Protoxin-II variants and methods of use

Patent metadata

Field	Value
Publication number	US-9624280-B2
Application number	US-201414505592-A
Country	US
Kind code	B2
Filing date	Oct 3, 2014
Priority date	Oct 3, 2013
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

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

First claim

We claim:

1. An isolated Protoxin-II variant comprising the sequence X 1 X 2 X 3 CX 4 X 5 WX 6 QX 7 CX 8 X 9 X 10 X 11 X 12 CCX 13 X 14 FX 15 CX 16 LWCX 17 KKLW (SEQ ID NO: 403), wherein
X 1 is G, P, A or deleted;
X 2 is P, A or deleted;
X 3 is S, Q, A, R or Y;
X 4 is Q, R, K, A or S;
X 5 is K, S, Q or R;
X 6 is M or F;
X 7 is T, S, R, K or Q;
X 8 is D or T;
X 9 is S, A or R;
X 10 is E, R, N, K, T or Q;
X 11 is R or K;
X 12 is K, Q, S or A;
X 13 is E, Q or D;
X 14 is G or Q;
X 15 is V or S;
X 16 is R or T; and
X 17 is K or R;
optionally having an N-terminal extension or a C-terminal extension,
wherein the polypeptide inhibits human Nav1.7 activity with an IC 50 value of about 1×10 −7 M or less,
wherein the IC 50 value is measured using a membrane depolarization 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 using bis-(1,3-diethylthiobarbituric acid)trimethine oxonol as an electron acceptor and Trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm.

2. The Protoxin-II variant of claim 1 , wherein the N-terminal extension comprises the amino acid sequence of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384 or 385.

3. The Protoxin-II variant of claim 1 , wherein the C-terminal extension comprises the amino acid sequence of SEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396 or 397.

4. The Protoxin-II variant of claim 2 or 3 , wherein the N-terminal and/or the C-terminal extension is conjugated to the Protoxin-II variant via a linker.

5. The Protoxin-II variant of claim 4 , wherein the linker comprises the amino acid sequence of SEQ ID NOs: 383, 392, 398, 399, 400, 401 or 402.

6. The isolated Protoxin-H variant of claim 1 comprising the amino acid sequence of SEQ ID NOs: 30, 40, 44, 52, 56, 56, 59, 65, 78, 109, 110, 111, 114, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 162, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 178, 179, 180, 182, 183, 184, 185, 186, 189, 190, 193, 195, 197, 199, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 224, 226, 227, 231, 232, 243, 244, 245, 247, 249, 252, 255, 258, 261, 263, 264, 265, 266, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 332, 334, 335, 336, 337, 339, 340, 341, 342, 346, 351, 358, 359, 364, 366, 367, or 368.

7. The isolated Protoxin-II variant of claim 1 that inhibits human Nav1.7 activity with an IC 50 value of about 3×10 −8 M or less.

8. The isolated Protoxin-II variant of claim 7 that inhibits human Nav1.7 activity with an IC 50 value of between about 3×10 −9 M to about 1×10 −9 M.

9. The isolated Protoxin-II variant of claim 7 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 KKLW (SEQ ID NO: 404), 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 5, 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.

10. The isolated Protoxin-II variant of claim 9 , comprising 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.

11. The isolated Protoxin-II variant of claim 7 , wherein the variant selectively inhibits human Nav1.7.

12. The isolated Protoxin-II variant of claim 11 , comprising the sequence GPX 1 CQKWMQX 2 CDX 3 X 4 RKCCX 5 GFX 6 CX 7 LWCX 8 KKLW (SEQ ID NO: 405); wherein
X 1 is Y, Q, A, S or R;
X 2 is T or S;
X 3 is S, R or A;
X 4 is E, T or N;
X 5 is E or Q;
X 6 is V or S;
X 7 is R or T; and
X 8 is K or R.

13. The isolated Protoxin-II variant of claim 12 , comprising the amino acid sequence of SEQ ID NOs: 56, 59, 65, 78, 111, 114, 117, 118, 119, 121, 122, 123, 129, 130, 133, 150, 190, 217, 281, 324, 325 or 326.

14. The isolated Protoxin-II variant of claim 12 , comprising the sequence GPQCQKWMQX 1 CDX 2 X 3 RKCCX 4 GFX 5 CX 6 LWCX 7 KKLW (SEQ ID NO: 406); wherein
X 1 is T or S;
X 2 is S, R or A;
X 3 is E, T or N;
X 4 is E or Q;
X 5 is V or S;
X 6 is R or T; and
X 7 is K or R.

15. An isolated Protoxin-II variant comprising an amino acid sequence that is at least 90%, identical to the amino acid sequence of SEQ ID NO: 78 (GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH), wherein a) the amino acid sequence has Q at position 1, Q at position 7 and F at position 19, when residue numbering is according to SEQ ID NO: 1; b) the polypeptide inhibits human Nav1.7 activity with an IC 50 value of about 30×10 −9 M or less, wherein the IC 50 value is measured using a membrane depolarization 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 using bis-(1,3-diethylthiobarbituric acid)trimethine oxonol as an electron acceptor and Trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm; and c) the polypeptide selectively inhibits Nav1.7.

16. The isolated Protoxin-II variant of claim 1 , 9 , 14 or 15 , having a free C-terminal carboxylic acid, amide, methylamide or butylamide group.

17. An isolated fusion protein comprising the Protoxin-II variant of claim 9 , 12 or 14 conjugated to a half-life extending moiety.

18. The fusion protein of claim 17 , wherein the half-life extending moiety is human serum albumin (HSA), albumin binding domain (ABD), Fc or polyethylene glycol (PEG).

19. An isolated polynucleotide encoding the Protoxin-II variant of claim 12 or 15 .

20. A vector comprising the isolated polynucleotide of claim 19 .

21. A host cell comprising the vector of claim 20 .

22. A method of producing an isolated Protoxin-II variant, comprising culturing the host cell of claim 21 and recovering the Protoxin-II variant produced by the host cell.

23. A pharmaceutical composition comprising the isolated Protoxin-II variant of claim 1 , 6 , 12 or 15 and a pharmaceutically acceptable excipient.

24. A method of reducing the perception of pain in a subject, comprising administering to a subject in need thereof an effective amount of the Protoxin-II variant of claim 12 or 15 to treat the pain.

25. The method of claim 24 , wherein the pain is chronic pain, acute pain, neuropathic pain, nociceptive pain, visceral pain, back pain, post-operative pain, thermal pain, phantom limb pain, or pain associated with inflammatory conditions, primary erythemalgia (PE), paraoxysmal extreme pain disorder (PEPD), ost

Assignees

• Janssen Biotech Inc

Inventors

• Flinspach Mack
• Wickenden Alan
• Fellows Ross
• Neff Robert
• Liu Yi
• Hagan Rebecca
• Xu Qinghao

Classifications

• C07K14/43518 Primary

  from spiders · CPC title

• C07K2319/31

  fusions, other than Fc, for prolonged plasma life, e.g. albumin · CPC title

• A61K38/1767

  from invertebrates · CPC title

• C07K14/47 Primary

  from mammals · CPC title

• C07K14/765

  Serum albumin, e.g. HSA · CPC title