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IL-15 FUSION PEPTIDES USED TO TREAT CANCER

20220372097 · 2022-11-24

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention is directed to a fusion polypeptide, the polypeptide comprising: a. an interleukin-15 (IL-15); and b. an IL-15 activity-promoting sequence, wherein said sequence: is between 10 and 60 amino acid residues in length; and increases CD8+ T-cell proliferation 5 by the IL-15. Also provided are nucleic acids encoding the fusion polypeptide, associated methods of producing the fusion polypeptide, pharmaceutical compositions and kits comprising the same, and therapeutic uses thereof.

    Claims

    1. A fusion polypeptide, the polypeptide comprising: a. an interleukin-15 (IL-15); and b. an IL-15 activity-promoting sequence, wherein said sequence: is between 10 and 60 amino acid residues in length; and increases CD8+ T-cell proliferation by the IL-15.

    2. The fusion polypeptide according to claim 1, wherein the IL-15 activity-promoting sequence does not increase receptor-independent binding of the polypeptide to a cell surface.

    3. The fusion polypeptide according to claim 1 or 2, wherein the IL-15 activity-promoting sequence is between 10 and 55 amino acid residues in length.

    4. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 activity-promoting sequence is between 15 and 55 amino acid residues in length.

    5. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 activity-promoting sequence is between 25 and 55 amino acid residues in length.

    6. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 activity-promoting sequence is between 30 and 55 amino acid residues in length.

    7. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 activity-promoting sequence is between 42 and 50 amino acid residues in length.

    8. The fusion polypeptide according to any one of the preceding claims, wherein the polypeptide comprises a N-terminal IL-15 and a C-terminal IL-15 activity-promoting sequence.

    9. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 activity-promoting sequence comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 4 or 9.

    10. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 activity-promoting sequence comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 4 or 9.

    11. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 activity-promoting sequence comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 4 or 9.

    12. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 activity-promoting sequence comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 4 or 9.

    13. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 activity-promoting sequence comprises SEQ ID NO: 4 or 9.

    14. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 activity-promoting sequence consists of SEQ ID NO: 4 or 9.

    15. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 is a human IL-15.

    16. The fusion polypeptide according to any one of the preceding claims, wherein the IL-15 comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2 or 3.

    17. The fusion polypeptide according to any one of the preceding claims comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 5, 10 or 28.

    18. The fusion polypeptide according to any one of the preceding claims comprising a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 5, 10 or 28.

    19. The fusion polypeptide according to any one of the preceding claims comprising a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 5, 10 or 28.

    20. The fusion polypeptide according to any one of the preceding claims comprising a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 5, 10 or 28.

    21. The fusion polypeptide according to any one of the preceding claims comprising SEQ ID NO: 5, 10 or 28.

    22. The polypeptide according to any one of the preceding claims, wherein a membrane binding agent is conjugated to the IL-15 activity-promoting sequence.

    23. The fusion polypeptide according to claim 22, wherein the membrane binding agent comprises an aliphatic acyl group.

    24. The fusion polypeptide according to claim 23, wherein the aliphatic acyl group is myristoyl.

    25. The fusion polypeptide according to any one of claims 22-24, wherein the membrane binding element further comprises a hydrophilic peptide.

    26. The fusion polypeptide according to claim 25, wherein the hydrophilic peptide comprises a peptide sequence having at least 70% sequence identity to SEQ ID NO: 6.

    27. The fusion polypeptide according to claim 25 or 26, wherein the hydrophilic peptide comprises a peptide sequence having at least 80% sequence identity to SEQ ID NO: 6.

    28. The fusion polypeptide according to any one of claims 25-27, wherein the hydrophilic peptide comprises a peptide sequence having at least 90% sequence identity to SEQ ID NO: 6.

    29. The fusion polypeptide according to any one of claims 25-28, wherein the hydrophilic peptide comprises a peptide sequence having at least 95% sequence identity to SEQ ID NO: 6.

    30. The fusion polypeptide according to any one of claims 25-29, wherein the hydrophilic peptide comprises SEQ ID NO: 6.

    31. The fusion polypeptide according to any one of claims 25-30, wherein the hydrophilic peptide consists of SEQ ID NO: 6.

    32. The fusion polypeptide according to any one of claims 22-31, wherein the membrane binding element is conjugated to a cysteine residue or a lysine residue of the IL-15 activity-promoting sequence.

    33. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide is conjugated to N-(α,εbis-myristoyllysine)SSKSPSKKDDKKPGDC (SEQ ID NO: 31) via a di-sulphide bond.

    34. A fusion polypeptide, the polypeptide comprising: a. an interleukin-15 (IL-15); and b. a peptide, wherein the peptide is between 10 and 60 amino acid residues in length and has at least 70% sequence identity to SEQ ID NO: 4 or 9.

    35. The fusion polypeptide according to claim 34, wherein the peptide is between 10 and 55 amino acid residues in length.

    36. The fusion polypeptide according to claim 34 or 35, wherein the peptide is between 15 and 55 amino acid residues in length.

    37. The fusion polypeptide according to any one of claims 34-36, wherein the peptide is between 25 and 55 amino acid residues in length.

    38. The fusion polypeptide according to any one of claims 34-37, wherein the peptide is between 30 and 55 amino acid residues in length.

    39. The fusion polypeptide according to any one of claims 34-38, wherein the peptide is between 42 and 50 amino acid residues in length.

    40. The fusion polypeptide according to any one of claims 34-39, wherein the polypeptide comprises a N-terminal IL-15 and a C-terminal peptide.

    41. The fusion polypeptide according to any one of claims 34-40, wherein the peptide comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 4 or 9.

    42. The fusion polypeptide according to any one of claims 34-41, wherein the peptide comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 4 or 9.

    43. The fusion polypeptide according to any one of claims 34-42, wherein the peptide comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 4 or 9.

    44. The fusion polypeptide according to any one of claims 34-43, wherein the peptide comprises SEQ ID NO: 4 or 9 (preferably consists of SEQ ID NO: 4 or 9).

    45. The fusion polypeptide according to any one of claims 34-44, wherein the IL-15 is a human IL-15.

    46. The fusion polypeptide according to any one of claims 34-45, wherein the IL-15 comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2 or 3.

    47. The fusion polypeptide according to any one of claims 34-46 comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 5, 10 or 28.

    48. The fusion polypeptide according to any one of claims 34-47 comprising a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 5, 10 or 28.

    49. The fusion polypeptide according to any one of claims 34-48 comprising a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 5, 10 or 28.

    50. The fusion polypeptide according to any one of claims 34-49 comprising a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 5, 10 or 28.

    51. The fusion polypeptide according to any one of claims 34-50 comprising SEQ ID NO: 5, 10 or 28.

    52. The polypeptide according to any one of claims 34-51, wherein a membrane binding agent is conjugated to the peptide.

    53. The fusion polypeptide according to claim 52, wherein the membrane binding agent comprises an aliphatic acyl group.

    54. The fusion polypeptide according to claim 53, wherein the aliphatic acyl group is myristoyl.

    55. The fusion polypeptide according to any one of claims 52-54, wherein the membrane binding element further comprises a hydrophilic peptide.

    56. The fusion polypeptide according to claim 55, wherein the hydrophilic peptide comprises a peptide sequence having at least 70% sequence identity to SEQ ID NO: 6.

    57. The fusion polypeptide according to claim 55 or 56, wherein the hydrophilic peptide comprises a peptide sequence having at least 80% sequence identity to SEQ ID NO: 6.

    58. The fusion polypeptide according to any one of claims 55-57, wherein the hydrophilic peptide comprises a peptide sequence having at least 90% sequence identity to SEQ ID NO: 6.

    59. The fusion polypeptide according to any one of claims 55-58, wherein the hydrophilic peptide comprises a peptide sequence having at least 95% sequence identity to SEQ ID NO: 6.

    60. The fusion polypeptide according to any one of claims 55-59, wherein the hydrophilic peptide comprises SEQ ID NO: 6.

    61. The fusion polypeptide according to any one of claims 55-60, wherein the hydrophilic peptide consists of SEQ ID NO: 6.

    62. The fusion polypeptide according to any one of claims 52-61, wherein the membrane binding element is conjugated to a cysteine residue or a lysine residue of the peptide.

    63. The fusion polypeptide according to any one of claims 34-62, wherein the fusion polypeptide is conjugated to N-(α,εbis-myristoyllysine)SSKSPSKKDDKKPGDC (SEQ ID NO: 31) via a di-sulphide bond.

    64. A fusion polypeptide, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 5, 10 or 28.

    65. The fusion polypeptide according to claim 64, wherein the IL-15 is a human IL-15.

    66. The fusion polypeptide according to claim 64 or 65, wherein the IL-15 comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 2 or 3.

    67. The fusion polypeptide according to any one of claims 64-66 comprising a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 5, 10 or 28.

    68. The fusion polypeptide according to any one of claims 64-67 comprising a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 5, 10 or 28.

    69. The fusion polypeptide according to any one of claims 64-68 comprising a polypeptide sequence comprising SEQ ID NO: 5, 10 or 28.

    70. The fusion polypeptide according to any one of claims 64-69 consisting of SEQ ID NO: 5, 10 or 28.

    71. The polypeptide according to any one of claims 64-70, wherein a membrane binding agent is conjugated to the polypeptide.

    72. The fusion polypeptide according to claim 71, wherein the membrane binding agent comprises an aliphatic acyl group.

    73. The fusion polypeptide according to claim 72, wherein the aliphatic acyl group is myristoyl.

    74. The fusion polypeptide according to any one of claims 71-73, wherein the membrane binding element further comprises a hydrophilic peptide.

    75. The fusion polypeptide according to claim 74, wherein the hydrophilic peptide comprises a peptide sequence having at least 70% sequence identity to SEQ ID NO: 6.

    76. The fusion polypeptide according to claim 74 or 75, wherein the hydrophilic peptide comprises a peptide sequence having at least 80% sequence identity to SEQ ID NO: 6.

    77. The fusion polypeptide according to any one of claims 74-76, wherein the hydrophilic peptide comprises a peptide sequence having at least 90% sequence identity to SEQ ID NO: 6.

    78. The fusion polypeptide according to any one of claims 74-77, wherein the hydrophilic peptide comprises a peptide sequence having at least 95% sequence identity to SEQ ID NO: 6.

    79. The fusion polypeptide according to any one of claims 74-78, wherein the hydrophilic peptide comprises SEQ ID NO: 6.

    80. The fusion polypeptide according to any one of claims 74-79, wherein the hydrophilic peptide consists of SEQ ID NO: 6.

    81. The fusion polypeptide according to any one of claims 71-80, wherein the membrane binding element is conjugated to a cysteine residue or a lysine residue of the polypeptide.

    82. The fusion polypeptide according to any one of claims 64-81, wherein the fusion polypeptide is conjugated to N-(α,εbis-myristoyllysine)SSKSPSKKDDKKPGDC (SEQ ID NO: 31) via a di-sulphide bond.

    83. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 24.

    84. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide is encoded by a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 24.

    85. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 24.

    86. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide is encoded by a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 24.

    87. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO: 8 or SEQ ID NO: 24.

    88. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide is encoded by a nucleotide sequence consisting of SEQ ID NO: 8 or SEQ ID NO: 24.

    89. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide comprises a polypeptide sequence having at last 70% sequence identity to SEQ ID NO: 7.

    90. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide comprises a polypeptide sequence having at last 80% sequence identity to SEQ ID NO: 7.

    91. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide comprises a polypeptide sequence having at last 90% sequence identity to SEQ ID NO: 7.

    92. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide comprises a polypeptide sequence having at last 95% sequence identity to SEQ ID NO: 7.

    93. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide comprises SEQ ID NO: 7.

    94. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide consists of SEQ ID NO: 7.

    95. A nucleic acid encoding a fusion polypeptide according to any one of claims 1-94.

    96. The nucleic acid according to claim 95, wherein the nucleic acid comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 24.

    97. The nucleic acid according to claim 95 or 96, wherein the nucleic acid comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 24.

    98. The nucleic acid according to any one of claims 95-97, wherein the nucleic acid comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 24.

    99. The nucleic acid according to any one of claims 95-98, wherein the nucleic acid comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 24.

    100. The nucleic acid according to any one of claims 95-99, wherein the nucleic acid comprises SEQ ID NO: 8 or SEQ ID NO: 24.

    101. The nucleic acid according to any one of claims 95-100, wherein the nucleic acid comprises a nucleotide sequence consists of SEQ ID NO: 8 or SEQ ID NO: 24.

    102. A method for producing a fusion polypeptide, the method comprising: a. expressing the nucleic acid sequence according to any one of claims 95-101 in a host cell; and b. isolating the fusion polypeptide.

    103. A fusion polypeptide obtainable by the method of claim 102.

    104. A pharmaceutical composition comprising the fusion polypeptide according to any one of claim 1-94 or 103 and a pharmaceutically acceptable carrier, excipient, adjuvant, and/or salt.

    105. A kit comprising: a. the fusion polypeptide according to any one of claim 1-94 or 103 or the pharmaceutical composition according to claim 104; and b. instructions for use of the same (e.g. in treating cancer).

    106. A fusion polypeptide according to any one of claim 1-94 or 103 or a pharmaceutical composition according to claim 104 or a kit according to claim 105 for use in treating cancer.

    107. A method of treating cancer, the method comprising administering a fusion polypeptide according to any one of claim 1-94 or 103 or a pharmaceutical composition according to claim 104 or a kit according to claim 105 to a subject.

    108. Use of a fusion polypeptide according to any one of claim 1-94 or 103 or a pharmaceutical composition according to claim 104 or a kit according to claim 105 in the manufacture of a medicament for treating cancer.

    109. The fusion polypeptide, pharmaceutical composition, or kit for use, method or use according to any one of claims 106-108, wherein the cancer is a solid tumour cancer.

    110. The fusion polypeptide, pharmaceutical composition, or kit for use, method or use according to any one of claims 106-109, wherein the cancer is one or more selected from: prostate cancer, colon cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, pancreatic cancer, brain cancer, head cancer, neck cancer, lymphoma, and neuronal cancer.

    111. The fusion polypeptide, pharmaceutical composition, or kit for use, method, or use according to any one of claims 106-110, wherein the fusion polypeptide or composition is administered intratumourally.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0199] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

    [0200] FIG. 1 shows: (A) the ability of various Th1 cytokines (compared to phosphate buffered saline (PBS) control) to expand and activate Natural Killer (NK) cells and CD8 T cells in co-cultures of peripheral blood mononuclear cells (PBMCs) and prostate cancer cells (PC3 and LNCaP); and (B) NK and CD8 T cell cytotoxic capabilities by way of perforin expression and apoptotic and necrotic cell death of tumour cells by way of Annexin-FITC and propidium iodide (PI) staining.

    [0201] FIG. 2 shows the activity of modified IL-15 (containing an activity-promoting sequence) compared to unmodified wild-type IL-15 in a CTLL-2 assay as described in the Examples.

    [0202] FIG. 3 shows visualisation of tailed IL-15 by gel electrophoresis followed by silver nitrate staining, western blot analysis and UV light visualisation of fluorescently labelled tail compound PTL3146. The band circled represents the main tailed protein moiety. Lane 4 contains the newly prepared modified IL-15, which is pure on silver stain and anti-IL-15 western blot. Lane key: 1=marker; 2, 4=modified IL-15; and 3=membrane-anchored modified IL-15 with a FAM-labelled tail.

    [0203] FIG. 4 shows cell membrane binding of membrane-anchored modified IL-15 (“tailed Il-15”) and modified IL-15 (“untailed IL-15”) by flow cytometry on: (A) Jurkat cells after 30 minutes and 24 hours; and (B) sheep red blood cells.

    [0204] FIG. 5 shows the activity of modified IL-15 (“untailed”) vs. membrane-anchored modified IL-15 (“tailed”) and wild-type unmodified IL-15. Proliferation was measured by a CTLL-2 assay as described in the Examples at an absorbance of 490 nm for n=3 experiments.

    [0205] FIG. 6 shows comparison of NK expansion in a PBMC population treated with IL-2 (100 units per ml), wild-type IL-15, modified IL-15 (“untailed IL-15”) and membrane-anchored modified IL-15 (“tailed IL-15”) (2.5 ng/ml each). A shows representative dot blots from Flow cytometry analysis. Top left quadrant on the dot blots represents NK cells (CD56+CD3−). B shows a graph (human PBMCs) showing expansion of human NK cells by the tested IL-15 polypeptides. Control=PBS only.

    [0206] FIG. 7 shows killing of PC3 cells co-cultured with human NK cells in the presence of IL-2, wild-type IL-15 (IL-15 pep.), modified IL-15 (“untailed IL-15”) and membrane-anchored modified IL-15 (“tailed IL-15”). Cell killing is represented by positive staining of the cells with propidium iodide (Pl). n=2, *p<0.05 by one-way ANOVA and post-hoc test Newman-Keuls. Control=PBS only.

    [0207] FIG. 8 shows the effect of IL-15 on growth of TRAMP-C2 prostate tumour xenografts. Mice with TRAMP-C2 tumours of approximately 100 mm.sup.3 were injected intratumourally vehicle (100 μl PBS, n=10), 10 μg untailed IL-15 (n=10) or tailed IL-15 (n=10), or intraperitoneally with untailed IL-15 (n=6) at days 0 and 3. (A) Tumour volumes up to day 14 post-treatment. (B) Survival curves of treated mice post-treatment. Survival endpoint was when tumours reach a maximum diameter of 15 mm. No side effects were caused by any of the treatments (*p<0.05, **p<0.01, ***P<0.001 by two-way ANOVA with Dunnett multiple comparisons post-test).

    [0208] FIG. 9 shows ex vivo histopathological assessment of TRAMP-C2 prostate tumours. Tumours were excised at experimental endpoints, snap frozen and subsequently sectioned at 10 μm sections. (a) Composite images of H&E-stained sections indicating necrotic regions and magnified regions of the same images. (b) Composite images from sections stained with NK1.1 (NK cell) and CD3 antibodies. (c) Composite images from sections stained CD8 and KLRA1 (NK cell) antibodies. (d) Composite images from sections stained with CD4 antibody. Nuclei in all fluorescent sections were stained with DAPI.

    [0209] FIG. 10 shows a quantitation of the “vehicle” and “tailed IL-15” histopathological assessment of FIG. 9: a) Necrosis b) CD8+ staining, c) CD4+ staining, d) CD3+ staining and e) NK1.1 (NK cell) staining. Quantitation was based on results obtained from at least 6 animals in each group.

    [0210] FIG. 11 shows proliferation of CTLL-2 cells after incubation with varying concentrations of wild-type IL-15 and modified IL-15 polypeptides SEQ ID NO: 28, SEQ ID NO: 10, and SEQ ID NO: 12 as measured by IL-15 ELISA. Proliferation was measured by MTS assay at an absorbance of 490 nm. N=2.*=p<0.05 by T-Test for SEQ ID NO: 28 vs SEQ ID NO: 10. SEQ ID NO: 28 is significantly more active vs SEQ ID NO: 12 and wild-type IL-15 at all concentrations (p<0.05 by 1 way ANOVA and Tukey test).

    [0211] FIG. 12 shows binding of FITC labelled SEQ ID NO: 28 and wild-type IL-15 to CTLL-2 cells as analysed by flow cytometry using a FACs Calibur (BD Biosciences).

    [0212]

    TABLE-US-00007 SEQUENCE LISTING Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon is optional. (Full-Length Interleukin-15) SEQ ID NO: 1 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKI EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANN SLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Mature Interleukin-15 - Amino Acids 49-162) SEQ ID NO: 2 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Mature Interleukin-15) SEQ ID NO: 3 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Activity-Promoting Seguence) SEQ ID NO: 4 GSGSRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTHHHHHHC (Fusion Polypeptide) SEQ ID NO: 5 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGSRGKSLTSKVP PTVQKPTTVNVPTTEVSPTSQKTTTHHHHHHC (Hydrophilic Peptide) SEQ ID NO: 6 SSKSPSKKDDKKPGDC (Fusion Polypeptide Comprising a Membrane Binding Element) SEQ ID NO: 7 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGSRGKSLTSKVP PTVQKPTTVNVPTTEVSPTSQKTTTHHHHHHC* N-(α,ε bis-myristoyl lysine)SSKSPSKKDDKKPGDC* *indicates the location of the di-sulphide bond between the activity-promoting peptide and membrane binding element. (Nucleic Acid Seguence Encoding SEQ ID NO: 28) SEQ ID NO: 8 AACTGGGTGA ACGTTATCTC GGACCTGAAA AAAATCGAAG ACCTGATCCA AAGCATGCAC ATTGACGCTA CGCTGTATAC GGAAAGCGAT GTGCATCCGT CGTGCAAAGT TACCGCGATG AAATGTTTTC TGCTGGAACT GCAGGTCATT TCGCTGGAAA GCGGCGATGC GAGTATCCAC GACACCGTTG AAAACCTGAT TATCCTGGCC AACAATTCCC TGAGCTCTGG CAATGTGACG GAATCAGGTT GCAAAGAATG TGAAGAACTG GAAGAGAAAA ACATCAAAGA ATTCCTGCAG TCTTTCGTCC ATATTGTGCA AATGTTCATC AATACGAGTG GCTCCGGTTC ACGTGGTAAA TCTCTGACCA GTAAAGTTCC GCCGACGGTC CAAAAACCGA CCACGGTGAA CGTTCCGACC ACCGAAGTCT CTCCGACCAG TCAGAAAACC ACCACCCACC ATCACCATCA TCATTGC (Activity-Promoting Seguence 2) SEQ ID NO: 9  GSGSHHHHHHC (Fusion Polypeptide 2) SEQ ID NO: 10 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSL SSGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGSHHHHHHC (Comparative Fusion Sequence) SEQ ID NO: 11 GSGSRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHHHHHHHC (Comparative Fusion Polypeptide) SEQ ID NO: 12 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSL SSGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGSRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQK TTTKTTTPNAQATRSTPVSRTTKHHHHHHHC  (Fusion Polypeptide 2 Comprisinq a Membrane Bindinq Element) SEQ ID NO: 13 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSL SSGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGSHHHHHHC* N-(α,ε bis-myristoyl lysine) SSKSPSKKDDKKPGDC* *indicates the location of the di-sulphide bond between the activity-promoting peptide and membrane binding element. (Nucleic Acid Sequence Encodinq SEQ ID NO: 28 plus Met) SEQ ID NO: 24 ATGAACTGGGTGA ACGTTATCTC GGACCTGAAA AAAATCGAAG ACCTGATCCA AAGCATGCAC ATTGACGCTA CGCTGTATAC GGAAAGCGAT GTGCATCCGT CGTGCAAAGT TACCGCGATG AAATGTTTTC TGCTGGAACT GCAGGTCATT TCGCTGGAAA GCGGCGATGC GAGTATCCAC GACACCGTTG AAAACCTGAT TATCCTGGCC AACAATTCCC TGAGCTCTGG CAATGTGACG GAATCAGGTT GCAAAGAATG TGAAGAACTG GAAGAGAAAA ACATCAAAGA ATTCCTGCAG TCTTTCGTCC ATATTGTGCA AATGTTCATC AATACGAGTG GCTCCGGTTC ACGTGGTAAA TCTCTGACCA GTAAAGTTCC GCCGACGGTC CAAAAACCGA CCACGGTGAA CGTTCCGACC ACCGAAGTCT CTCCGACCAG TCAGAAAACC ACCACCCACC ATCACCATCA TCATTGC (Full-Lenqth Interleukin-15 Variant) SEQ ID NO: 25 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKI EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANN SLSSGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Mature Interleukin-15 - Amino Acids 49-162 Variant) SEQ ID NO: 26 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Mature Interleukin-15 Variant) SEQ ID NO: 27 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Fusion Polypeptide) SEQ ID NO: 28 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGSRGKSLTSKVPP TVQKPTTVNVPTTEVSPTSQKTTTHHHHHHC (Fusion Polypeptide Comprising a Membrane Binding Element) SEQ ID NO: 29 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGSRGKSLTSKVPP TVQKPTTVNVPTTEVSPTSQKTTTHHHHHHC* N-(α,ε bis-myristoyl lysine) SSKSPSKKDDKKPGDC* *indicates the location of the di-sulphide bond between the activity-promoting peptide and membrane binding element. (Membrane Binding Element) SEQ ID NO: 31 N-(α, εbis-myristoyllysine) SSKSPSKKDDKKPGDC* *indicates the location of the di-sulphide bond between the activity-promoting peptide and membrane binding element.

    EXAMPLES

    Example 1

    [0213] Cytokine Selection

    [0214] Non-adherent PBMCs were cultured for 7 days with irradiated PC3 cells in an 8:1 ratio and stimulated with IL-2, IFN-gamma, IL-12, IL-15 or IL-21 used at ED.sub.50 doses (25 ng/ml for IFN gamma, IL-12, IL-15 and IL-21, and 100 units/ml for IL-2). Expansion of effector cells was measured using anti-CD3, CD56, CD4, CD8, CD25 and FOXP3 antibodies. Results were analysed on a FACSCalibur. NK and CD8 T cell cytotoxic capabilities were assessed by measuring perforin. Apoptotic and necrotic cell death was assessed by staining tumour cells with Annexin-FITC, and propidium iodide using an Annexin/PI kit (Invitrogen).

    [0215] The results showed that IL-15 is superior to other selected Th1 cytokines at activating and expanding NK, NKT and CD8 T cells in co-cultures of PBMCs and prostate cancer cells (FIG. 1). IL-15 was therefore selected for further characterisation and testing as an appropriate therapeutic for treating cancer.

    Example 2

    [0216] Modified IL-15 (Fusion Polypeptide of the Invention)

    [0217] The mature form of human IL-15 was fused to an extended C-terminal sequence shown as SEQ ID NO: 4 and recombinantly expressed in E. coli.

    [0218] The modified form of IL-15 was tested using a CTLL-2 assay (Soman G, Yang X, Jiang H, et al. MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of Interleukin-15: Assay qualification, standardization and statistical analysis. Journal of immunological methods. 2009; 348(1-2):83-94). Briefly, CTLL-2 cells (a mouse CD8 T cell line) were grown in the presence of IL-15. Said cells only proliferate when exposed to Interleukin-2 or Interleukin-15. The cells were cultured at a concentration of 1×10.sup.4 cells/ml in 96 well plates for 48 hours in the presence of a range of doses of IL-15. At the 48 hour time point cells were stained with MTS (5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-2-thiazolyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt), which correlated with the numbers of cells detected.

    [0219] Surprisingly, the modified form of IL-15 was found to have improved activity when compared to the unmodified wild-type IL-15 (see FIG. 2). Thus, the extended C-terminal sequence was found to promote IL-15 activity. Without wishing to be bound by theory, it is believed that the IL-15 activity-promoting sequence may stabilise the interaction of IL-15 with its receptor, thus stimulating CLL-2 cell proliferation.

    Example 3

    [0220] Preparation of Membrane-Anchored IL-15

    [0221] In an attempt to further improve the therapeutic utility of the modified IL-15, it was decided to introduce an additional modification to localise the polypeptide to cell membranes. To achieve this, cytotopic modification was employed. This procedure employs the use of a hydrophobic membrane-insertive myristoyl group, linked by hydrophilically charged amino-acids and a C-terminal-activated disulphide (the combination of these is referred to as the “tail”), which is attached to a protein or peptide directly (through free thiol groups) or indirectly (through thiolated lysine residues) in the latter structure. The reaction creates stable amphipathic compounds which can be tethered to the phosphatidyl-serine rich regions of cell membranes. The tethering process is driven by two non-covalent interactions: one hydrophobic (myristoyl) and one electrostatic (based on lysine residues). Therefore, such agents can localise in any tissue into which they are injected.

    [0222] The modified IL-15 of Example 2 was conjugated to a tail compound, PTL3146 N-(α,ε bis-myristoyl lysine) SSKSPSKKDDKKPGD(S-2-pyridyldithio)-C-acid (SEQ ID NO: 30) (MW of 3 KDa) using a standard procedure: after a mild reduction step (incubation with 100 μM TCEP overnight at room temperature), modified IL-15 was incubated with PTL3146 for an hour at room temperature at a 3:1 molar ratio, followed by overnight dialysis in 1 litre of PBS at 4° C. to remove excess tail.

    [0223] The attachment of the tail to modified IL-15 was confirmed using gel electrophoresis of the untailed and tailed protein using a tail labelled with the fluorophore FAM (Carboxyfluorescein), and western blot analysis using an antibody to IL-15 that recognises active protein (FIG. 3).

    Example 4

    [0224] Confirmation of Binding of Membrane-Anchored IL-15 to Cell Membranes

    [0225] To test the ability of the membrane-anchored IL-15 (tailed IL-15) of Example 3 to bind to cell membranes, assays using sheep red blood erythrocytes or Jurkat cells were employed. These cell types were chosen as they do not have receptors or proteins that can bind IL-15. Binding of tailed IL-15 to these cells was assessed by flow cytometric analysis using a Phycoerythrin (PE) labelled antibody to IL-15. Briefly, the relevant IL-15 polypeptides were incubated with either Jurkat cells or Sheep Red Blood Cells (Cat. Number ABIN770405, antibodies-online). Cells were centrifuged and resuspended in 4 ml of PBS containing 2% FCS to a final concentration of 2×10.sup.6 cells/ml. After dilution, cells were centrifuged at 1800 rpm for 5 minutes at room temperature and the supernatant was discarded. Cells were incubated at room temperature for 20 minutes with 2 μg of either tailed or untailed IL-15. Unbound IL-15 was removed by washing the cells with PBS containing 2% FCS followed by a centrifugation at 1800 rpm for 5 minutes at room temperature. Supernatant was removed and cells were incubated in the dark for 20 minutes at 4° C. with 2 μl of mouse anti-human IL15 PE conjugated antibody (Cat. Number IC2471P, R&D Systems). The washing step was repeated twice, and cells were resuspended in 400 μl PBS containing 2% FCS and analysed by Flow Cytometry.

    [0226] FIG. 4 shows that no binding was seen with untailed IL-15 either on sheep red blood cells (b) or Jurkat cells (a). In contrast membrane-anchored IL-15 (tailed IL-15) exhibited high levels of cell binding, with similar results obtained with 30 min or 24 h incubation of tailed IL-15 on Jurkat cells (b) showing that it can be retained on cell membranes through the tail portion of the molecule for a significant period of time. Internalisation is therefore slow allowing significant cell-surface binding and presentation for activity.

    Example 5

    [0227] Study of the Activity of Membrane-Anchored IL-15 In Vitro

    [0228] The activity of the membrane-anchored modified IL-15 of Example 3 (tailed IL-15) was compared to the non-anchored modified IL-15 of Example 2 (untailed IL-15) and unmodified wild-type control IL-15 using a CTLL2 assay:

    [0229] a) murine CTLL-2 cells (LGC standards, UK [cat no. ATCC® TIB-214™]) were cultured at a concentration of 5×10.sup.5 cells/ml in 96 well plates (5×10.sup.4 cells per well in a volume of 100 ul) for 72 hours in the presence of tailed IL-15, untailed IL-15, or antibody only, or in the absence of any IL-15 polypeptide or antibody (unstained) at 37° C.;

    [0230] b) cells were incubated with MTS (5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-2-thiazolyI)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) (Promega [CellTiter 96® AQueous One Solution Cell Proliferation Assay]) for 3-4 hours (at the 72 hour time point); and

    [0231] c) the number of cells was quantified by colorimetry at an absorbance of 490 nm.

    [0232] FIG. 5 shows that, consistent with the results of Example 2, the non-anchored modified IL-15 (untailed) was significantly more active than wild-type IL-15. However, membrane-anchored modified IL-15 (tailed) was, advantageously, more active than either the untailed or wild-type.

    [0233] The activity of the tailed IL-15 was also confirmed using human and murine NK lymphocytes, which were incubated with tailed and untailed IL-15 to induce their expansion. After 7 days of culture, the NK cell population was analysed by flow cytometry showing that the tailed IL-15 has a greater ability to expand human NK cells (** p<0.05 compared with untailed IL-15 or wild-type IL-15 by one-way ANOVA and Newman-Keuls post-test, n=5) (FIG. 6).

    Example 6

    [0234] Killing of Prostate Cancer Cells by Modified IL-15 and Membrane-Anchored Modified IL-15

    [0235] Both the modified IL-15 (untailed) and membrane-anchored modified IL-15 (tailed) advantageously activated NK cell mediated killing of human prostate cancer cells when compared to the unmodified wild-type (IL-15 pep.) and IL-2 (see FIG. 7). These data confirm that both the modified IL-15 containing the IL-15 activity-promoting sequence (without a membrane-anchor) and the membrane-anchored modified IL-15 are efficacious against cancer cells, especially prostate cancer cells, thereby confirming therapeutic efficacy.

    Example 7

    [0236] Study of the Activity of Modified IL-15 Polypeptides In Vivo

    [0237] The efficacy of modified IL-15 polypeptides of the invention to inhibit tumour growth was further confirmed in an in vivo subcutaneous prostate cancer model in C57BL/6 mice. Male 6-8 week-old C57BL/6 mice were subcutaneously injected with 5×10.sup.6 TRAMP-C2 tumour cells in sterile PBS. When tumours reached 100 mm.sup.3, the mice were injected intratumourally with sterile PBS (Vehicle, n=10), modified IL-15 “untailed IL-15” (n=10), membrane-anchored modified IL-15 “tailed IL-15” (n=10), or with modified IL-15 “untailed IL-15” intraperitoneally (i.p.) (n=6). Tumour growth was measured up to 3 times per week until tumours reached a maximum diameter of 15 mm, at which stage animals were culled.

    [0238] Intratumoural injection of membrane-anchored modified IL-15 “tailed IL-15” and modified IL-15 “untailed IL-15” led to a reduction (50% and 32%, respectively) of tumour growth on day 14 compared with vehicle injection. Intraperitoneal injection of modified IL-15 “untailed IL-15” reduced tumour growth by 16% compared with vehicle (FIG. 8A).

    [0239] Both membrane-anchored modified IL-15 “tailed IL-15” and modified IL-15 “untailed IL-15” increased survival. Membrane-anchored modified IL-15 “tailed IL-15” significantly increased survival to 28 days compared with 17 days in the vehicle group. Modified IL-15 “untailed IL-15” increased survival to 25 days when injected intratumourally and to 19 days when injected i.p (FIG. 8B).

    [0240] Histological analysis of the tumour tissue obtained from the animals showed increased necrosis as seen with H&E staining and increased infiltration of NK cells, CD4 and CD8 T in those animals treated with membrane-anchored modified IL-15 “tailed IL-15” and modified IL-15 “untailed IL-15” compared with PBS groups (FIG. 9). The results were particularly striking for membrane-anchored modified IL-15 “tailed IL-15”, as seen by the quantification provided in FIG. 10.

    Example 8

    [0241] Alternative Modified IL-15 Polypeptides

    [0242] Alternative C-terminal extensions were fused to IL-15 and their activity in the CTLL-2 assay compared to SEQ ID NO: 28 and wild-type IL-15.

    [0243] The first construct was formed by fusing IL-15 to an 11 amino acid sequence (SEQ ID NO: 9) yielding fusion polypeptide SEQ ID NO: 10. The second (comparative) construct was formed by fusing IL-15 to a 67 amino acid sequence (SEQ ID NO: 11) yielding comparative fusion polypeptide SEQ ID NO: 12.

    [0244] The fusion polypeptides were expressed and purified and subsequently tested in the CTLL-2 activity assay as per Example 1.

    Results

    [0245] The proteins were compared using the concentrations of protein as calculated using the IL-15 Elisa Max from Biolegend (London UK) according to the manufacturer's instructions. The IL-15 Elisa measures the IL-15 in the sample that is conformationally correct (i.e. that is recognised by an IL-15 antibody).

    [0246] FIG. 11 shows the activity of SEQ ID NO: 28 compared with SEQ ID NO: 10 and SEQ ID NO: 12, as well as unmodified wild-type IL-15 (Peprotech, UK) in a CTLL-2 assay. SEQ ID NO: 28 is significantly more active compared to the other three proteins, while the construct containing an 11 amino acid C-terminal extension (SEQ ID NO: 10) also showed improved activity versus wild-type IL-15 and comparative construct SEQ ID NO: 12. Thus the 11 amino acid residue sequence also functioned as an IL-15 activity promoting sequence, while the fusion comprising the 67 amino acid residue sequence displayed activity similar to that of wild-type IL-15.

    Example 9

    [0247] Binding of Modified IL-15 to its Receptor

    [0248] To compare binding of SEQ ID NO: 28 and wild-type IL-15 to CTLL-2 cells, the proteins were labelled with Fluorescein isothiocyanate. Briefly, 100 μg of protein prepared at a concentration of 4 mg/ml was dialysed against 200 mM carbonate buffer pH 9.3 for 2 hours; FITC solution prepared at 1 mg/ml was slowly added to IL-15, until an amount of 100 ng for every 1 μg of protein was achieved and IL-15 was then incubated for 2 h with slow rotation at 4° C. A PD10 column was then used to separate free FITC from bound FITC. The protein and FITC concentrations were measured by IL-15 ELISA and Abs Max 495 nm in a spectrophotometer. A 1% solution of BSA was added to the FITC labelled proteins to stabilise the conjugation.

    [0249] CTLL-2 cells were maintained by culturing with 10% TSTIM reagent (Thermofisher, UK). One hundred μl of cells at a concentration of 1×10.sup.6 cells per ml were aliquoted into 96 well plates with the 10% TSTIM reagent and then after 24 hrs, cells were washed twice with 0.2M glycine buffer/0.15 M NaCl (pH=3), followed by a 10 min incubation time, and then a PBS wash. Cells were then blocked for 15 mins with Fc Block (BD biosciences, UK) and then incubated for a further 30 mins with varying concentrations of FITC conjugated SEQ ID NO: 28 or FITC conjugated wild-type IL-15 at 4° C. in PBS containing 0.1% sodium azide. Cells were then washed with PBS and fixed with BD Cytofix (BD Biosciences, UK). Fluorescence intensity of the bound IL-15 was measured on a FACs Calibur flow cytometer (BD Biosciences, UK).

    [0250] Results are presented in FIG. 12, which shows that by adding the activity-promoting sequence to IL-15, the modified IL-15 of the invention exhibits improved binding to its receptor when compared to wild-type IL-15.

    [0251] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.