inhibition assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6  M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7; and
 (C) an isolated Protoxin-II variant, comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO: 422 (GPYCQKVVMQTCDSERKCCEGMVCRLWCKKKLL-COOH) wherein the amino sequence comprises: 
 
   a Q or W at position 7, when residue numbering is according to SEQ ID NO: 1;   a L, F, Y, or W at position 30, when residue numbering is according to SEQ ID NO: 1, wherein the variant comprises 30 or 32 amino acids; and   a potency comprising an IC 50  value of about 30×10 −9  M or less, wherein the IC 50  value is measured using a veratridine-induced depolarization inhibition assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6  M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7; and wherein the conservative amino acid substitutions are selected from the group consisting of: (1) a substitution of one acidic amino acid for another acidic amino acid, wherein the acidic amino acids are aspartic acid and glutamic acid; (2) a substitution of one basic amino acid for another basic amino acid, wherein the basic amino acids are lysine, arginine, and histidine; (3) a substitution of one nonpolar amino acid for another nonpolar amino acid, wherein the nonpolar amino acids are alanine, valine, leucine, isoleucine, phenylalanine, methionine, and tryptophan; and (4) a substitution of one uncharged polar amino acid for another uncharged polar amino acid, wherein the uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine; or
 (ii) a fusion protein selected from the group consisting of:
 (A) a fusion protein comprising the isolated Protoxin-II variant of  claim 1  and a moiety selected from the group consisting of a half-life extending moiety selected from the group consisting of human serum albumin (HSA), an albumin binding domain (ABD), and an Fc fragment; 
 (B) a fusion protein comprising the isolated Protoxin-II variant of (a)(i)(B) and a moiety selected from the group consisting of a half-life extending moiety selected from the group consisting of human serum albumin (HSA), an albumin binding domain (ABD), and an Fc fragment; and 
 (C) a fusion protein comprising the isolated Protoxin-II variant of (a)(i)(C) and a moiety selected from the group consisting of a half-life extending moiety selected from the group consisting of human serum albumin (HSA), an albumin binding domain (ABD), and an Fc fragment; and 
 
 (iii) a conjugated polypeptide selected from the group consisting of:
 (A) a conjugated polypeptide comprising the isolated Protoxin-II variant of  claim 1  and a half-life extending moiety wherein the half-life extending moiety is polyethylene glycol (PEG); 
 (B) a conjugated polypeptide comprising the isolated Protoxin-II variant of (a)(i)(B) and a half-life extending moiety wherein the half-life extending moiety is polyethylene glycol (PEG); and 
 (C) a conjugated polypeptide comprising the isolated Protoxin-II variant of (a)(i)(C) and a half-life extending moiety wherein the half-life extending moiety is polyethylene glycol (PEG); and 
 
   (b) a pharmaceutically acceptable excipient.   
     
     
         17 . A method of treating Nav1.7-mediated pain in a subject, comprising administering to a subject in need thereof an effective amount of a Protoxin-II variant, fusion protein, or conjugated polypeptide to treat the pain, 
       wherein the Protoxin-II variant is the isolated Protoxin-II variant of  claim 1 ; 
       wherein the fusion protein is selected from the group consisting of:
 (d) a fusion protein comprising the isolated Protoxin-II variant of  claim 1  and a half-life extending moiety selected from the group consisting of a half-life extending moiety selected from the group consisting of human serum albumin (HSA), an albumin binding domain (ABD), and an Fc fragment; 
 (e) a fusion protein comprising an isolated Protoxin-II variant, comprising: a modified SEQ ID NO: 1, wherein amino acid numbering corresponds to amino acid numbering in SEQ ID NO: 1, comprising a W7Q substitution, a W30L substitution and a conserved M19 wherein the Protoxin-II variant is 30 or 32 amino acid residues and wherein, when the Protoxin-II variant is 32 amino acid residues, residues GP are at the amino-terminus of the Protoxin-II variant; and 
 wherein the variant possesses a potency comprising an IC 50  value of 3×10 8  M or less, wherein the IC 50  value is measured using a veratridine-induced depolarization inhibition assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6  M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7, and a moiety selected from the group consisting of a half-life extending moiety selected from the group consisting of human serum albumin (HSA), an albumin binding domain (ABD), and an Fc fragment; and 
 (f) a fusion protein comprising an isolated Protoxin-II variant, comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO: 422 (GPYCQKVVMQTCDSERKCCEGMVCRLWCKKKLL-COOH) wherein the amino sequence comprises: 
 a Q or W at position 7, when residue numbering is according to SEQ ID NO: 1; 
 a L, F, Y, or W at position 30, when residue numbering is according to SEQ ID NO: 1, wherein the variant comprises 30 or 32 amino acids; and 
 a potency comprising an IC 50  value of about 30×10 −9  M or less, wherein the IC 50  value is measured using a veratridine-induced depolarization inhibition assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6  M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7; and wherein the conservative amino acid substitutions are selected from the group consisting of: (1) a substitution of one acidic amino acid for another acidic amino acid, wherein the acidic amino acids are aspartic acid and glutamic acid; (2) a substitution of one basic amino acid for another basic amino acid, wherein the basic amino acids are lysine, arginine, and histidine; (3) a substitution of one nonpolar amino acid for another nonpolar amino acid, wherein the nonpolar amino acids are alanine, valine, leucine, isoleucine, phenylalanine, methionine, and tryptophan; and (4) a substitution of one uncharged polar amino acid for another uncharged polar amino acid, wherein the uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine, and a moiety selected from the group consisting of a half-life extending moiety selected from the group consisting of human serum albumin (HSA), an albumin binding domain (ABD), and an Fc fragment; and 
 
       wherein the conjugated polypeptide is selected from the group consisting of:
 (g) a conjugated polypeptide comprising the isolated Protoxin-II variant of  claim 1  and a half-life extending moiety wherein the half-life extending moiety is polyethylene glycol (PEG); 
 (h) a conjugated polypeptide comprising the isolated Protoxin-II variant of (e) and a half-life extending moiety wherein the half-life extending moiety is polyethylene glycol (PEG); and 
 (i) a conjugated polypeptide comprising the isolated Protoxin-II variant of (f) and a half-life extending moiety wherein the half-life extending moiety is polyethylene glycol (PEG). 
 
     
     
         18 . The method of  claim 17 , wherein the pain is chronic pain, acute pain, neuropathic pain, nociceptive pain, visceral pain, back pain, postoperative pain, thermal pain, phantom limb pain, or pain associated with inflammatory conditions, primary erythemalgia (PE), paroxysmal extreme pain disorder (PEPD), osteoarthritis, rheumatoid arthritis, lumbar discectomy, pancreatitis, fibromyalgia, painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN), trigeminal neuralgia (TN), spinal cord injuries or multiple sclerosis. 
     
     
         19 . The method of  claim 17 , wherein the Protoxin-II variant, fusion protein, or conjugated polypeptide is administered peripherally. 
     
     
         20 . The method of  claim 19 , wherein the Protoxin-II variant, fusion protein, or conjugated polypeptide is administered locally to a joint, spinal cord, surgical wound, sites of injury or trauma, peripheral nerve fibers, urogenital organs, or inflamed tissues. 
     
     
         21 . The method of  claim 17 , wherein the subject is a human. 
     
     
         22 . A method of treating Nav1.7-mediated pain in a subject, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of  claim 16  to treat the pain. 
     
     
         23 . The method of  claim 22 , wherein the pain is chronic pain, acute pain, neuropathic pain, nociceptive pain, visceral pain, back pain, postoperative pain, thermal pain, phantom limb pain, or pain associated with inflammatory conditions, primary erythemalgia (PE), paroxysmal extreme pain disorder (PEPD), osteoarthritis, rheumatoid arthritis, lumbar discectomy, pancreatitis, fibromyalgia, painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN), trigeminal neuralgia (TN), spinal cord injuries or multiple sclerosis. 
     
     
         24 . The method of  claim 22 , wherein the pharmaceutical composition is administered peripherally. 
     
     
         25 . The method of  claim 24 , wherein the pharmaceutical composition is administered locally to a joint, spinal cord, surgical wound, sites of injury or trauma, peripheral nerve fibers, urogenital organs, or inflamed tissues. 
     
     
         26 . The method of  claim 22 , wherein the subject is a human.