IP Library › Granted Patent US 9,790,261
Granted Patent B2
US 9,790,261 · App. 14/739,796 · Granted Oct 17, 2017

Codon optimized IL-15 and IL-15R-alpha genes for expression in mammalian cells

Inventors: Barbara K. Felber (Rockville, MD); George N. Pavlakis (Rockville, MD)
Assignee: THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES
C07K14/5443C07K14/535C07K14/7155C07K2319/02
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 9,790,261
App. No.
14/739,796
Granted
Oct 17, 2017
Kind
B2
Abstract

The present invention provides for nucleic acids improved for the expression of interleukin-15 (IL-15) in mammalian cells. The invention further provides for methods of expressing IL-15 in mammalian cells by transfecting the cell with a nucleic acid sequence encoding an improved IL-15 sequence. The present invention further provides expression vectors, and IL-15 and IL-15 receptor alpha combinations (nucleic acid and protein) that increase IL-15 stability and potency in vitro and in vivo. The present methods are useful for the increased bioavailability and biological effects of IL-15 after DNA, RNA or protein administration in a subject (e.g. a mammal, a human).

Claims (29)

1. An expression vector comprising:

an IL-15 nucleic acid sequence encoding an interleukin-15 (IL-15) polypeptide, wherein the IL-15 nucleic acid sequence has at least 85% sequence identity to nucleotides 145-489 SEQ ID NO:3; and

an IL-15 receptor α (IL-15Rα) nucleic acid sequence encoding an IL-15Rα polypeptide;

wherein the IL-15 nucleic acid sequence and the IL-15Rα nucleic acid sequence are each operably linked to a promoter.

2. The expression vector of claim 1 , wherein the IL-15 nucleic acid sequence has non-native nucleic acid bases at 80% or more of the 80 nucleotide positions 156, 159, 162, 165, 168, 169, 174, 177, 180, 183, 186, 189, 192, 195, 198, 204, 207, 210, 213, 216, 217, 219, 222, 228, 231, 237, 246, 252, 255, 258, 261, 277, 283, 285, 291, 294, 297, 300, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 351, 354, 363, 364, 369, 372, 375, 384, 387, 390, 393, 396, 402, 405, 414, 423, 426, 429, 432, 435, 438, 442, 450, 453, 456, 459, 462, 468, 483 of SEQ ID NO:3.

3. The expression vector of claim 1 , wherein the IL-15 nucleic acid sequence has non-native nucleic acid bases at 90% or more of the 80 nucleotide positions 156, 159, 162, 165, 168, 169, 174, 177, 180, 183, 186, 189, 192, 195, 198, 204, 207, 210, 213, 216, 217, 219, 222, 228, 231, 237, 246, 252, 255, 258, 261, 277, 283, 285, 291, 294, 297, 300, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 351, 354, 363, 364, 369, 372, 375, 384, 387, 390, 393, 396, 402, 405, 414, 423, 426, 429, 432, 435, 438, 442, 450, 453, 456, 459, 462, 468, 483 and 486 of SEQ ID NO:3.

4. The expression vector of claim 1 , wherein the IL-15 nucleic acid sequence has non-native nucleic acid bases at 95% or more of the 80 nucleotide positions 156, 159, 162, 165, 168, 169, 174, 177, 180, 183, 186, 189, 192, 195, 198, 204, 207, 210, 213, 216, 217, 219, 222, 228, 231, 237, 246, 252, 255, 258, 261, 277, 283, 285, 291, 294, 297, 300, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 351, 354, 363, 364, 369, 372, 375, 384, 387, 390, 393, 396, 402, 405, 414, 423, 426, 429, 432, 435, 438, 442, 450, 453, 456, 459, 462, 468, 483 and 486 of SEQ ID NO:3.

5. The expression vector of claim 1 , wherein the IL-15 nucleic acid sequence comprises a guanine (g) or a cytosine (c) nucleotide at nucleotide positions 156, 159, 162, 165, 168, 169, 174, 177, 180, 183, 186, 189, 192, 195, 198, 204, 207, 210, 213, 216, 217, 219, 222, 228, 231, 237, 246, 252, 255, 258, 261, 277, 283, 285, 291, 294, 297, 300, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 351, 354, 363, 364, 369, 372, 375, 384, 387, 390, 393, 396, 402, 405, 414, 423, 426, 429, 432, 435, 438, 442, 450, 453, 456, 459, 462, 468, 483 and 486 of SEQ ID NO:3.

6. The expression vector of claim 1 , wherein the IL-15 nucleic acid sequence comprises at least 50% GC content.

7. The expression vector of claim 1 , wherein the IL-15 nucleic acid sequence has at least 95% sequence identity to nucleotides 145-489 of SEQ ID NO:3.

8. The expression vector of claim 1 , wherein the IL-15 nucleic acid sequence encodes the region of SEQ ID NO:2 corresponding to mature IL-15.

9. The expression vector of claim 8 , wherein the IL-15 nucleic acid sequence has at least 90% identity to SEQ ID NO:3.

10. The expression vector of claim 1 , wherein the IL-15 nucleic acid sequence comprises nucleotides 145-489 of SEQ ID NO:3.

11. The expression vector of claim 1 , wherein the expression vector comprises a nucleic acid sequence encoding a signal peptide-propeptide (SIG-PRO) or a signal peptide (SIG) from a heterologous protein fused to the nucleic acid sequence encoding the IL-15 polypeptide and/or a nucleic acid sequence encoding a signal peptide-propeptide (SIG-PRO) or a signal peptide (SIG) from a heterologous protein fused to the nucleic acid sequence encoding the IL-15Rα polypeptide.

12. The expression vector of claim 11 , wherein the heterologous protein is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), tissue plasminogen activator (tPA), growth hormone, and an immunoglobulin.

13. The expression vector of claim 1 , wherein the IL-15 nucleic acid sequence and the IL-15Rα nucleic acid sequence are each operably linked to different promoters.

14. The expression vector of claim 1 , wherein the IL-15Rα nucleic acid sequence comprises a codon optimized nucleic acid sequence encoding human IL-15Rα.

15. The expression vector of claim 1 , wherein the IL-15Rα nucleic acid sequence has at least 95% sequence identity to SEQ ID NO:47 or at least 95% sequence identity to SEQ ID NO:49.

16. The expression vector of claim 15 , wherein the IL-15Rα nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO:47 or SEQ ID NO:49.

17. A host cell comprising the expression vector of claim 1 .

18. The host cell of claim 17 , wherein the host cell is a mammalian host cell.

19. The host cell of claim 18 , wherein the mammalian host cell is human.

20. The host cell of claim 18 , wherein the host cell is a HEK 293-T, COS, CHO, HeLa, NIH3T3, RD or PC12 cell.

21. The host cell of claim 19 , wherein the host cell is a HEK293 cell.

22. A method of producing IL-15 and IL-15Rα, the method comprising culturing the host cell of claim 17 under conditions in which IL-15 and IL-15Rα are expressed as a heterodimer.

23. The method of claim 22 , wherein the IL-15 nucleic acid sequence is operably linked to a human cytomegalovirus (CMV) promoter and IL-15Rα nucleic acid sequence is operably linked to a simian virus CMV promoter.

24. The method of claim 22 , further comprising isolating a complex comprising IL-15 and IL-15Rα.

25. A host cell comprising an IL-15 nucleic acid sequence encoding an interleukin-15 (IL-15) polypeptide, wherein the IL-15 nucleic acid sequence has at least 85% sequence identity to nucleotides 145-489 SEQ ID NO:3; and an IL-15receptor α(IL-15Rα) nucleic acid sequence encoding an IL-15Rα polypeptide.

26. A method of producing IL-15 and IL-15Rα, the method comprising culturing the host cell of claim 25 under conditions in which IL-15 and IL-15Rαare expressed as a heterodimer.

Continuity (4)
Division 12160263
Provisional Application 60812566 · Jun 9, 2006
Provisional Application 60758819 · Jan 13, 2006
Related Publication 20160102128A1 · Apr 14, 2016