Protoxin-II variants and methods of use

• US 10,463,714 B2
• Filed: 04/04/2016
• Issued: 11/05/2019
• Est. Priority Date: 04/02/2015
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Flinspach, Mack, Wickenden, Alan, Fellows, Ross, Xu, Qinghao, Piekarz, Andrew
• Primary Examiner(s): Weidner, Adam
• Application Number:
  US15/090,328
• Publication Number:
  US 20160287666A1
• Time in Patent Office: 1,310 Days
• Field of Search: None
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/1767 from invertebrates
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/43518 from spiders
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/50 containing protease site

Abstract

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

Claims

1. An isolated Protoxin-II variant that inhibits human Nav1.7 activity, the Protoxin-Ill variant comprising:

a W30L substitution; and

a second substitution comprising at least one of position Y1X, W7Q, S11X, E12R, E12N, E12K, E12T, E12Q, E12Y, E12 glutamyl-4-aminobutane, K14X, E17X, G18X, M19F, R22X and L29V, when residue numbering is according to SEQ ID NO;

1,

wherein X is individually selected from a natural amino acid and a non-natural amino acid.

20. A pharmaceutical composition comprising:

at least one of the isolated Protoxin-II variant of claim 2 an isolated fusion protein, wherein the isolated fusion protein comprises the Protoxin-II variant of claim 1 conjugated to a half-life extending moiety; and

a pharmaceutically acceptable excipient.

2. An isolated Protoxin-II variant that inhibits human Nav1.7 activity, wherein the Protoxin-II variant comprises a modification of SEQ ID NO:

1, the modification comprising at least one of;

a) W7Q and Q3Y substitutions,

b) W7Q and D10 asparagyl-4-aminobutane substitutions,

c) W7Q and S11I substitutions,

d) W7Q and S11V substitutions,

e) W7Q and E12Y substitutions,

f) W7Q and E12 glutamyl-4-aminobutane substitutions,

g) W7Q and K14F substitutions,

h) W7Q and E17L substitutions,

i) W7Q and E17N substitutions,

j) W7Q and E17 glutamyl-4-aminobutane substitutions,

k) W7Q and G18P substitutions,

l) W7Q and R22N-omega methyl-L-arginine substitutions,

m) W7Q and L29V substitutions, and

n) W7Q and W30L substitutions,

wherein residue numbering is according to SEQ ID NO;

1.

3. The isolated Protoxin-II variant of claim 2, comprising the sequence X₁X₂X₃CX₄X₅WX₆QX₇CX₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆CX₁₇LWCX₁₈KKLX₁₉ (SEQ ID NO:

432),

X₁ is G, P, A or deleted;

X₂ is P, A or deleted;

X₃ is S, Q, A, R or Y;

X₄ is Q, R, K, A, S or Y;

X₅ is K, S, Q or R;

X₆ is M or F;

X₇ is T, S, R, K or Q;

X₈ is D, T, or asparagyl-4-aminobutane;

X₉ is S, A, R, I or V;

X₁₀ is E, R, N, K, T, Q, Y or glutamyl-4-aminobutane;

X₁₁ is R or K;

X₁₂ is K, Q, S, A or F;

X₁₃ is E, Q, D, L, N, or glutamyl-4-aminobutane;

X₁₄ is G, Q or P;

X₁₅ is M or F;

X₁₆ is V or S;

X₁₇ is R, T or N-omega methyl-L-arginine; and

X₁₈ is K or R;

X₁₉ is W or L; and

optionally having an N-terminal extension or a C-terminal extension.

4. The Protoxin-II variant of claim 2, further comprising:

a substitution at at least one of residue Y1, S11, E12, K14, E17, G18, L29 and W30,

wherein said substitution comprises at least one of a natural amino acid and a non-natural amino acid.

5. The Protoxin-II variant of claim 2, comprising:

a W7Q substitution;

a W30L substitution; and

a third substitution at at least one of residue Y1, S11, E12, K14, E17, G18, M19 and L29; and

wherein said third substitution comprises at least one of a natural amino acid and a non-natural amino acid.

6. The Protoxin-II variant of claim 2, comprising:

a substitution W30L

a substitution WQ7; and

at least 90% identity to SEQ ID NO;

422 (GPYCQKWMQTCDSERKCCEGMVCRLWCKKKLL-COOH), when residue numbering is according to SEQ ID NO;

1.

7. The Protoxin-Ill variant of claim 2, comprising the sequence X₁X₂X₃CQKWMQTCDX₄X₅RX₆CCX₇X₈X₉VCRLWCKKKX₁₀X₁₁ (SEQ ID NO:

737);

wherein

X₁ is G, P, A or deleted;

X₂ is P, A or deleted;

X₃ is S, Q, A, R or Y;

X₄ is S, A, R, I or V;

X₅ is E, R, N, K, T, Q, Y or glutamyl-4-aminobutane;

X₆ is K, Q, S, A or F;

X₇ is E, Q, D, L, N or glutamyl-4-aminobutane;

X₈ is G, Q or P;

X₉ is M or F;

X₁₀ is L, V; and

X₁₁ is W or L.

8. The isolated Protoxin-II variant of claim 2, wherein the variant inhibits Nav1.7 activity by at least 25%, when the Nav1.7 activity is measured using a patch clamp assay using an extracellular solution containing 137 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM glucose, and 10 mM HEPES, pH=7.4 and osmolarity=315 mOsm, an intracellular solution containing 135 mM CsF, 10 mM CsCl, 5 mM EGTA, 5 mM NaCl and 10 mM HEPES, pH=7.3 and osmolarity=290 mOsm and a holding potential of −

75 mV.

9. The isolated Protoxin-II variant of claim 2, comprising the amino acid sequence GPQCX₁X₂WX₃QX₄CX₅X₆X₇X₈X₉CCX₁₀X₁₁FX₁₂CX₁₃LWCX₁₄KKLL (SEQ ID NO:

433), wherein

X₁ is Q, R, K, A or S;

X₂ is K, S, Q or R;

X₃ is M or F;

X₄ is T, S, R, K or Q;

X₅ is D or T;

X₆ is S, A or R;

X₇ is E, R, N, K, T or Q;

X₈ is R or K;

X₉ is K, Q, S or A;

X₁₀ is E, Q or D;

X₁₁ is G or Q;

X₁₂ is V or S;

X₁₃ is R or T; and

X₁₄ is K or R.

10. The isolated Protoxin-II variant of claim 2, comprising the amino acid sequence of SEQ ID NOs:

417,418, 419, 420, 421, 422, 423, 425, 426, 427, 428, 443, 455, 470, 519, 521, 585, 589, 592, 596, 599, 612, 613, 614, 615, 624, 644, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680,681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 694, 695, 696, 708, 709, 710,711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 732, or 733.

11. The isolated Protoxin-II variant of claim 2, further comprising at least one of a free C-terminal carboxylic acid, amide, methylamide or butylamide group.

12. The isolated Protoxin-II variant of claim 2, that inhibits human Nav1.7 activity with an IC₅₀ value of about 3×

10<sup>−

8</sup> M or less, wherein the IC₅₀ value is measured using a veratridine-induced depolarization inhibition assay using fluorescence resonance energy transfer (FRET) in the presence of 25×

10<sup>−

6</sup> M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7.

13. The isolated Protoxin-II variant of claim 12 inhibits human Nav1.7 activity with an IC₅₀ value of between about 3×

10<sup>−

8</sup> M to about 1×

10<sup>−

9</sup> M.

14. An isolated fusion protein comprising the Protoxin-II variant of claim 2 conjugated to a half-life extending moiety.

15. The fusion protein of claim 14, wherein the half-life extending moiety is human serum albumin (HSA), albumin binding domain (ABD), Fc or polyethylene glycol (PEG).

16. The Protoxin-II variant of claim 2, further comprising:

at least one linker; and

at least one of an N-terminal extension conjugated via the linker and a C-terminal extension conjugated via the linker.

17. The Protoxin-II variant of claim 16, 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.

18. The Protoxin-II variant of claim 16, wherein the C-terminal extension comprises at least one of SEQ ID NOs:

374, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396 and 397.

19. The Protoxin-II variant of claim 16, wherein the linker comprises at least one of SEQ ID NOs:

383, 392, 398, 399, 400, 401 and 402.

21. An isolated polynucleotide encoding the Protoxin-Ill variant of claim 2.

22. A vector comprising the isolated polynucleotide of claim 21.

23. A host cell comprising the vector of claim 22.

24. A method of producing an isolated Protoxin-II variant, comprising culturing the host cell of claim 23 and recovering the Protoxin-II variant produced by the host cell.

25. A method of treating pain in a subject, comprising:

administering to a subject perceiving pain as a result of Nav1.7 channel activity a therapeutically effective amount of a therapeutic composition comprising at least one of the Protoxin-II variant of claim 2 and an isolated fusion protein,

wherein the isolated fusion protein comprises the Protoxin-II variant of claim 2 conjugated to a half-life extending moiety, and

wherein the therapeutically effective amount treats the pain by at least partially preventing the perception of the pain.

26. The method of claim 25, wherein the subject is a human.

27. The method of claim 25, wherein pain is chronic pain, acute pain, neuropathic pain, cancer 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), osteoarthritis, rheumatoid arthritis, lumbar discectomy, pancreatitis, fibromyalgia, painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN), trigeminal neuralgia (TN), spinal cord injuries or multiple sclerosis.

28. The method of claim 27, wherein the Protoxin-Ill variant is administered peripherally.

29. The method of claim 28, wherein the Protoxin-II variant is administered locally to a joint, spinal cord, surgical wound, sites of injury or trauma, peripheral nerve fibers, urogenital organs, or inflamed tissues.