U.S. patent application number 15/760200 was filed with the patent office on 2019-05-16 for compositions and methods relating to the treatment of diseases.
The applicant listed for this patent is Alfacyte Ltd. Invention is credited to William Stimson.
Application Number | 20190144519 15/760200 |
Document ID | / |
Family ID | 57003529 |
Filed Date | 2019-05-16 |
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United States Patent
Application |
20190144519 |
Kind Code |
A9 |
Stimson; William |
May 16, 2019 |
COMPOSITIONS AND METHODS RELATING TO THE TREATMENT OF DISEASES
Abstract
The present invention relates to compositions and methods for
promoting the induction of a cell-mediated immune response (such as
that mediated by Th1 cells) and the suppression of a humoral or
allergic immune response (such as that mediated by Th2 and Th17
cells). In particular, the invention relates to compositions and
methods for preventing or treating allergy, such as food allergy,
and associated allergic diseases, and conditions where an
exaggerated Th17 response plays a detrimental role, such as
inflammatory responses and autoimmune diseases. The invention
further extends to the use of the compositions of the invention in
the treatment and/or prophylaxis of allergy and associated allergic
diseases and also of cancer.
Inventors: |
Stimson; William; (Glasgow,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alfacyte Ltd |
Lanarkshire |
|
GB |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20180273597 A1 |
September 27, 2018 |
|
|
Family ID: |
57003529 |
Appl. No.: |
15/760200 |
Filed: |
September 14, 2016 |
PCT Filed: |
September 14, 2016 |
PCT NO: |
PCT/GB2016/052841 PCKC 00 |
371 Date: |
March 14, 2018 |
Current U.S.
Class: |
424/85.7 |
Current CPC
Class: |
C07K 19/00 20130101;
C07K 14/56 20130101; A61P 29/00 20180101; C07K 2319/00 20130101;
A61K 38/212 20130101; A61K 39/0011 20130101; A61P 37/08 20180101;
A61K 39/35 20130101; A61P 35/00 20180101; A61P 37/00 20180101 |
International
Class: |
C07K 14/56 20060101
C07K014/56; A61K 38/21 20060101 A61K038/21; A61K 39/35 20060101
A61K039/35; A61K 39/00 20060101 A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 15, 2015 |
GB |
1516303.3 |
Sep 16, 2015 |
GB |
1516437.9 |
Claims
1. A method for the treatment and/or prophylaxis of a condition
where an enhancement of a Th1-mediated immune response and
suppression of a Th2/Th17-mediated immune response are desired,
said method comprising the step of: (i) administering to a subject
in need thereof a therapeutically effective amount of at least one
interferon alpha subtype selected from IFN-.alpha.10, IFN-.alpha.14
and a hybrid thereof, wherein the hybrid includes the primary
interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14.
2. The method as claimed in claim 1 wherein the condition where an
enhancement of a Th1-mediated immune response and suppression of a
Th2/Th17-mediated immune response are desired is selected from the
group consisting of an autoimmune disease, an inflammatory disease,
allergy or an associated allergic condition and cancer.
3. The method as claimed in claim 2 wherein the inflammatory
disease is inflammatory bowel disease.
4. The method as claimed in claim 3 wherein the inflammatory bowel
disease is ulcerative colitis or Crohn's disease.
5. The method as claimed in claim 2 wherein the condition where an
enhancement of a Th1-mediated immune response and suppression of a
Th2/Th17-mediated immune response are desired is allergy or an
associated allergic condition suitably food allergy or an
associated condition.
6. The method as claimed in claim 2 wherein the cancer is hepatic
cell cancer, lung cancer, non-small cell lung cancer, ovarian
cancer, breast cancer, skin cancer, melanoma, genitourinary cancer,
prostate cancer, renal cell cancer, or bladder cancer.
7. The method as claimed in any one of claims 1 to 6 wherein the
method includes a step of administering to the subject a
therapeutically effective amount of a vaccine composition for
treatment or prophylaxis of the condition where an enhancement of a
Th1-mediated immune response and suppression of a Th2/Th17-mediated
immune response are desired.
8. The method as claimed in claim 7 wherein the vaccine composition
comprises at least one allergen capable of mediating a Th2/Th17
immune response.
9. The method as claimed in claim 8 wherein the at least one
allergen is a food allergen or a tumour antigen, for example tumour
specific antigen or tumour-associated antigen.
10. The method as claimed in any one of claims 7 to 9 wherein the
vaccine composition is administered sequentially, separately or
simultaneously with the at least one interferon alpha subtype.
11. The method as claimed in any one of claims 1 to 10 wherein the
at least one interferon alpha subtype is administered orally.
12. The method as claimed in any one of claims 1 to 11 wherein the
at least at least one interferon alpha subtype is IFN-.alpha.10,
IFN-.alpha.14, or an IFN-.alpha.10 IFN-.alpha.14hybrid wherein the
hybrid includes the primary Interferon receptor binding sites of
IFN-.alpha.10 and IFN-.alpha.14 and has improved binding to
interferon receptor 1 and interferon receptor 2 in comparison to
IFN-.alpha.10 or IFN-.alpha.14.
13. The method as claimed in any one of claims 1 to 12 wherein the
hybrid comprises the amino acid sequence set forth by SEQ ID NO: 1
or a fragment or variant thereof.
14. The method as claimed in any one of claims 1 to 13 wherein the
at least one interferon alpha subtype is a hybrid protein
comprising at least part of IFN-.alpha.10 and IFN-.alpha.14 wherein
the hybrid includes at least 1, at least 2, at least 3, at least 4,
at least 5, at least 6 of the amino acids corresponding to primary
interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14.
15. The method as claimed in any one of claims 1 to 14 wherein the
at least one interferon alpha subtype is a recombinant form of
IFN-.alpha.10 and/or IFN-.alpha.14 or a hybrid thereof.
16. The method as claimed in any one of claims 1 to 15 wherein the
hybrid comprises at least one, at least two, at least three, at
least four or at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144.
17. An interferon alpha subtype hybrid comprising the primary
interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14.
18. An interferon alpha subtype comprising the amino acid sequence
of SEQ ID NO: 1 or a fragment or variant thereof.
19. At least one interferon alpha subtype selected from the group
consisting of IFN-.alpha.10, IFN-.alpha.14 and a hybrid thereof
wherein the hybrid comprises the primary interferon receptor
binding sites of IFN-.alpha.10 and IFN-.alpha.14 for use in the
treatment and/or prophylaxis of a condition where an enhancement of
a Th1-mediated immune response and suppression of a
Th2/Th17-mediated immune response are desired.
20. The at least one interferon alpha subtype for use as claimed in
claim 17 wherein the condition where an enhancement of a
Th1-mediated immune response and suppression of a Th2/Th17-mediated
immune response are desired is selected from the group consisting
of an autoimmune disease, an inflammatory disease, allergy or an
associated allergic condition and cancer.
21. The at least one interferon alpha subtype for use as claimed in
claim 20 wherein the inflammatory disease is inflammatory bowel
disease.
22. The at least one interferon alpha subtype for use as claimed in
claim 21 wherein the inflammatory bowel disease is ulcerative
colitis or Crohn's disease.
23. The at least one interferon alpha subtype for use as claimed in
claim 20 wherein the condition where an enhancement of a
Th1-mediated immune response and suppression of a Th2/Th17-mediated
immune response are desired is allergy or an associated allergic
condition preferably wherein the allergy is a food allergy or an
associated allergic condition.
24. The at least one interferon alpha subtype for use as claimed in
claim 20 wherein the cancer is hepatic cell cancer, lung cancer,
non-small cell lung cancer, ovarian cancer, breast cancer, skin
cancer, melanoma, genitourinary cancer, prostate cancer, renal cell
cancer, or bladder cancer.
25. The at least one interferon alpha subtype for use as claimed in
any one of claims 19 to 24 wherein the at least one interferon
alpha subtype is provided for simultaneous, separate or sequential
administration with a vaccine composition for treatment or
prophylaxis of the condition where an enhancement of a Th1-mediated
immune response and suppression of a Th2/Th17-mediated immune
response are desired.
26. The at least one interferon alpha subtype for use as claimed in
claim 25 wherein the vaccine composition comprises at least one
allergen capable of mediating a Th2/Th17 immune response there
against.
27. The at least one interferon alpha subtype for use as claimed in
claim 26 wherein the at least one allergen is a food allergen or a
tumour antigen, for example a tumour specific antigen or a
tumour-associated antigen.
28. The at least one interferon alpha subtype for use as claimed in
any one of claims 19 to 27 wherein the at least one Interferon
alpha subtype is provided for administration orally.
29. The at least one interferon alpha subtype for use as claimed in
any one of claims 19 to 28 wherein the at least one interferon
alpha subtype is IFN-.alpha.10, IFN-.alpha.14, or an IFN-.alpha.10
IFN-.alpha.14 hybrid wherein the hybrid includes the primary
interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14 and has improved binding to interferon receptor 1 and
interferon receptor 2 in comparison to IFN-.alpha.10 or
IFN-.alpha.14.
30. The at least one interferon alpha subtype for use as claimed in
any one of claims 19 to 29 wherein wherein the hybrid comprises the
amino acid sequence set forth by SEQ ID NO: 1 or a fragment or
variant thereof.
31. The at least one interferon alpha subtype for use as claimed in
any one of claims 19 to 30 wherein the at least one interferon
alpha subtype is a hybrid protein comprising at least part of
IFN-.alpha.10 and IFN-.alpha.14 wherein the hybrid includes at
least 1, at least 2, at least 3, at least 4, at least 5, at least 6
of the amino acids corresponding to primary interferon receptor
binding sites of IFN-.alpha.10 and IFN-.alpha.14.
32. The at least one interferon alpha subtype for use as claimed in
any one of claims 19 to 31 wherein the at least one interferon
alpha subtype is a recombinant form of IFN-.alpha.10 and/or
IFN-.alpha.14 or a hybrid thereof.
33. The at least one interferon alpha subtype for use as claimed in
any one of claims 19 to 32 wherein the hybrid comprises at least
one, at least two, at least three, at least four or at least five
mutations selected from amino acids at positions 94, 101, 102, 109
or 144.
34. Use of at least one interferon alpha subtype selected from the
group consisting of IFN-.alpha.10, IFN-.alpha.14 and a hybrid
thereof including the primary interferon receptor binding sites of
IFN-.alpha.10 and IFN-.alpha.14 in the preparation of a medicament
for the treatment and/or prophylaxis of a condition where an
enhancement of a Th1-mediated immune response and suppression of a
Th2/Th17-mediated immune response are desired.
35. The use as claimed in claim 34 wherein the condition where an
enhancement of a Th1-mediated immune response and suppression of a
Th2/Th17-mediated immune response are desired is selected from the
group consisting of an autoimmune disease, an inflammatory disease,
allergy or an associated allergic condition or cancer.
36. The use as claimed in claim 35 wherein the inflammatory disease
is inflammatory bowel disease.
37. The use as claimed in claim 36 wherein the inflammatory bowel
disease is ulcerative colitis or Crohn's disease.
38. The use as claimed in claim 35 wherein the condition where an
enhancement of a Th1-mediated immune response and suppression of a
Th2/Th17-mediated immune response are desired is allergy or an
associated allergic condition, preferably wherein the allergy is a
food allergy or associated allergic condition.
39. The use as claimed in claim 35 wherein the cancer is hepatic
cell cancer, lung cancer, non-small cell lung cancer, ovarian
cancer, breast cancer, skin cancer, melanoma, genitourinary cancer,
prostate cancer, renal cell cancer, or bladder cancer.
40. The use as claimed in any one of claims 34 to 39 wherein the at
least one interferon alpha subtype is provided for simultaneous,
separate or sequential administration with a vaccine
composition.
41. The use as claimed in claim 40 wherein the vaccine composition
comprises at least one allergen capable of mediating a Th2/Th17
immune response there against.
42. The use as claimed in claim 41 wherein the at least one
allergen is a food allergen, or a tumour antigen, for example a
tumour specific antigen or a tumour-associated antigen.
43. The use as claimed in any one of claims 35 to 42 wherein the at
least one interferon alpha subtype is provided for administration
orally.
44. The use as claimed in any one of claims 34 to 43 wherein the at
least one interferon alpha subtype is IFN-.alpha.10, IFN-.alpha.14,
or an IFN-.alpha.10 IFN-.alpha.14 hybrid wherein the hybrid
includes the primary interferon receptor binding sites of
IFN-.alpha.10 and IFN-.alpha.14 and has improved binding to
interferon receptor 1 and interferon receptor 2 in comparison to
IFN-.alpha.10 or IFN-.alpha.14.
45. The use as claimed in any one of claims 34 to 44 wherein the at
least one interferon alpha subtype is a IFN-.alpha.10 IFN-.alpha.14
hybrid and the hybrid comprises the amino acid sequence set forth
by SEQ ID NO: 1 or a fragment or variant thereof.
46. The use as claimed in any one of claims 34 to 45 wherein the at
least one interferon alpha subtype is a hybrid protein comprising
at least part of IFN-.alpha.10 and IFN-.alpha.14 wherein the hybrid
includes at least 1, at least 2, at least 3, at least 4, at least
5, at least 6 of the amino acids corresponding to primary
interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14.
47. The use as claimed in any one of claims 34 to 46 wherein the at
least one interferon alpha subtype is a recombinant form of
IFN-.alpha.10 and/or IFN-.alpha.14.
48. The use as claimed in any one of claims 34 to 47 wherein the
hybrid comprises at least one, at least two, at least three, at
least four or at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144.
49. A composition comprising: (i) a vaccine; and (ii) at least one
interferon alpha subtype selected from IFN-.alpha.10, IFN-.alpha.14
and a hybrid thereof wherein the hybrid includes the primary
interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14.
50. The composition claimed in claim 49 wherein the vaccine
comprises at least one allergen capable of mediating a Th2/Th17
immune response there against.
51. The composition as claimed in claim 50 wherein the at least one
allergen is a food allergen or a tumour antigen, for example a
tumour specific antigen or a tumour-associated antigen.
52. The composition as claimed in any one of claims 49 to 51
wherein the composition is provided for administration orally.
53. The composition as claimed in any one of claims 49 to 52
wherein the at least one interferon alpha subtype is IFN-.alpha.10,
IFN-.alpha.14, or an IFN-.alpha.10 IFN-.alpha.14hybrid wherein the
hybrid includes the primary interferon receptor binding sites of
IFN-.alpha.10 and IFN-.alpha.14 and has improved binding to
interferon receptor 1 and interferon receptor 2 in comparison to
IFN-.alpha.10 or IFN-.alpha.14.
54. The composition as claimed in any one of claims 49 to 53
wherein the at least one interferon alpha subtype is a hybrid of
IFN-.alpha.10 and IFN-.alpha.14.
55. The composition as claimed in any one of claims 49 to 53
wherein the hybrid comprises the amino acid sequence set forth by
SEQ ID NO: 1 or a fragment or variant thereof.
56. The composition as claimed in any one of claims 49 to 55
wherein the at least one interferon alpha subtype is a hybrid
protein comprising at least part of IFN-.alpha.10 and IFN-.alpha.14
wherein the hybrid includes at least 1, at least 2, at least 3, at
least 4, at least 5, at least 6 of the amino acids corresponding to
primary interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14.
57. The composition as claimed in any one of claims 49 to 56
wherein the at least one interferon alpha subtype is a recombinant
form of IFN-.alpha.10 and/or IFN-.alpha.14 or a hybrid thereof.
58. The composition as claimed in any one of claims 49 to 57 for
use in treating or preventing a condition where an enhancement of a
Th1-mediated immune response and suppression of a Th2/Th17-mediated
immune response are desired
59. The composition of claim 58 wherein the condition is selected
from the group consisting of autoimmune disease, inflammatory
disease, allergy or an associated allergic condition or cancer.
60. The composition of claim 59 wherein the cancer is hepatic cell
cancer, lung cancer, non-small cell lung cancer, ovarian cancer,
breast cancer, skin cancer, melanoma, genitourinary cancer,
prostate cancer, renal cell cancer, or bladder cancer.
61. The use as claimed in any one of claims 49 to 60 wherein the
hybrid comprises at least one, at least two, at least three, at
least four or at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144.
62. A pharmaceutical composition, wherein the pharmaceutical
composition comprises a vaccine and at least one interferon alpha
subtype selected from IFN-.alpha.10, IFN-.alpha.14 and a hybrid
thereof wherein the hybrid includes the primary interferon receptor
binding sites of IFN-.alpha.10 and IFN-.alpha.14, along with a
pharmaceutically acceptable excipient, diluent or carrier.
63. The pharmaceutical composition as claimed in claim 61 wherein
the vaccine comprises at least one allergen capable of mediating a
Th2 immune response.
64. A method for the treatment and/or prophylaxis of a condition
mediated by enhanced expression of IL-17, said method comprising
the step of: (i) administering to a subject in need thereof a
therapeutically effective amount of at least one interferon alpha
subtype selected from IFN-.alpha.10, IFN-.alpha.14 and a hybrid
thereof wherein the hybrid includes the primary interferon receptor
binding sites of IFN-.alpha.10 and IFN-.alpha.14.
65. The method as claimed in claim 64 wherein the condition
mediated by enhanced expression of IL-17 is inflammatory bowel
disease.
66. The method as claimed in claim 65 wherein the inflammatory
bowel disease is ulcerative colitis or Crohn's disease.
67. The method as claimed in any one of claims 64 to 66 wherein the
at least one interferon alpha subtype is administered orally.
68. The method as claimed in any one of claims 64 to 66 wherein the
at least at least one interferon alpha subtype is IFN-.alpha.10,
IFN-.alpha.14, or an IFN-.alpha.10 IFN-.alpha.14hybrid wherein the
hybrid includes the primary interferon receptor binding sites of
IFN-.alpha.10 and IFN-.alpha.14 and has improved binding to
interferon receptor 1 and interferon receptor 2 in comparison to
IFN-.alpha.10 or IFN-.alpha.14.
69. The method as claimed in any one of claims 64 to 68 wherein the
hybrid comprises the amino acid sequence set forth by SEQ ID NO: 1
or a fragment or variant thereof.
70. The method as claimed in any one of claims 64 to 69 wherein the
at least one interferon alpha subtype is a hybrid protein
comprising at least part of IFN-.alpha.10 and IFN-.alpha.14 wherein
the hybrid includes at least 1, at least 2, at least 3, at least 4,
at least 5, at least 6 of the amino acids corresponding to primary
interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14.
71. The method as claimed in any one of claims 64 to 70 wherein the
at least one interferon alpha subtype is a recombinant form of
IFN-.alpha.10 and/or IFN-.alpha.14 or a hybrid thereof.
72. The use as claimed in any one of claims 64 to 70 wherein the
hybrid comprises at least one, at least two, at least three, at
least four or at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144.
73. At least one interferon alpha subtype selected from
IFN-.alpha.10, and IFN-.alpha.14 and a hybrid thereof for use in
the treatment and/or prophylaxis of a condition mediated by
enhanced expression of IL-17.
74. The at least one interferon alpha subtype for use as claimed in
claim 73 wherein the condition mediated by enhanced expression of
IL-17 is inflammatory bowel disease preferably wherein the
inflammatory bowel disease is ulcerative colitis or Crohn's
disease.
75. The at least one interferon alpha subtype for use as claimed in
claim 73 wherein the condition is cancer, preferably wherein the
cancer is hepatic cell cancer, lung cancer, non-small cell lung
cancer, ovarian cancer, breast cancer, skin cancer, melanoma,
genitourinary cancer, prostate cancer, renal cell cancer, or
bladder cancer.
76. The at least one interferon alpha subtype for use as claimed in
any one of claims 73 to 75 wherein the at least one interferon
alpha subtype is administered orally.
77. The at least one interferon alpha subtype for use as claimed in
any one of claims 73 to 76 wherein the at least one interferon
alpha subtype is IFN-.alpha.10, IFN-.alpha.14, or an IFN-.alpha.10
IFN-.alpha.14 hybrid wherein the hybrid includes the primary
interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14 and has improved binding to interferon receptor 1 and
interferon receptor 2 in comparison to IFN-.alpha.10 or
IFN-.alpha.14.
78. The at least one interferon alpha subtype for use as claimed in
any one of claims 73 to 77 wherein wherein the hybrid comprises the
amino acid sequence set forth by SEQ ID NO: 1 or a fragment or
variant thereof.
79. The at least one interferon alpha subtype for use as claimed in
any one of claims 73 to 78 wherein the at least one interferon
alpha subtype is a hybrid protein comprising at least part of
IFN-.alpha.10 and IFN-.alpha.14 wherein the hybrid includes at
least 1, at least 2, at least 3, at least 4, at least 5, at least 6
of the amino acids corresponding to primary interferon receptor
binding sites of IFN-.alpha.10 and IFN-.alpha.14.
80. The at least one interferon alpha subtype for use as claimed in
any one of claims 73 to 79 wherein the at least one interferon
alpha subtype is a recombinant form of IFN-.alpha.10 and/or
IFN-.alpha.14 or a hybrid thereof.
81. The at least one interferon alpha subtype for use as claimed in
any one of claims 73 to 80 wherein the hybrid comprises at least
one, at least two, at least three, at least four or at least five
mutations selected from amino acids at positions 94, 101, 102, 109
or 144.
82. Use of at least one interferon alpha subtype selected from
IFN-.alpha.10, IFN-.alpha.14and a hybrid thereof wherein the hybrid
includes the primary interferon receptor binding sites of
IFN-.alpha.10 and IFN-.alpha.14 in the preparation of a medicament
for the treatment and/or prophylaxis of a condition mediated by
enhanced expression of IL-17.
83. The use as claimed in claim 82 wherein the condition mediated
by enhanced expression of IL-17 is inflammatory bowel disease,
preferably, wherein the inflammatory bowel disease is ulcerative
colitis or Crohn's disease.
84. The use as claimed in claim 82 wherein the condition is cancer,
preferably wherein the cancer is hepatic cell cancer, lung cancer,
non-small cell lung cancer, ovarian cancer, breast cancer, skin
cancer, melanoma, genitourinary cancer, prostate cancer, renal cell
cancer, or bladder cancer.
85. The use as claimed in any one of claims 82 to 84 wherein the at
least one interferon alpha subtype is administered orally.
86. The use as claimed in any one of claims 82 to 85 wherein the at
least one interferon alpha subtype is IFN-.alpha.10, IFN-.alpha.14,
or an IFN-.alpha.10 IFN-.alpha.14 hybrid wherein the hybrid
includes the primary interferon receptor binding sites of
IFN-.alpha.10 and IFN-.alpha.14 and has improved binding to
interferon receptor 1 and interferon receptor 2 in comparison to
IFN-.alpha.10 or IFN-.alpha.14.
87. The use as claimed in any one of claims 82 to 86 wherein the at
least one interferon alpha subtype is a IFN-.alpha.10 IFN-.alpha.14
hybrid and the hybrid comprises the amino acid sequence set forth
by SEQ ID NO: 1 or a fragment or variant thereof.
88. The use as claimed in any one of claims 82 to 87 wherein the at
least one interferon alpha subtype is a hybrid protein comprising
at least part of IFN-.alpha.10 and IFN-.alpha.14 wherein the hybrid
includes at least 1, at least 2, at least 3, at least 4, at least
5, at least 6 of the amino acids corresponding to primary
interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14.
89. The use as claimed in any one of claims 82 to 88 wherein the at
least one interferon alpha subtype is a recombinant form of
IFN-.alpha.10 and/or IFN-.alpha.14.
90. The use as claimed in any one of claims 82 to 88 wherein the
hybrid comprises at least one, at least two, at least three, at
least four or at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions and methods
for promoting the induction of a cell-mediated immune response
(such as that mediated by Th1 cells) and the suppression of a
humoral or allergic immune response (such as that mediated by Th2
and Th17 cells). In particular, the invention relates to
compositions and methods for preventing or treating allergy, such
as food allergy, and associated allergic diseases, and conditions
where an exaggerated Th17 response plays a detrimental role, such
as inflammatory responses and autoimmune diseases. The invention
further extends to the use of the compositions of the invention in
the treatment and/or prophylaxis of allergy and associated allergic
diseases and also of cancer.
BACKGROUND TO THE INVENTION
[0002] It is hypothesised that in certain circumstances, the Th1
response or the Th2/Th17 response can cause disease. An
over-reactive Th1 response can generate organ-specific autoimmune
disease such as arthritis, multiple sclerosis, or Type I diabetes,
while an over-reactive Th2/Th17 response may underlie allergy and
atrophy. It is currently believed that Th17 cells play a major role
in host defence against pathogens and an exaggerated Th17 response
may lead to severe inflammatory responses and autoimmune
diseases--inflammatory bowel diseases (IBD), namely, ulcerative
colitis (UC) and Crohn's disease (CD), are chronic inflammatory
processes of the gastrointestinal tract. In these diseases a
disturbed and exaggerated immune response, mainly towards the
endogenous microflora, plays a major role. IL-17 expression is
increased in both UC and CD. Type I IFNs have been studied in
clinical trials in patients with UC and demonstrated efficacy in
selected studies. As anti- viral cytokines, it is now known that
Type I IFNs can regulate the development of Th17 cells.
[0003] It is known that different pathogens induce different
IFN-.alpha. subtypes in vitro and that IFN-.alpha. subtypes have
different antiviral, antiproliferative and immunomodulatory
activities. Infection via a variety of routes, including orally,
has been shown to induce different subtype profiles. IFN-.alpha.
subtypes bind to the same receptor, activate common signaling
pathways and are expected to have the same biological functions.
All IFN-.alpha. subtypes have anti-viral activities, by definition,
although their absolute efficacy in this context may vary
considerably. In addition, many other biological properties have
been described, but with varying potencies, including
immunomodulatory and anti-proliferative activities. The pleiotropic
effects appear to be due to differential interaction with the
receptor chains and signaling through different intracellular
pathways to an array of effector molecules. The Type IFN receptor
consists of two chains, IFNR1 and IFNR2. There is a range of
binding affinities for each of the 12 IFN-.alpha. subtypes with the
different receptor chains.
[0004] IFN-.alpha. may have a key role in the regulation of the Th1
response. It has been shown that IFN-.alpha. treatment promotes Th1
cell differentiation indirectly (largely via IFN-.gamma.), but also
appears to suppress Th2 cell development through the suppression of
IL-4 and IL-13 gene expression. IFN-.alpha. therefore is able to
re-establish a Th1/Th2 population balance in diseases and
infections that promote a Th2 cell imbalance. In recent years, it
became evident that besides its anti-viral effects, several
immunomodulatory functions are exerted by IFN-.alpha.. IFN-.alpha.
can impact on dendritic cell differentiation and controls the
expression of various pro-inflammatory cytokines such as IL-8 or
IL-18 and induces several anti-inflammatory mediators such as IL-1
receptor antagonist (IL-1Ra), soluble TNF receptor p55, IL-10 and
IL-18 binding protein. However, the mechanisms of actions of
IFN-.alpha., and in particular individual IFN-.alpha. subtypes, are
still only partly understood.
[0005] In patients with allergy or allergic disease, a
Th2-predominant immune response is generated. Th2 cells secrete
IL-4 and IL-13 driving B cells to produce Immunoglobulin E (IgE)
antibodies specific to an allergen. An allergen is an antigen
capable of stimulating a type-I hypersensitivity reaction in atopic
individuals mainly through Immunoglobulin E (IgE)-mediated
responses. Following that, IgE binds to its high affinity receptor
on mast cells, skin cells and mucosal tissues. Upon exposure to the
allergen, mast cells release their contents, which include
histamine, leukotrienes and prostaglandins. This causes allergic
symptoms including, but not limited to, red eyes, itchiness, runny
nose, eczema, urticaria, angioedema, shortness of breath, wheezing,
coughing an asthma attack, abdominal pain, vomiting diarrhoea or
even anaphylaxis.
[0006] Allergic diseases are among the most common form of chronic
illness. The World Health Organisation estimates that over 20
percent of the world population is affected and Europe alone has
over 80 million sufferers (Global Allergy and Asthma European
Network, 2008). An allergic reaction is usually caused by
hypersensitivity of the immune system to an allergen, causing a
misdirected immune response. Mild allergies, such as hay fever, are
very common in the human population. Severe allergies can be caused
by dietary allergens, such as food, by environmental allergens,
such as the venom of stinging insects, by medication or can be
genetically determined.
[0007] Food allergy is a major health concern, which is estimated
to affect around 6% of young children and 3-4% of adults in Western
societies. Food allergy is hypothesised to result from a breakdown
in oral tolerance to ingested antigens or allergens. Food allergies
and associated allergic diseases include, but are not limited to,
dairy (milk) allergy, including Heiner syndrome, egg allergy, soya
allergy, fish (shellfish) allergy, peanut and tree nut allergy,
sesame and other seed allergy, gluten (wheat) and grains allergy,
fruit and vegetable allergy, caffeine allergy, oral allergy
syndrome, alcohol allergy, pollen food allergy syndrome,
eosinophilic gastroenteritis, IgE mediated gastrointestinal food
allergy and C1 esterase deficiency.
[0008] Management and treatment of allergic disease is usually via
three general approaches: (i) avoidance of the allergen; (ii)
medications that target disease symptoms and (iii) conventional
immunotherapy, known as desensitisation, which aims to enhance the
Th1 response in established disease. However, these approaches are
far from ideal. Avoidance of allergens is not always possible,
medications that target disease symptoms, such as anti-histamines,
provide only short-term relief and desensitisation involves the use
of the actual allergen, which can result in potentially frequent
harmful side-effects. The possibility of anaphylaxis is never
completely eliminated in patients suffering from allergic diseases
and this causes a great deal of stress to the patient and their
families.
[0009] Interferon subtypes IFN-.alpha.10 and IFN-.alpha.14 and
hybrids thereof are discussed in PCT Publication Number
WO2014/037717 and PCT Application Number PCT/GB2015/050717. In
particular IFN-.alpha.10-IFN-.alpha.14 hybrids are disclosed that
contain sequences characteristic of the IFN-.alpha.10 and
IFN-.alpha.14 subtype binding sites based on a consensus backbone
sequence of all 12 alpha-interferons, for example
TABLE-US-00001 (SEQ ID NO: 3)
CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKAQAISVLHEM
##STR00001## RKYFQRITLYLIERKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD.
It would be advantageous to provide alternative hybrids and further
compositions and methods that provide alternative immunotherapeutic
approaches.
[0010] The present inventor submits that it would be desirable to
develop an improved immunotherapeutic approach which involves safer
use of an allergen, as lower doses may be employed, and provides
longer-term protection against the allergic reaction. Since allergy
results from over-reactivity of Th2/Th17 cells and a corresponding
lack of activity of the Th1 response, a medication that is able to
modify and balance a misdirected Th2/Th17 response would be
beneficial in preventing the allergic reaction. Such a medication
would further be suitable to treat diseases and conditions where an
exaggerated Th17 response plays a role, such as inflammatory bowel
diseases. Additionally, the inventor considers the ability to
enhance of a Th1-mediated immune response and suppress a
Th2/Th17-mediated immune response would be useful in the provision
of compositions that mediate immune response in subjects with
cancer.
SUMMARY OF THE INVENTION
[0011] The present invention relates to the action of cytokines
that promote the induction of a cell-mediated immune response (such
as that mediated by Th1 cells) and cytokines that suppress a
humoral or allergic immune response (such as that mediated by Th2
and Th17 cells). The present invention relates to hybrids of
specific interferon (IFN) subtypes, and in particular to hybrids of
IFN-.alpha.10 and IFN-.alpha.14.
[0012] Following extensive experimentation, the present inventor
has made the surprising discovery that the administration of a
specific interferon alpha (IFN-.alpha.) subtype which is a hybrid
of IFN-.alpha.10 and IFN-.alpha.14, preferably wherein the hybrid
includes the primary interferon receptor binding sites of
IFN-.alpha.10 and IFN-.alpha.14 as part of a composition to
modulate the immune system, such as a vaccine, for example
comprising an allergen, can result in enhanced activation of the
Th1 immune response and suppression of the Th2/Th17 immune
response. In particular, the inventor has developed hybrids of
IFN-.alpha.10 and IFN-.alpha.14 containing higher affinity binding
sites derived from each of IFN-.alpha.10 and IFN-.alpha.14 for the
interferon receptors IFNR1 and IFNR2. In particular, the inventor
demonstrates that it is advantageous to provide
IFN-.alpha.10-IFN-.alpha.14 hybrids with high affinity binding
sites derived from IFN-.alpha.10 and IFN-.alpha.14 subtypes that
are not based on a consensus sequence of all 12 IFN-.alpha.
subtypes which provide SEQ ID NO:3. The hybrids of the present
invention are not based on a consensus backbone sequence of all 12
interferon-alphas. Instead, they derive the sequence
characteristics of IFN-.alpha.10 and IFN-.alpha.14 subtypes without
the sequence characteristics of the other 10 interferon-alpha
subtypes.
[0013] In particular, the inventor has discovered that
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprising or
consisting of at least one mutation selected from amino acids at
positions 94, 101, 102, 109 or 144, (numbering as used in FIG. 16),
in particular 94, 109 or 144 or combinations thereof, preferably at
least two mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least three mutations selected
from amino acids at positions 94, 101, 102, 109 or 144, more
preferably at least four mutations selected from amino acids at
positions 94, 101,102,109 or 144 or more preferably at least five
mutations selected from amino acids at positions 94, 101, 102, 109
or 144 or in particular SEQ ID NO:1 result in higher affinity
binding of the interferon receptors IFNR1 and IFNR2. In
embodiments, the IFN-.alpha.10-IFN-.alpha.14 hybrid sequence can be
SEQ ID NO:3 further comprising the mutation(s) discussed above.
These hybrid sequences can be used in all aspects and embodiments
of the invention.
[0014] The inventor has surprisingly discovered that administration
of the novel IFN-.alpha.10-IFN-.alpha.14 hybrids result in a
greater reduction of IL-17 compared to previous
IFN-.alpha.10-IFN-.alpha.14 hybrids. The inventor has discovered
that administration of the novel IFN-.alpha.10-IFN-.alpha.14
hybrids result in a 10%, preferably a 20%, preferably a 30%,
preferably a 40% and more preferably a 50% greater reduction of
IL-17 compared to previous IFN-.alpha.10-IFN-.alpha.14 hybrids.
[0015] This has led to the identification by the inventor of
improved therapeutic compositions which have utility in the
treatment and/or prophylaxis of allergy and allergic diseases and
diseases and conditions where an exaggerated Th17 response plays a
role and also to cancer. In particular, the inventor has identified
that the administration of at least one food allergen which is
capable of mediating a Th2/Th17 immune response with a hybrid of
IFN-.alpha.10 and IFN-.alpha.14 preferably wherein the hybrid
includes the primary interferon receptor binding sites of
IFN-.alpha.10 and IFN-.alpha.14 can be used in the treatment of
food allergy and associated allergic diseases.
[0016] Moreover, the inventor has identified that the
administration of a tumour antigen, either a tumour associated or a
tumour specific antigen, in combination with a specific interferon
alpha (IFN-.alpha.) subtype which is a hybrid of IFN-.alpha.10 and
IFN-.alpha.14, preferably wherein the hybrid includes the primary
interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14 as part of a composition to modulate the immune
system, such as a vaccine, can be used in the treatment of cancer.
Suitably, the cancer may be hepatic cancer, lung cancer, in
particular non-small cell lung cancer, ovarian cancer, breast
cancer, skin cancer, melanoma or genitourinary cancer.
[0017] Suitably, the tumour associated antigen may be selected from
a prostate tumour, a renal cell tumour and a bladder tumour.
[0018] Accordingly a first aspect of the present invention provides
a method for the treatment and/or prophylaxis of a condition where
an enhancement of a Th1-mediated immune response and suppression of
a Th2/Th17-mediated immune response are desired, said method
comprising the step of: [0019] (i) administering to a subject in
need thereof a therapeutically effective amount of at least one
interferon alpha subtype which is a hybrid of IFN-.alpha.10 and
IFN-.alpha.14 wherein the hybrid includes the primary interferon
receptor binding sites of IFN-.alpha.10 and IFN-.alpha.14.
[0020] Whilst not wishing to be bound by theory, the inventor
believes that proteins comprising the amino acid sequence of
IFN-.alpha.10 have greater affinity to interferon receptor 2
(IFNR2) and proteins comprising the amino acid sequence of
IFN-.alpha.14 have greater affinity to interferon receptor 1
(IFNR1). Thus, substitution of a protein comprising an
IFN-.alpha.10 amino acid sequence with amino acids of IFN-.alpha.14
which allow binding to interferon receptor 1 or substitution of a
protein comprising an IFN-.alpha.14 amino acid sequence with amino
acids of IFN-.alpha.10 which allow binding to interferon receptor 2
is considered to provide a IFN-.alpha.10 IFN-.alpha.14 hybrid
protein which should have stronger binding affinity to both
interferon receptors 1 and 2 than IFN-.alpha.10 or IFN-.alpha.14
alone. By including the primary interferon receptor binding sites
of IFN-.alpha.10 and IFN-.alpha.14 is meant that the hybrid
comprises amino acids selected from IFN-.alpha.10 and substituted
into an IFN-.alpha.14 amino acid sequence to improve the ability of
an IFN-.alpha.14 subtype to bind to an interferon receptor 2 and/or
that the hybrid comprises amino acids selected from IFN-.alpha.14
and substituted into an IFN-.alpha.10 amino acid sequence to
improve the ability of an IFN-.alpha.10 subtype to bind to an
interferon receptor 1.
[0021] Suitably, several amino acid substitutions of protein
comprising an IFN-.alpha.10 amino acid sequence with amino acids of
IFN-.alpha.14 determined to be involved in binding to interferon
receptor 1 may enhance the binding of the protein to interferon
receptor 1. Suitably, an amino acid substitution of protein
comprising an IFN-.alpha.14 amino acid sequence with amino acids of
IFN-.alpha.10 determined to be involved in binding to interferon
receptor 2 may enhance the binding of the protein to interferon
receptor 2.
[0022] In embodiments the IFN-.alpha.10-IFN-.alpha.14 hybrid can
substantially have the amino-acid sequence of IFN-.alpha.10, but be
modified in a region between amino residues 80 to 150, or suitably
between amino acid residues 84 to 144, or suitably amino acid
residues 92 to 115 or suitably between amino acid residues 90 to
110, (utilizing the numbering of the IFN-.alpha.10 sequence
providing in FIG. 16) to provide the amino acids provided by the
IFN-.alpha.14 sequence. It is considered the amino acid residues in
these regions or parts of these regions provide for the binding of
IFN-.alpha.14 to interferon receptor 1. In particular, the hybrid
sequence may include at least one, at least two, at least three, at
least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10 or at least 11 modifications of the IFN-.alpha.10
sequence to provide the corresponding residues of the IFN-.alpha.14
sequence (suitably substituted residues are noted in bold in FIG.
9) or a conserved mutation thereof. In embodiments, eleven
modifications are provided as indicated by the amino acids noted in
bold in FIG. 9. In embodiments, the IFN-.alpha.10-IFN-.alpha.14
hybrid sequence may include at least one mutation selected from
amino acids at positions 94, 101, 102, 109 or 144, preferably at
least two mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least three mutations selected
from amino acids at positions 94, 101, 102, 109 or 144, more
preferably at least four mutations selected from amino acids at
positions 94, 101, 102, 109 or 144 or more preferably at least five
mutations selected from amino acids at positions 94, 101, 102, 109
or 144. In alternative embodiments, IFN-.alpha.14 can be utilised
as a backbone structure of the hybrid and the residues which differ
between the IFN-.alpha.10 and IFN-.alpha.14 sequences at the N and
C terminal regions of the sequences can be provided in the hybrid
sequence as those present in the IFN-.alpha.10 sequence. Suitably
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10 or at
least 11 substitutions of the IFN-.alpha.14 N-terminal sequence may
be made to provide the hybrid sequence to provide residues from
IFN-.alpha.10 at those amino acid positions wherein the amino acids
are not shared/common between IFN-.alpha.10 and IFN-.alpha.14.
Suitably, at least 1, at least 2, or 3 substitutions are provided
at the IFN-.alpha.14 C terminal sequence to provide residues from
IFN-.alpha.10 to the hybrid sequence at those amino acid positions
which are not shared/common between IFN-.alpha.10 and
IFN-.alpha.14. In embodiments at least 1, at least 2, at least 3,
at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10 or at least 11 substitutions from the
N-terminal sequence and at least 1, at least 2, or 3 substitutions
from the C-terminal sequence of the IFN-.alpha.14 are made to
provide residues from IFN-.alpha.10 to the hybrid at those amino
acid positions which have amino acids that are not shared/common
between IFN-.alpha.10 and IFN-.alpha.14.
[0023] In embodiments, the hybrid comprises or consists of an amino
acid sequence SEQ ID NO: 1 or a functionally active fragment or
variant thereof.
[0024] In certain embodiments, the method includes a step of
administering to the subject a therapeutically effective amount of
a vaccine composition for treatment or prophylaxis of the condition
where an enhancement of a Th1-mediated immune response and
suppression of a Th2/Th17-mediated immune response are desired. The
vaccine composition may be administered sequentially, separately or
simultaneously with the at least one interferon alpha subtype.
[0025] By functionally active is meant an IL-.alpha.10 IL-.alpha.14
hybrid peptide comprising the primary interferon binding sites of
IFN-.alpha.10 and IFN-.alpha.14 wherein the administration of
peptide to a subject or expression of peptide in a subject promotes
enhancement of Th1 mediated immune response and suppression of a
Th2/Th17 mediated immune response. Further, functional activity may
be indicated by the ability of a hybrid peptide to enhance a Th1
mediated immune response and to suppress a Th2/Th17 mediated
response.
[0026] A fragment can comprise at least 50, preferably 100 and more
preferably 150 or greater contiguous amino acids from SEQ ID NO: 1
and which is functionally active. Suitably, a fragment may be
determined using, for example, C-terminal serial deletion of cDNA
such as SEQ ID NO: 2 or SEQ ID NO: 3. Said deletion constructs may
then be cloned into suitable plasmids. The activity of these
deletion mutants may then be tested for biological activity as
described herein.
[0027] By variant is meant an amino acid sequence which is at least
70% homologous to SEQ ID NO: 1, more preferably at least 80%
homologous to SEQ ID NO: 1, more preferably at least 90% homologous
to SEQ ID NO: 1, even more preferably at least 95% homologous to
SEQ ID NO: 1, even more preferably at least 96% homologous to SEQ
ID NO: 1, even more preferably at least 97% homologous to SEQ ID
NO: 1 and most preferably at least 98% homology with SEQ ID NO: 1.
A variant encompasses a polypeptide sequence of SEQ ID NO: 1 which
includes substitution of amino acids, especially a substitution(s)
which is/are known for having a high probability of not leading to
any significant modification of the biological activity or
configuration, or folding, of the protein. These substitutions,
typically known as conserved substitutions, are known in the art.
For example the group of arginine, lysine and histidine are known
interchangeable basic amino acids. Suitably, in embodiments amino
acids of the same charge, size or hydrophobicity may be substituted
with each other. Suitably, any substitution may be selected based
on analysis of amino acid sequence alignments of interferon alpha
subtypes to provide amino acid substitutions to amino acids which
are present in other alpha subtypes at similar or identical
positions when the sequences are aligned. Hybrids, and variants and
fragments thereof may be generated using suitable molecular biology
methods as known in the art.
[0028] In certain embodiments, the vaccine composition comprises at
least one antigen. In certain embodiments, the vaccine composition
comprises at least one allergen capable of mediating a Th2/Th17
immune response, for example, a food allergen.
[0029] In aspects and embodiments of the invention the antigen can
be a tumour antigen, for example a tumour specific antigen or a
tumour associated antigen, in particular a tumour antigen can be of
a hepatic carcinoma, lung cancer, in particular non-small cell lung
cancer, ovarian cancer, breast cancer, skin cancer, melanoma or of
a genitourinary cancer. In particular an antigen of a genitourinary
cancer can include an antigen from a prostate cancer, renal cell
carcinoma, or bladder cancer. Suitably, an antigen may be a tumour
specific antigen or tumour associated antigen provided in an
existing cancer vaccine in use or development which would benefit
from an adjuvant that enhances T-cell immunity, in particular that
enhances a Th1 response or provides an enhancement of a Th1
mediated immune response and suppression of a Th2/Th17-mediated
immune response. Suitably a tumour specific or tumour-associated
antigen may be obtained from a tumour of a subject to be treated.
In embodiments only a tumour-associated antigen can be used.
[0030] In embodiments a tumour antigen, in particular an associated
antigen may be an antigen for a prostate cancer antigen, in
particular prostate-specific antigen. Suitably a method of
providing a prostate specific antigen or a prostate cancer antigen
with the interferon-alpha subtypes of the invention maybe used to
treat prostate cancer, specifically castration-resistant prostate
cancer.
[0031] As will be appreciated by a physician, the subjects who will
benefit most from such treatments may be those with minimal
disease, as there may be less chance of increasing tumour
suppression of the immune system, additionally or alternatively
such treatments may benefit subjects with advanced disease who may
have significant tumour immune suppression and may benefit more
from the use of vaccines in combination with other forms of
treatment. Suitably the use of vaccines including tumour antigens,
in particular tumour associated antigen may be in combination with
other forms of immunotherapy, for example Sunitinib (Sutent by
Pfizer) a tyrosine kinase inhibitor.
[0032] In embodiments specific tumour antigens, in particular
tumour-associated antigens may be selected from the antigens
utilised in the prostate cancer vaccines TroVax and Prostvac.
[0033] In embodiments a tumour antigen, in particular a
tumour-associated antigen can be selected from renal cell
carcinoma. Suitably a tumour antigen, for example a
tumour-associated antigen for renal cell carcinoma may be selected
from a heat shock protein or proteins of renal tumour cell lysates,
in particular the antigen used in the potential vaccine
MVA-5T4.
[0034] Suitably a tumour antigen may be MUC1 from melanoma.
[0035] In embodiments, a tumour antigen, for example a
tumour-associated antigen can be selected from bladder cancer.
Suitably a tumour-associated antigen may be selected from Bacille
Calmette-Guerin (BCG) vaccine, human leukocyte antigen--A*2402
restricted epitope peptides, immucin peptide (a 21mer synthetic
vaccine composed of the entire signal peptide of the MUCI protein)
human chorionic gonadotropin-colony stimulating factor, or human
chorionic gonadotropin-beta.
[0036] In certain embodiments, the method therefore includes a step
of administering to the subject a therapeutically effective amount
of at least one allergen capable of mediating a Th2/Th17 immune
response, for example, a food allergen or tumour antigen, for
example a tumour associated antigen. The allergen maybe
administered sequentially, separately or simultaneously with the at
least one interferon alpha subtype.
[0037] Typically, the subject is a mammal, in particular a human.
In certain embodiments, the subject can be suffering from a
condition where an enhancement of a Th1-mediated immune response
and suppression of a Th2/Th17-mediated immune response are
desired.
[0038] According to a second aspect of the present invention, there
is provided at least one interferon alpha subtype which is a hybrid
of IFN-.alpha.10 and IFN-.alpha.14 wherein the hybrid comprises the
primary interferon binding sites of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, in particular SEQ ID NO:1 or a
fragment or variant thereof for use in the treatment and/or
prophylaxis of a condition where an enhancement of a Th1-mediated
immune response and suppression of a Th2/Th17-mediated immune
response are desired.
[0039] In certain embodiments, the at least one interferon alpha
subtype, in particular a hybrid IFN-.alpha.10 and IFN-.alpha.14
subtype, for example SEQ ID NO: 1, as described herein is provided
for simultaneous, separate or sequential administration with a
vaccine composition for treatment or prophylaxis of the condition
where an enhancement of a Th1-mediated immune response and
suppression of a Th2/Th17-mediated immune response are desired. In
certain embodiments, the at least one interferon alpha subtype is
provided for simultaneous, separate or sequential administration
with at least one allergen capable of mediating a Th2/Th17 immune
response there against, for example, a food allergen, or a tumour
antigen, in particular a tumour-associated antigen.
[0040] According to a third aspect of the present invention, there
is provided use of at least one interferon alpha subtype which is a
hybrid of IFN-.alpha.10 and IFN-.alpha.14 wherein the hybrid
comprises the primary interferon binding sites of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, in particular wherein the
hybrid can be SEQ ID No: 1 or a variant or fragment thereof in the
preparation of a medicament for the treatment and/or prophylaxis of
a condition where an enhancement of a Th1-mediated immune response
and suppression of a Th2/Th17-mediated immune response are
desired.
[0041] In certain embodiments, the at least one interferon alpha
subtype is provided for simultaneous, separate or sequential
administration with a vaccine composition for treatment or
prophylaxis of the condition where an enhancement of a Th1-mediated
immune response and suppression of a Th2/Th17-mediated immune
response are desired. In certain embodiments, the at least one
interferon alpha subtype is provided for simultaneous, separate or
sequential administration with at least one allergen capable of
mediating a Th2/Th17 immune response there against, for example, a
food allergen, or a tumour antigen, in particular a tumour
associated antigen.
[0042] According to a further aspect of the present invention,
there is provided a composition comprising: [0043] (i) a vaccine
for treatment or prophylaxis of a condition where an enhancement of
a Th1-mediated immune response and suppression of a
Th2/Th17-mediated immune response are desired; and [0044] (ii) at
least one interferon alpha subtype which is a hybrid of
IFN-.alpha.10 and IFN-.alpha.14, in particular wherein the hybrid
comprises the primary interferon binding sites of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101,102,109 or
144, preferably at least two mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, more preferably at least three
mutations selected from amino acids at positions 94, 101, 102,109
or 144, more preferably at least four mutations selected from amino
acids at positions 94, 101, 102, 109 or 144 or more preferably at
least five mutations selected from amino acids at positions 94,
101, 102, 109 or 144, in particular wherein the hybrid can be SEQ
ID NO:1 or a variant or fragment, as described herein.
[0045] In certain embodiments, the vaccine comprises at least one
allergen capable of mediating a Th2/Th17 immune response, for
example, a food allergen or a tumour antigen, in particular a
tumour-associated antigen.
[0046] A further aspect of the present invention provides a
pharmaceutical composition for enhancement of a Th1 mediated immune
response and suppression of a Th2/Th17-mediated immune response,
wherein the composition comprises a vaccine for treatment or
prophylaxis of a condition where an enhancement of a Th1-mediated
immune response and suppression of a Th2/Th17-mediated immune
response are desired and at least one interferon alpha subtype
which is a hybrid of IFN-.alpha.10 and IFN-.alpha.14, in particular
wherein the hybrid comprises the primary interferon binding sites
of IFN-.alpha.10 and IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least Five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, in particular wherein the
hybrid can be SEQ ID NO:1 or a fragment or variant thereof along
with a pharmaceutically acceptable excipient, diluent or
carrier.
[0047] In certain embodiments, the vaccine comprises at least one
allergen capable of mediating a Th2/Th17 immune response, for
example, a food allergen or tumour antigen, in particular a
tumour-associated antigen.
[0048] In a further aspect, the present invention extends to
improvements in the efficacy of vaccines, for example, anti-allergy
or allergic disease vaccines or tumour or cancer vaccines, in
particular genitourinary cancer vaccines, for example prostate
cancer, renal cancer and or bladder cancer. A composition which
comprises a vaccine for treatment or prophylaxis of a condition
where an enhancement of a Th1-mediated immune response and
suppression of a Th2/Th17-mediated immune response are desired,
such as at least one allergen capable of mediating a Th2/Th17
immune response, and at least one interferon alpha subtype which is
a hybrid of IFN-.alpha.10 and IFN-.alpha.14 in particular wherein
the hybrid comprises the primary interferon binding sites of
IFN-.alpha.10 and IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, in particular SEQ ID NO:1 or a
variant or fragment thereof, has been surprisingly identified by
the inventor as providing an unexpectedly efficacious composition
for the treatment and/or prophylaxis of diseases, such as allergy
or associated allergic diseases.
[0049] Accordingly, a further aspect of the present invention
provides a vaccine composition comprising;
(i) a vaccine for treatment or prophylaxis of a condition where an
enhancement of a Th1-mediated immune response and suppression of a
Th2/Th17-mediated immune response are desired; and (ii) at least
one interferon alpha subtype which is a hybrid of IFN-.alpha.10 and
IFN-.alpha.14 in particular wherein the hybrid comprises the
primary interferon binding sites of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, in particular can be SEQ ID
NO:1 or a variant or fragment thereof.
[0050] In certain embodiments, the vaccine comprises at least one
allergen capable of mediating a Th2/Th17 immune response, for
example, a food allergen or a tumour antigen, in particular a
tumour-associated antigen.
[0051] A further aspect of the present invention provides a vaccine
composition for use in the treatment and/or prophylaxis of allergy
or cancer, in particular genitourinary cancer, for example prostate
cancer, renal cancer or bladder cancer, where an enhancement of a
Th1-mediated immune response and the suppression of a
Th2/Th17-mediated immune response are desired, said vaccine
composition comprising;
(i) at least one allergen capable of mediating a Th2/Th17 immune
response; and (ii) at least one interferon alpha subtype which is a
hybrid IFN-.alpha.10 and IFN-.alpha.14 subtype, in particular
wherein the hybrid comprises the primary interferon binding sites
of IFN-.alpha.10 and IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102,109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, in particular wherein the
hybrid can be SEQ ID NO:1 or a variant or fragment, as described
herein.
[0052] A further aspect of the present invention provides for the
use of a vaccine composition comprising at least one allergen
capable of mediating a Th2/Th17 immune response and at least one
interferon alpha subtype which is a hybrid of IFN-.alpha.10 and
IFN-.alpha.14 in particular wherein the hybrid comprises the
primary interferon binding sites of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102,109 or 144, in particular wherein the hybrid
can be SEQ ID NO:1 or a variant or fragment thereof, in the
preparation of a medicament for the treatment and/or prophylaxis of
allergy or associated allergic diseases, or cancer, in particular
genitourinary cancer, for example prostate cancer, renal cancer or
bladder cancer.
[0053] A further aspect of the present invention provides a method
for the treatment and/or prophylaxis of allergy or associated
allergic diseases or of cancer, in particular genitourinary cancer
for example prostate cancer, renal cancer or bladder cancer the
method comprising the step of: [0054] (i) administering a
therapeutically effective amount of a vaccine composition or an
immunogenic composition which comprises at least one allergen
capable of mediating a Th2/Th17 immune response and at least one
interferon alpha subtype which is a hybrid of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the hybrid comprises the
primary interferon binding sites of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, in particular wherein the
hybrid can be SEQ ID NO: 1 or a fragment or variant thereof to a
subject in need thereof.
[0055] According to a further aspect of the present invention,
there is provided a method for the treatment and/or prophylaxis of
a condition mediated by enhanced expression of IL-17, said method
comprising the step of: [0056] (i) administering to a subject in
need thereof a therapeutically effective amount of at least one
interferon alpha subtype which is a hybrid of IFN-.alpha.10 and
IFN-.alpha.14 in particular wherein the hybrid comprises the
primary interferon binding sites of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, in particular SEQ ID NO:1 or a
fragment or variant thereof.
[0057] According to a further aspect of the present invention,
there is provided at least one interferon alpha subtype comprising
or consisting of an IFN-.alpha.10 and IFN-.alpha.14 hybrid in
particular wherein the hybrid comprises the primary interferon
binding sites of IFN-.alpha.10 and IFN-.alpha.14, in particular
wherein the IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises
at least one mutation selected from amino acids at positions 94,
101, 102, 109 or 144, preferably at least two mutations selected
from amino acids at positions 94, 101, 102, 109 or 144, more
preferably at least three mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, more preferably at least four
mutations selected from amino acids at positions 94, 101, 102, 109
or 144 or more preferably at least five mutations selected from
amino acids at positions 94, 101, 102, 109 or 144, in particular
SEQ ID NO:1 or a variant or fragment thereof for use in the
treatment and/or prophylaxis of a condition mediated by enhanced
expression of IL-17.
[0058] Suitably, in aspects and embodiments of the invention, the
hybrid may comprise or consist of the amino acid sequence of SEQ ID
NO:1.
[0059] According to a further aspect of the present invention,
there is provided use of at least one interferon alpha subtype
IFN-.alpha.10 and IFN-.alpha.14 hybrid in particular wherein the
hybrid comprises the primary interferon binding sites of
IFN-.alpha.10 and IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least Five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, in particular SEQ ID NO:1 or a
variant or fragment thereof, in the preparation of a medicament for
the treatment and/or prophylaxis of a condition mediated by
enhanced expression of IL-17.
[0060] According to a further aspect of the present invention,
there is provided a method for modulating an immune response, said
method comprising the step of: [0061] (i) administering to a
subject in need thereof a therapeutically effective amount of at
least one interferon alpha subtype IFN-.alpha.10 and IFN-.alpha.14
hybrid, wherein the hybrid comprises the primary interferon binding
sites of IFN-.alpha.10 and IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, and in particular can be SEQ ID
NO:1 or a variant or fragment thereof.
[0062] Suitably, in aspects and embodiments of the invention, the
administration or use of at least one interferon alpha subtype
comprising or consisting of an IFN-.alpha.10 and IFN-.alpha.14
hybrid in particular wherein the hybrid comprises the primary
interferon binding sites of IFN-.alpha.10 and IFN-.alpha.14, in
particular wherein the IFN-.alpha.10-IFN-.alpha.14 hybrid sequence
comprises at least one mutation selected from amino acids at
positions 94, 101, 102, 109 or 144, preferably at least two
mutations selected from amino acids at positions 94, 101, 102, 109
or 144, more preferably at least three mutations selected from
amino acids at positions 94, 101, 102, 109 or 144, more preferably
at least four mutations selected from amino acids at positions 94,
101, 102, 109 or 144 or more preferably at least five mutations
selected from amino acids at positions 94, 101, 102, 109 or 144, in
particular SEQ ID NO:1 or a variant or fragment thereof results in
the full or partial inhibition of IL-17 and/or the full or partial
activation of IFN-.gamma..
[0063] According to a further aspect of the present invention,
there is provided at least one interferon alpha subtype
IFN-.alpha.10 and IFN-.alpha.14 hybrid, wherein the hybrid
comprises the primary interferon binding sites of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, and in particular can be SEQ ID
NO:1 or a variant or fragment thereof for use in modulating an
immune response.
[0064] According to a further aspect of the present invention,
there is provided use of at least one interferon alpha subtype
hybrid IFN-.alpha.10 and IFN-.alpha.14 subtype, wherein the hybrid
comprises the primary interferon binding sites of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, and in particular SEQ ID NO:1
or a variant or fragment thereof in the preparation of a medicament
for modulating an immune response.
[0065] In certain embodiments of the aspects of the invention
outlined above, the at least one interferon alpha subtype is
provided for simultaneous, separate or sequential administration
with a vaccine for treatment or prophylaxis of the condition where
an enhancement of a Th1-mediated immune response and suppression of
a Th2/Th17-mediated immune response are desired, for example, a
vaccine for the treatment or prophylaxis of a condition mediated by
enhanced expression of IL-17, e.g. an inflammatory disease or
condition or an autoimmune disease, such as inflammatory bowel
disease (IBD), ulcerative colitis (UC) or Crohn's disease (CD),
cancer, suitably hepatic cancer, lung cancer, in particular
non-small cell lung cancer, ovarian cancer, breast cancer, skin
cancer, melanoma or genitourinary cancer, in particular
genitourinary cancer, for example prostate cancer, renal cancer or
bladder cancer. In certain embodiments, the vaccine composition
comprises at least one antigen. In certain embodiments, the vaccine
comprises at least one allergen capable of mediating a Th2/Th17
immune response there against, for example, a food allergen.
[0066] In certain embodiments the antigen can be a tumour antigen
in particular a tumour specific and/or a tumour-associated
antigen.
[0067] In certain embodiments of the aspects of the invention
outlined above, the at least one IFN-.alpha. subtype comprises,
consists of or is an IFN-.alpha.10 IFN-.alpha.14 hybrid such as a
fusion protein, or recombinant protein or the like which includes
the primary interferon receptor binding sites of IFN-.alpha.10 and
IFN-.alpha.14, and in particular which comprises or consists of the
amino acid sequence SEQ ID NO:1 or a variant or fragment thereof.
In embodiments the IFN-.alpha.10 IFN-.alpha.14 hybrid can be
glycosylated. Suitably the IFN-.alpha.10 IFN-.alpha.14 hybrid can
be glycosylated in a similar fashion to IFN-.alpha.14.
[0068] In certain embodiments of the aspects of the invention
outlined above, the at least one allergen is at least one food
allergen or a tumour specific or tumour-associated tumour allergen,
for example a prostate cancer allergen, a renal cancer allergen and
or bladder cancer allergen. In certain embodiments, the at least
one allergen is a dietary allergen such as food, an environmental
allergen such as the venom of stinging insects, or a
medication.
[0069] In a further aspect of the invention there is provided a
recombinant polypeptide comprising or consisting of SEQ ID NO:1 or
a fragment or variant thereof. Nucleic acid sequences derived from
the amino acid sequence SEQ ID NO:1 are provided as SEQ ID NO:2.
These nucleic acid sequences can form additional aspects to the
invention.
[0070] In certain embodiments of the aspects of the invention
outlined above, the at least one food allergen is selected from the
group consisting of, but not limited to, corn, garlic, oats,
coffee, chocolate, pickle, wheat or gluten and their products or
derivatives which include durum wheat, spelt (triticum spelta),
kamut (triticum poloncium), couscous, bran, wheat bran, wheat germ,
wheat gluten, farina, rusk, semolina, durum wheat semolina, flour,
wholewheat flour, wheat flour, wheat starch, starch, modified
starch, hydrolysed starch, food starch, edible starch, vegetable
starch, vegetable gum, vegetable protein, cereal filler, cereal
binder, cereal protein; tree nuts (including almonds, cashews,
macademia, walnut and brazil nuts); seeds, including sesame,
sunflower and poppy seeds; dairy derived antigens, such as milk or
milk derivatives, including cheese and yoghurt; fish or shellfish
or their derivatives, including from the mollusc phylum (gastropod
class: snails and abalone; bivalve class: clam, mussel and oyster;
cephalopod class: octopus, squid and scallop), arthropod phylum
(crustacean family: crab, lobster, shrimp, prawn and crayfish) or
chordate phylum (cartilaginous family: ray and shark; bony fish:
cod, salmon and tuna); eggs or egg derivatives; monosodium
glutamate (MSG); sulphites or sulphur dioxide; legume allergies to
the leguminosae family, which includes peanut, soya (soybean or
soya derivatives), bean seeds, peas, green beans, lentils, carob
and liquorice; other vegetable allergies such as potato; fruit
allergies to the rosaceae family, which includes apple, pear,
cherry, peach and plum; fruit allergies to the cucurbitaceae
family, which includes cucumber, melon, watermelon, zucchini and
pumpkin; and other fruit allergies such as those developed against
kiwi, banana, avocado, tomatoes, strawberries and raspberries.
[0071] In certain embodiments, the vaccine or vaccine composition
can be a vaccine composition for the treatment or prophylaxis of a
condition mediated by enhanced expression of IL-17, e.g. an
inflammatory disease or condition or an autoimmune disease, such as
inflammatory bowel disease (IBD), ulcerative colitis (UC) or
Crohn's disease (CD), or cancer, suitably hepatic cancer, lung
cancer, non-small cell lung cancer, ovarian cancer, breast cancer,
skin cancer, melanoma or genitourinary cancer, in particular
genitourinary cancer, for example prostate cancer, renal cancer or
bladder cancer. In certain embodiments, the vaccine or vaccine
composition can be a vaccine composition for the treatment or
prophylaxis of an inflammatory disease or condition or an
autoimmune disease, such as inflammatory bowel disease (IBD),
ulcerative colitis (UC) or Crohn's disease (CD).
[0072] In certain embodiments of the aspects of the invention
outlined above, the condition where an enhancement of a
Th1-mediated immune response and the suppression of a
Th2/Th17-mediated immune response arc desired can be a condition
mediated by enhanced expression of IL-17, e.g. an inflammatory
disease or condition or an autoimmune disease, such as inflammatory
bowel disease (IBD), ulcerative colitis (UC) or Crohn's disease
(CD).
[0073] In certain embodiments of the aspects of the invention
outlined above, the condition where an enhancement of a
Th1-mediated immune response and the suppression of a
Th2/Th17-mediated immune response are desired can be an
inflammatory disease, in particular an inflammatory disease which
is mediated by an exaggerated or overactive Th17 immune response.
In certain embodiments of the aspects of the invention outlined
above, the condition where an enhancement of a Th1-mediated immune
response and the suppression of a Th2/Th17-mediated immune response
are desired can be an autoimmune disease, in particular an
autoimmune disease which is mediated by an exaggerated or
overactive Th17 immune response. For example, in certain
embodiments the condition can be inflammatory bowel disease (IBD),
such as ulcerative colitis (UC) or Crohn's disease (CD). In certain
embodiments, the condition can be selected from the group
consisting of asthma, allergic rhinitis, atopic dermatitis and food
allergy. In certain embodiments, the condition is cancer, in
particular a genitourinary cancer, in particular prostate cancer,
bladder cancer or renal cancer.
[0074] In certain embodiments of the aspects of the invention
outlined above, the condition where an enhancement of a
Th1-mediated immune response and the suppression of a
Th2/Th17-mediated immune response are desired is an allergy or
associated allergic diseases and conditions caused thereby, or
cancer wherein an immune response is desired against a
tumour-associated antigen, in particular a tumour associated
antigen of prostate cancer, renal cancer or bladder caner. In
particular, in certain embodiments the condition is a food allergy
including food associated or derived allergies and associated
allergic diseases and conditions caused thereby.
[0075] In certain embodiments, the food allergy associated allergic
diseases or conditions include, but are not limited to, milk/dairy
allergy, including Heiner syndrome, egg allergy, soya allergy, fish
(shellfish) allergy, peanut and tree nut allergy, sesame and other
seed allergy, wheat and grains allergy, fruit and vegetable
allergy, caffeine allergy, oral allergy syndrome, alcohol allergy,
pollen food allergy syndrome, eosinophilic gastroenteritis, IgE
mediated gastrointestinal food allergy and C1 esterase
deficiency.
[0076] In certain embodiments of the present invention, the method
of administration is oral administration. In certain embodiments,
the method of administration is sublingual or buccal
administration. In certain embodiments, the method of
administration involves placing a lozenge under the patient's
tongue. In certain embodiments, the route of administration is
ocular or by means of introduction into the nasal cavity, by way of
nasal administration. Also it may be introduced by oral
administration (swallowing) of a capsule or similar device into the
small intestine/duodenum such that the capsule does not dissolve in
the stomach, but bypasses same and delivers/releases the interferon
alpha subtype only into the small intestine/duodenum.
DETAILED DESCRIPTION OF THE INVENTION
[0077] The inventor of the present invention has surprisingly
discovered that administering an IFN-.alpha. subtype which is a
hybrid of IFN-.alpha.10 and IFN-.alpha.14 subtypes, for example SEQ
ID NO:1, as described herein results in the enhancement of a Th1 T
cell mediated immune response and the suppression of a Th2/Th17 T
cell mediated immune response and can therefore skew the immune
response towards a cell-mediated (Th1) path, whilst simultaneously
suppressing the allergic (Th2/Th17) response. Surprisingly, this
effect is enhanced when the IFN-.alpha. subtype is administered
orally.
[0078] In particular, the inventor discovered that
IFN-.alpha.10-IFN-.alpha.14 hybrids that contain sequences
characteristic of the IFN-.alpha.10 and IFN-.alpha.14 subtype
binding sites that are not based on a consensus sequence of all 12
IFN-.alpha. subtypes resulted in a protein with higher affinity
binding sites for the two interferon receptors, IFNR1 and IFNR2.
This finding can be applied to provide an improved method and
improved adjuvant composition for treating and/or preventing
conditions where the enhancement of a Th1 T cell mediated immune
response and/or the suppression of a Th2/Th17 T cell mediated
immune response are desired, for example, inflammatory, autoimmune
or allergy conditions, or cancer (including malignant conditions),
in particular genitourinary cancers, in particular prostate cancer,
renal cancer or bladder cancer. In particular, the hybrid of
IFN-.alpha.10 and IFN-.alpha.14, in particular wherein the hybrid
comprises the primary interferon binding sites of IFN-.alpha.10 and
IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, and in particular SEQ ID NO:1
or a fragment or variant thereof may be used as an adjuvant in
vaccines to boost immune response to antigens and direct the immune
response towards a Th1 immune response.
[0079] The inventor has also discovered that a combination of a
vaccine composition or a food or tumour specific or
tumour-associated antigen allergen which is capable of mediating a
Th2/Th17 immune response and an IFN-.alpha. subtype which is a
hybrid of IFN-.alpha.10 and IFN-.alpha.14, in particular a hybrid
comprising the primary interferon binding sites of IFN-.alpha.10
and IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, and in particular SEQ ID NO:1
or a fragment or variant thereof can result in the activation of a
Th1 T cell mediated immune response and the suppression of a
Th2/Th17 T cell mediated immune response.
[0080] Tumour progression in normal immunocompetent subjects may
reflect a failure of the immune system to recognize the tumour
antigens or a subversion of the anti-tumour immune response through
induction and activation of regulatory T cells. In subjects with
hepatic choriocarcinoma (HCC) studies of IL-17 .alpha. cells have
suggested a potential pro-tumour role for IL-17. Increased IL-17
producing cell density within the tumours of HCC patients
correlates with both microvessel density and poor prognosis.
Further, in subjects with non-small cell lung and ovarian cancer,
higher levels of IL-17 within the tumour correlated with higher
blood vessel density and shorter survival. Additionally IL-17 has
been suggested to have pro-angiogenic roles and this has not been
restricted to particular cell populations. Moreover, it has been
shown that IL-17A or IL-17A producing cells are elevated in the
environment of breast tumours and correlate with poor
prognosis.
[0081] Isolation of tumour infiltrating lymphocytes (TILS) from
breast cancer biopsies revealed these cells secreted significant
amounts of IL-17A, and that recombinant IL-17A recruits the MAPK
pathway by upregulating phosphorylated ERK 1/2 in human breast
cancer lines thereby promoting proliferation and resistance to
conventional chemotherapeutic agents such as Docetaxel. IL-17A has
also been indicated to stimulate migration and invasion of breast
cancer cells. Importantly IL-17A-neutralizing antibodies abrogated
these effects, demonstrating the pathophysiological role of IL-17A
as a potential therapeutic target for breast cancer. The inventor
has surprisingly discovered that administration of the novel
IFN-.alpha.10-IFN-.alpha.14 hybrid result in a greater reduction of
IL-17 compared to previous IFN-.alpha.10-IFN-.alpha.14 hybrid. The
inventor has discovered that administration of the novel
IFN-.alpha.10-IFN-.alpha.14 hybrid results in a 10%, preferably a
20%, preferably a 30%, preferably a 40% and more preferably a 50%
greater reduction of IL-17 compared to previous
IFN-.alpha.10-IFN-.alpha.14 hybrid. The determination by the
inventor or means thus to activate a Th1 T cell mediated immune
response and suppress a Th2/Th17 T cell mediated immune response is
therefore significant and of utility in cancer. Thus, the present
invention may be used for the treatment and prophylaxis of any
known cancerous or malignant condition.
[0082] In addition, the inventor has discovered that the
administration or use of at least one interferon alpha subtype
comprising or consisting of an IFN-.alpha.10 and IFN-.alpha.14
hybrid in particular wherein the hybrid comprises the primary
interferon binding sites of IFN-.alpha.10 and IFN-.alpha.14, in
particular wherein the IFN-.alpha.10-IFN-.alpha.14 hybrid sequence
comprises at least one mutation selected from amino acids at
positions 94, 101, 102, 109 or 144, preferably at least two
mutations selected from amino acids at positions 94, 101, 102, 109
or 144, more preferably at least three mutations selected from
amino acids at positions 94, 101, 102, 109 or 144, more preferably
at least four mutations selected from amino acids at positions 94,
101, 102, 109 or 144 or more preferably at least five mutations
selected from amino acids at positions 94, 101, 102, 109 or 144, in
particular SEQ ID NO:1 or a variant or fragment thereof results in
the full or partial inhibition of IL-17 and/or the full or partial
activation of IFN-.gamma..
[0083] Moreover, the inventor has surprisingly discovered that
orally administering the antigen and IFN-.alpha. subtype which is a
hybrid of IFN-.alpha.10 and IFN-.alpha.14 in particular a hybrid
comprising the primary interferon binding sites of IFN-.alpha.10
and IFN-.alpha.14, in particular wherein the
IFN-.alpha.10-IFN-.alpha.14 hybrid sequence comprises at least one
mutation selected from amino acids at positions 94, 101, 102, 109
or 144, preferably at least two mutations selected from amino acids
at positions 94, 101, 102, 109 or 144, more preferably at least
three mutations selected from amino acids at positions 94, 101,
102, 109 or 144, more preferably at least four mutations selected
from amino acids at positions 94, 101, 102, 109 or 144 or more
preferably at least five mutations selected from amino acids at
positions 94, 101, 102, 109 or 144, and in particular SEQ ID NO:1
or a fragment or variant thereof in combination as discussed herein
can result in the activation of a Th1 T cell mediated immune
response and the suppression of a Th2/Th17 T cell mediated immune
response. A standard flu vaccine was mixed with a low dose of
leukocyte-derived interferon alpha (LDA1) and orally administered
to mice. The inventor noted that without the interferon, a small
anti-flu antibody response was recorded in mice, which was
approximately 50 times less than with an injected vaccine. With
interferon-alpha, the response from the orally delivered vaccine
was exactly the same as the injected vaccine. A series of buccal
immunisations using a standard protein antigen and two interferons,
LDA1 and an isolated subtype IFN-.alpha.14, surprisingly resulted
in oral immunisation of mice to which the composition was
administered. However, the inventor surprisingly noted that while
the LDA1 gave a balanced response, IFN-.alpha.14 mediated only a
significant humoral response. The production of IgG1 is indicative
of a Th2 response (humoral immunity) and the production of IgG2a is
indicative of a Th1 response (cell-mediated immunity).
[0084] The inventor, whilst not wishing to be bound by theory, has
identified that the oral administration of a food allergen capable
of mediating a Th2/Th17 immune response and an interferon alpha
subtype which is a hybrid of IFN-.alpha.10 and IFN-.alpha.14 can
skew the immune response towards a cell-mediated (Th1) path, whilst
simultaneously suppressing the allergic (Th2/Th17) response.
Accordingly, the inventor has surprisingly shown that the
co-administration of an allergen such as a food derived antigen
that is causative of allergy or associated allergic diseases in a
subject with certain interferon subtypes modulates the resulting
immune response and skews it away from the Th2/Th17 response which
would have been expected to develop against the allergen or
antigen. This surprising finding provides an unexpected approach to
treat or prevent allergic responses or diseases which occur in
subjects as a result of allergens such as food-derived allergens or
tumour associated antigens.
DEFINITIONS
Subject
[0085] As herein defined, a "subject" includes and encompasses
mammals such as humans, primates and livestock animals (e.g. sheep,
pigs, cattle, horses, donkeys); laboratory test animals such as
mice, rabbits, rats and guinea pigs; and companion animals such as
dogs and cats.
Treatment/Therapy
[0086] The term "treatment" is used herein to refer to any regimen
that can benefit a human or non-human animal. The treatment may be
in respect of any existing inflammatory, autoimmune, allergic or
allergy-associated condition and the treatment may be prophylactic
(preventative treatment). Treatment may include curative or
alleviative effects. Reference herein to "therapeutic" and
"prophylactic" treatment is to be considered in its broadest
context. The term "therapeutic" does not necessarily imply that a
subject is treated until total recovery. Similarly, "prophylactic"
does not necessarily mean that the subject will not eventually
contract a disease condition. Accordingly, therapeutic and/or
prophylactic treatment includes amelioration of the symptoms of a
particular allergic condition or preventing or otherwise reducing
the risk of developing a particular allergic condition. The term
"prophylactic" may be considered as reducing the severity or the
onset of a particular condition. "Therapeutic" may also reduce the
severity of an existing condition.
Administration
[0087] The active ingredients used in the present invention (e.g.
vaccine or allergen and IFN-.alpha.10, IFN-.alpha.14 or a hybrid
thereof) in particular a hybrid IFN-.alpha.10 and IFN-.alpha.14
subtype, for example SEQ ID NO: 1, as described herein can be
administered separately to the same subject, optionally
sequentially, or can be co-administered simultaneously as a
pharmaceutical, immunogenic or vaccine composition. In certain
embodiments, the vaccine or allergen is co-administered with the
interferon alpha subtype. The pharmaceutical composition will
generally comprise a suitable pharmaceutical excipient, diluent or
carrier selected depending on the intended route of
administration.
[0088] The active ingredients can be administered to a patient in
need of treatment via any suitable route. The precise dose will
depend upon a number of factors, as is discussed below in more
detail.
[0089] One suitable route of administration is parenterally
(including subcutaneous, intramuscular, intravenous, by means of,
for example a drip patch). Other suitable routes of administration
include (but, are not limited to) oral, ocular, nasal, topical
(including buccal and sublingual), infusion, intradermal or
administration via oral or nasal inhalation, by means of, for
example, a nebuliser or inhaler, or by an implant. Preferable
routes of administration include (but, are not limited to) oral,
buccal and sublingual. The compositions of the invention may also
be administered in such a manner that they are directed to, or
released in, specific areas of the gut intestinal tract (such as
the small intestine/duodenum). Typically such release will occur
after passage through the stomach, this targeted release being
achievable through the use of coatings and the like.
[0090] For intravenous injection, the active ingredient will be in
the form of a parenterally acceptable aqueous solution which is
pyrogen-free and has suitable pH, isotonicity and stability. Those
of relevant skill in the art are well able to prepare suitable
solutions using, for example, isotonic vehicles such as sodium
chloride injection, Ringer's injection, Lactated Ringer's
injection. Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
[0091] The compositions of the present invention for oral
administration may be in tablet, capsule, lozenge, powder or liquid
form. Oral administration may involve placing a lozenge under the
tongue of the patient A tablet may comprise a solid carrier such as
gelatin or an adjuvant. Liquid pharmaceutical compositions
generally comprise a liquid carrier such as water, petroleum,
animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0092] The compositions of the present invention may also be
administered via microspheres, liposomes, other microparticulate
delivery systems or sustained release formulations placed in
certain tissues including blood. Suitable examples of sustained
release carriers include semipermeable polymer matrices in the form
of shared articles, e.g. suppositories or microcapsules. Examples
of the techniques and protocols mentioned above and other
techniques and protocols which may be used in accordance with the
invention can be found in Remington's Pharmaceutical Sciences, 18th
edition, Gennaro, A.R., Lippincott Williams & Wilkins; 20th
edition (Dec. 15, 2000) ISBN 0-912734-04-3 and Pharmaceutical
Dosage Forms and Drug Delivery Systems; Ansel, H.C. et al. 7.sup.th
Edition ISBN 0-683305-72-7, the entire disclosures of which are
herein incorporated by reference.
Pharmaceutical Compositions
[0093] As described above, the present invention extends to a
pharmaceutical composition for the treatment of inflammatory
diseases, autoimmune diseases and allergy such as food allergy and
associated allergic diseases and, in particular, for the induction
of a Th1 immune response and the suppression or inhibition of a
Th2/Th17 immune response.
[0094] Pharmaceutical compositions according to the present
invention, and for use in accordance with the present invention,
may comprise, in addition to an active ingredient, a
pharmaceutically acceptable excipient, carrier, buffer stabiliser
or other materials well known to those skilled in the art. Such
materials should be non-toxic and should not interfere with the
efficacy of the active ingredient. The precise nature of the
carrier or other material will depend on the route of
administration, which may be, for example, oral, intravenous,
intranasal or via oral or nasal inhalation. The formulation may be
a liquid, for example, a physiologic salt solution containing
non-phosphate buffer at pH 6.8-7.6, or a lyophilised or
freeze-dried powder.
Dose
[0095] The composition is preferably administered to an individual
in a "therapeutically effective amount" or a "desired amount", this
being sufficient to show benefit to the individual. As defined
herein, the term an "effective amount" means an amount necessary to
at least partly obtain the desired response, or to delay the onset
or inhibit progression or halt altogether the onset or progression
of a particular condition being treated. The amount varies
depending upon the health and physical condition of the subject
being treated, the taxonomic group of the subject being treated,
the degree of protection desired, the formulation of the
composition, the assessment of the medical situation and other
relevant factors. It is expected that the amount will fall in a
relatively broad range, which may be determined through routine
trials. Prescription of treatment, e.g. decisions on dosage etc.,
is ultimately within the responsibility and at the discretion of
general practitioners, physicians or other medical doctors, and
typically takes account of the disorder to be treated, the
condition of the individual patient, the site of delivery, the
method of administration and other factors known to practitioners.
The optimal dose can be determined by physicians based on a number
of parameters including, for example, age, sex, weight, severity of
the condition being treated, the active ingredient being
administered and the route of administration. A broad range of
doses may be applicable. Considering oral administration to a human
patient, for example, from about 10 .mu.g to about 1000 .mu.g of
agent may be administered per human dose, optionally for 3 to 4
doses. Dosage regimes may be adjusted to provide the optimum
therapeutic response and reduce side effects. For example, several
divided doses may be administered daily, weekly, monthly or other
suitable time intervals or the dose may be proportionally reduced
as indicated by the exigencies of the situation.
[0096] Unless otherwise defined, all technical and scientific terms
used herein have the meaning commonly understood by a person who is
skilled in the art in the field of the present invention.
Autoimmune Disease
[0097] The term "autoimmune disease" as used herein is understood
to mean any disease or condition which is caused by a body's
tissues being attacked by its own immune system.
[0098] Throughout the specification, unless the context demands
otherwise, the terms "comprise" or "include", or variations such as
"comprises" or "comprising", "includes" or "including" will be
understood to imply the inclusion of a stated integer or group of
integers, but not the exclusion of any other integer or group of
integers.
[0099] The present invention will now be exemplified with reference
to the following non-limiting Figures and examples which are
provided for the purpose of illustration and are not intended to be
construed as being limiting on the present invention. Other
embodiments of this invention will be apparent to those of ordinary
skill in the art in view of this description.
BRIEF DESCRIPTION OF THE FIGURES
[0100] FIG. 1 shows a graph of IgG subtype (IgG1 and IgG2a)
production in BALB-c mice immunised with ovalbumin and different
subtypes of IFN-.alpha..
[0101] FIG. 2 shows a graph of the percentage of IgG subtype (IgG1
and IgG2a) produced in BALB-c mice immunised with ovalbumin and
different subtypes of IFN-.alpha..
[0102] FIG. 3 shows a graph of IgG2a production in BALB-c mice
immunised with ovalbumin and MULTIFERON.TM., glycosylated
IFN-.alpha.14 and non-glycosylated IFN-.alpha.14 administered via
intraperitoneal injection.
[0103] FIG. 4 shows a graph of IgG1 production in BALB-c mice
immunised with ovalbumin and MULTIFERON.TM., glycosylated
IFN-.alpha.14 and non-glycosylated IFN-.alpha.14 administered via
intraperitoneal injection.
[0104] FIG. 5 shows a graph of IgG2a production in BALB-c mice
immunised with ovalbumin and MULTIFERON.TM., glycosylated
IFN-.alpha.14 and non-glycosylated IFN-.alpha.14 administered
orally.
[0105] FIG. 6 shows a graph of IgG1 production in BALB-c mice
immunised with ovalbumin and MULTIFERON.TM., glycosylated
IFN-.alpha.14 and non-glycosylated IFN-.alpha.14 administered
orally.
[0106] FIG. 7 shows inhibition of human PBMC interleukin-17 (IL-17)
secretion with lipopolysaccharide (LPS) alone and with LPS and
increasing concentrations of IFN-.alpha.2a (black), IFN-.alpha.10
(white) or IFN-14 (grey).
[0107] FIG. 8 shows the inhibition of Interleukin-4 (IL4)-induced
CD4+ Th2 cell development using increasing concentrations of
IFN-.alpha.2a (black), IFN-.alpha.10 (white) or IFN-14 (grey).
[0108] FIG. 9 shows the IFN-.alpha.10 and IFN-.alpha.14 hybrid
amino acid sequence which contains the 2 interferon receptor
(IFNaR1 and IFNaR2) binding sites.
[0109] Based on the protein sequence SEQ ID NO:1 using online
webservices (for example
http://www.ebi.ac.uk/Tools/st/emboss_backtranseq/), a nucleic acid
sequence can be obtained. FIG. 10 provides the reverse translation
of the protein sequence SEQ ID NO:1 when an E. coli codon usage
table is used.
[0110] FIG. 11 shows the alignment of a previously described
IFNalpha10 and IFNalpha14 hybrid amino acid sequence with the
IFNalpha10 and IFNalpha14 hybrid amino acid sequence as presently
claimed.
[0111] FIG. 12 indicates the effect caused by rIFN-.alpha.14 on the
production of IL-17 in whole human blood incubated with one
microgram E.coli lipopopolysaccharide (LPS) for 48 hours. The
.alpha.-14 gave a significant suppression of IL-17 secretion.
IL-.alpha.-2 and .alpha.-10 showed no significant suppression.
[0112] FIG. 13 shows the effect caused by rIFN-.alpha.14 on the
production of IL-17 from human peripheral blood mononuclear cells
incubated with 10 micrograms E. coli lipopolysaccharide (LPS) for
48 hours. The .alpha.-14 caused a significant suppression of IL-17
secretion with and without LPS activation. IL- .alpha.-2 and
.alpha.-10 showed no significant changes in the IL-17
concentrations (results not shown).
[0113] FIG. 14 shows the effect caused by rIFN-.alpha.10,
rIFN-.alpha.14 and rIFN-.alpha.2 on IL-17 production by whole human
blood incubated with PHA for 5 days. The .alpha.-14 is an extremely
potent inhibitor (P<0.001 at 1,000 IU/ml) of IL-17 compared with
the commonly available .alpha.-2; the .alpha.-10 is more than
20.times. less active in this context
[0114] FIG. 15 shows the effect caused by rIFN-.alpha.10,
rIFN-.alpha.14 and rIFN-.alpha.2 on IFN-gamma production by whole
human blood incubated with PHA for 5 days. The .alpha.-10 is the
most potent interferon--alpha in this context causing enhanced
secretion of IFN-gamma--critical for both and innate and adaptive
immunity against viruses, intracellular bacterial infections and in
the control/elimination of tumours.
[0115] FIG. 16 illustrate a sequence alignment of IFN-alpha10 (SEQ
ID NO:4) and IFN-alpha14 (SEQ ID NO:5) amino acid sequences and the
hybrid sequence SEQ ID NO: 1 discussed herein.
[0116] FIG. 17 shows the effect caused by rIFN-.alpha.14 and
rIFN-.alpha.2 on IL-17 production by whole human blood incubated
with PHA for 5 days. The .alpha.-14 is an extremely potent
inhibitor of IL-17 compared with the commonly available
IFN-.alpha.2 (EC50s for the two subtypes are 100 and 10,000
IU/ml).
[0117] FIG. 18 shows the induction of Interferon-gamma by
Interferon-alpha subtypes and in particular demonstrates the effect
caused by IFN-.alpha.10, IFN-.alpha.14 and IFN-.alpha.2 on
IFN-gamma production by whole human blood incubated with PHA for 5
days. The .alpha.-10 is the most potent interferon--alpha in this
context causing enhanced secretion of IFN-gamma.
[0118] FIG. 19 shows the effect of the IFN-.alpha.10-IFN-.alpha.14
hybrid of the present invention (SEQ ID NO:1) on IL-17 production
from whole human blood compared to the effect on IL-17 of a
previously disclosed IFN-.alpha.10-IFN-.alpha.14 hybrid. The hybrid
of the present invention demonstrates an greater reduction in
IL-17.
EXAMPLE 1
Identification of Interferon-Alpha Subtypes that are Immunological
Adjuvants
[0119] 50 .mu.g ovalbumin and 10.sup.5 IU of interferon subtypes
IFN-.alpha.14, IFN-.alpha.2, IFN-.alpha.21, IFN-.alpha.10, an IFN
"mix" (including IFN-.alpha.1, IFN-.alpha.8, IFN-.alpha.21 and
possibly IFN-.alpha.l7), IFN-.alpha.8, Intron A, MULTIFERON.TM. and
IFN-.alpha.1 in 50 .mu.l were administered via intraperitoneal
injection three times per week to BALB-c female mice, in groups of
10.
[0120] The serum concentrations of IgG1 mg/ml (Th2
response--humoral immunity to the ovalbumin antigen) and IgG2a
mg/ml (Th1 response--cell-mediated immunity to the ovalbumin
antigen) were measured by ELISA.
[0121] FIGS. 1 and 2 show the anti-ovalbumin IgG subtype production
in BALB-c mice treated with IFN-.alpha.14, IFN-.alpha.2,
INF-.alpha.21, IFN-.alpha.10, a "mix" of IFN-.alpha.1,
IFN-.alpha.8, IFN-.alpha.21 and possibly IFN-.alpha.17),
IFN-.alpha.8, Intron A, MULTIFERON.TM., ovalbumin only, ovalbumin
plus human serum albumin (used as a carrier in interferon
preparations) and IFN-.alpha.1.
[0122] The inventor demonstrated that IFN-.alpha.10 and
IFN-.alpha.14 enhanced the production of IgG2a antibodies
significantly which is indicative of an enhanced Th1 immune
response. The inventor also demonstrated that IFN-.alpha.10 in
particular showed low production of IgG1 antibody which is
indicative of suppressing a Th2/Th17 immune response.
EXAMPLE 2
Identification of Antibody Response in BALB-c Mice After
Administration of a Composition Comprising a Flu Vaccine and a Low
Dose of Leukocyte Derived Interferon-Alpha (LDA1)
[0123] The standard flu vaccine was mixed with a low dose (10.sup.5
IU) of leukocyte derived interferon alpha (LDA1). Without the
interferon, a small anti-flu antibody response was recorded in
mice, approximately 50 times less than with an injection. With
interferon-alpha, the response from the orally delivered vaccine
was exactly the same as the injected vaccine. A series of buccal
immunisations were carried out using a standard protein antigen
(ovalbumin). Two interferons were compared, namely, the LDA1 and an
isolated subtype, IFN-.alpha.14. Both produced a remarkable oral
immunisation of the mice, but whereas the LDA1 gave a balanced
response, the IFN-.alpha.14 gave only a significant humoral
response. The production of IgG1 is indicative of a Th2/Th17
response (humoral immunity) and the production of IgG2a is
indicative of a Th1 response (cell-mediated immunity).
EXAMPLE 3
The Identification of IFN-Alpha as an Oral Immunological
Adjuvant
[0124] 50 .mu.g ovalbumin and 10.sup.5 IU of interferon subtypes,
namely MULTIFERON.TM., glycosylated IFN-.alpha.14 and
non-glycosylated IFN-.alpha.14, in 50 .mu.l doses were administered
three times a week to BALB-c female mice via oral (buccal) and
intraperitoneal injection administration.
[0125] The controls used were antigen alone and Titermax--Titermax
is a mixture of compounds used in antibody generation and
vaccination to stimulate the immune system to recognise an antigen
given together with the mixture. Titermax is a recently developed
immune adjuvant deemed to be safe in animals.
[0126] Serum concentrations (mg/ml) of IgG1 (indicative of a
Th2/Th17 response) and IgG2a (indicative of a Th1 response)
anti-ovalbumin antibodies were quantitated by ELISA.
[0127] The production of IgG2a and IgG1 antibodies when
MULTIFERON.TM., glycosylated IFN-.alpha.14 and aglycosyl
IFN-.alpha.14 (CHO cell-derived) were administered both orally and
by injection were compared (see FIGS. 3, 4, 5 and 6).
[0128] The inventor demonstrated that IFN-.alpha.14 showed
pronounced immunological adjuvant activity both orally and by
injection. No significant difference was seen between the
glycosylated and non-glycosylated preparations.
[0129] The inventor also demonstrated that IFN-.alpha.14 only
enhanced IgG2a production associated with Th1 responses by the oral
route of administration. Hence IFN-.alpha.14 is an activator of
cell-mediated immunity when administered orally.
[0130] MULTIFERON.TM. enhanced both IgG1 and IgG2a responses when
administered both orally and by injection i.e. it induced both Th1
and Th2 responses significantly.
EXAMPLE 4
In Vitro Determination of the Inhibition of Humoral Immunity
(Th2/Th17) by Interferon-Alpha Subtypes--Analysis of Th17
Lymphocytes and Interleukin 17
[0131] A total of 2.times.10.sup.6 human PBMCs were stimulated with
lipopolysaccharide (LPS) in the absence or presence of increasing
concentrations of recombinant human alpha-IFN. Supernatants were
collected after 24 hours and IL-17 concentrations measured by
ELISA.
Human Cell Culture
[0132] Human peripheral blood was collected from healthy volunteers
and peripheral blood mononuclear cells (PBMCs) were obtained by
Lymphoprep gradient centrifugation (Pierce). For PBMC experiments,
2.times.10.sup.6 PBMCs per ml were seeded in 24-well plates and
stimulated with lipopolysaccharide (LPS) from Escherichia coli
055:B5 (Sigma) or 2.times.10.sup.6 PBMCs per mL were seeded into
24-well plates and stimulated with 5 mg/mL plate-bound anti-CD3
(clone: UCHT1) and 2.5 mg/ml. anti-CD28 (clone: CD28.2). Naive T
cells (CD4+CD45RA) were obtained by magnetically labeling and
depletion of non-helper T-cell and memory T-cells performed
according to manufacturer's instructions (Miltenyi Biotec). A total
of 1.times.10.sup.5 naive T-cells were primed in 96-well flat
bottom plates coated with anti-CD3 (clone UCHT1, 2.5 mg/mL) and
with anti-CD28 (clone CD28.2, 2.5 mg/mL) antibodies. After 48 h of
culture, 20 IU/mL recombinant human IL-2 (Peprotech) was added to
the culture.
[0133] For human Th17 differentiation, cells were supplemented with
neutralising anti-IL-4 and anti-IFN.gamma. antibodies (both from
Peprotech) and with 10 ng/mL recombinant IL-1.beta. and 50 ng/mL
recombinant IL-6 (both from Peprotech). Where required, recombinant
human IFN.alpha.10/14 was added to the culture. After 5 days of
culture, cells were washed, transferred into new plates and
expanded until day 12 in the presence of 20 IU/mL recombinant
IL-2.
ELISA and Intracellular Cytokine Staining
[0134] The IL-17 producing capacity of primed Th17 cells was
assessed by stimulation with 0.1 ng/ml LPS or alternatively can be
assessed by the stimulation of human cells with soluble 1 mg/mL
anti-CD3 (clone: OKT3) and phorbol-12-13-dibutyrate (PdBu).
Concentrations of human IL-17 in cell culture supernatants were
determined using commercially available antibody pairs and protein
standards (R&D Systems). Absorption was determined using an
ELISA reader at 450 nm. For intracellular staining of mouse
IFN.gamma. and IL-17, T-cells are stimulated with PMA and ionomycin
for 5 hours. Brefeldin A is added for the final 3 h of culture.
Intracellular staining can be performed with a BD Cytofix/Cytoperm
kit according to the manufacturer's instructions. Cells are
incubated with fluorescein isothiocyanate-labeled anti-IFN.gamma.
(clone: XMG1.2, BD Pharmingen) and Alexa Fluor 647-labeled
anti-mouse mouse IL-17A (clone: eBio17B7, eBioscience). After
washing, cells are immediately analysed using
Fluorescence-activated cell sorting (FACS).
Results
[0135] As shown by FIG. 7, inhibition of IL-17 was found to occur
in the order IFN.alpha.10>IFN.alpha.14>IFN.alpha.2a.
P<0.05 (FIG. 7).
EXAMPLE 5
In Vitro Determination of the Inhibition of Humoral Immunity
Th2/Th17) by Interferon-Alpha Subtypes--Analysis of Th2 Cells and
Associated Cytokines
CRTH2 Background
[0136] CRTH2 (Chemoattractant Receptor-homologous molecule
expressed on Th2 cells) is a G-protein coupled receptor expressed
by Th2 lymphocytes, eosinophils, and basophils. The receptor
mediates the activation and chemotaxis of these cell types in
response to prostaglandin D2 (PGD2), the major prostanoid produced
by mast cells. PGD2 is released through mast cell degranulation in
the initial phase of IgE-mediated reactions. This process is also
thought to occur at the site of inflammation, such as the nasal and
bronchial mucosa. Through interaction with CRTH2, PGD2 is thought
to mediate recruitment and activation of CRTH2-bearing cell types
to the site of the allergic reaction, in consequence amplifying and
maintaining the allergic inflammation. In the nasal and bronchial
mucosa, this pro-inflammatory cascade is thought to start during
the so-called late allergic response occurring 3 to 9 hours after
allergen challenge. The interaction between PGD2 and CRTH2 would,
therefore, contribute to the so-called "Th2 polarisation", with
consequent Th2 cytokine production and the typical eosinophilic and
basophilic characteristics of the inflammation.
IFN.alpha. Inhibits Human CD4+ Th2 Development
[0137] Purified human CD4+/CD45RA+ cells were activated with
plate-bound anti-CD3/anti-CD28 under defined cytokine conditions.
Induction of CRTH2 expression was assessed by flow cytometry. All
P<0.05, above 100 IU IFN compared with IL-4alone.
Human Subjects
[0138] Peripheral blood was collected from healthy adult donors and
cells purified as below.
T Cell Cultures and Analysis
[0139] Peripheral blood was obtained from healthy male adult donors
and naive CD4+/CD45RA+ T cells were purified (>92%) from buffy
coats by magnetic bead separation (BD Biosciences, USA). CD4+ cells
were activated with plate-bound anti-CD3/anti-CD28 and IL-2 (50
U/ml) in complete Iscove's Modified Dulbecco's Medium containing
10% FCS, in the presence of recombinant human recombinant IL-4
(R&D Systems, USA), at a concentration of 20 ng/ml for 7 days.
Flow cytometric analysis was performed with hCD294
(chemo-attractant receptor homologous molecule expressed on Th2
cells [CRTH2])-Alexa 647 (BD Biosciences).
Results
[0140] In humans, the PGD2 receptor, CRTH2, is selectively
expressed on Th2 cells and is induced by IL-4 during Th2
development. IL-4 promoted the development of cells expressing
CRTH2. However, as shown in FIG. 8 all the IFN-alphas markedly
blocked IL-4 driven CRTH2 expression, in a dose-dependent manner in
the order IFN.alpha.10>IFN.alpha.14>IFN.alpha.2a, thus
supporting the concept that these cytokines suppress Th2 (humoral)
immunity, but are recognised as potent activators of Th1-associated
immunity.
[0141] As shown in FIG. 12, the effect of rIFN-.alpha.14 on the
production of IL-17 in human blood incubated with LPS for 48 h was
tested.
[0142] Whole human blood was incubated without (open columns) or
with 1 .mu.g/ml LPS (cross hatched columns) in the absence and
presence of a range of concentrations of rIFN-.alpha.14 (0-1,000
IU/ml) for 48 h at 37.degree. C., in an atmosphere of 5% CO2 in
air, in a humidified incubator. Plasma was collected by
centrifugation and levels of IL-17 determined by ELISA.
[0143] FIG. 12 indicated a dose response to IFN-.alpha.14 wherein 1
mg=10.sup.-8 IU.
[0144] As shown in FIG. 13 the effect of rIFN-.alpha.14 on the
production of IL-17 in human PBMCs incubated with LPS for 48 h was
tested.
[0145] Human Peripheral Blood Mononuclear cells (PBMCs), a critical
component in the immune system, were isolated from whole human
blood by density gradient centrifugation. 2.times.10.sup.6 PBMCs
were incubated without (open columns) or with 10 .mu.g/ml LPS
(cross hatched columns) in the absence and presence of a range of
concentrations of rIFN-.alpha.14 (0-1,000 IU/ml) for 48 h at
37.degree. C., in an atmosphere of 5% CO2 in air, in a humidified
incubator. Levels of IL-17 in the supernatant were determined by
ELISA.
[0146] As indicated by FIG. 13, increasing concentrations of
rIFN-.alpha.14 was found to reduce the IL-17 both in untreated and
treated LPS cells.
[0147] As shown in FIG. 14, the effect of rIFN.alpha.10,
rIFN.alpha.14 and rIFN2 on IL-17production by whole blood incubated
with phytohaemagglutinin (PHA) for 5 days was tested.
[0148] Whole human blood was diluted 1/10 with RPMI 1640 culture
medium and incubated without or with 100 .mu.g/ml PHA in the
absence and presence of a range of concentrations of
rIFN-.alpha.14, rIFN-.alpha.10 and rIFN-.alpha.2 for 5 days at
37.degree. C., in an atmosphere of 5% CO2 in air, in a humidified
incubator. At the end of this period, supernatants were aspirated
and levels of IL-17 in supernatants measured by ELISA. Values
represent the mean .+-. sem, for n=3 incubations. Statistical
analysis and IC.sub.50 values were determined using GraphPad Prism
5 (GraphPad Software Inc., California, USA).
[0149] As indicated in FIG. 14 the provision of rIFN-.alpha.14 at
higher concentrations (100-1000 IU/ml) caused a greater decrease in
IL-17 than the provision of IFN-.alpha.2 or IFN-.alpha.10.
rIFN-.alpha.14 is considered to be the most potent interferon
tested at reducing IL-17 levels.
[0150] Similar results were achieved as shown in FIG. 17. Here the
effect of rIFN.alpha.14 and rIFN2 on IL-17 production by whole
blood incubated with phytohaemagglutinin (PHA) for 5 days was
tested using the same methodology. As indicated in FIG. 17 the
provision of rIFN-.alpha.14 at higher concentrations (100-1000
IU/ml) caused a greater decrease in IL-17 than the provision of
IFN-.alpha.2. rIFN-.alpha.14 is considered to be the most potent
interferon tested at reducing IL-17 levels.
[0151] As shown in FIG. 15, the effect of rIFN.alpha.10,
rIFN.alpha.14 and rIFN.alpha.2 on IFN-gamma production by whole
blood incubated with PHA for 5 days.
[0152] Whole human blood was diluted 1/10 with RPMI 1640 culture
medium and incubated without or with 100 .mu.g/ml PHA in the
absence and presence of a range of concentrations of
rIFN-.alpha.10, rIFN-.alpha.14 and rIFN-.alpha.2 for 5 days at
37.degree. C., in an atmosphere of 5% CO.sub.2 in air, in a
humidified incubator. At the end of this period supernatants were
aspirated and levels of IFN-gamma in supernatants measured by
ELISA. Values represent the mean .+-. sem, for n=3 incubations,
plasma was collected by centrifugation and levels of IFN-gamma
determined by ELISA. Values represent the mean .+-. sem, for n=3
incubations.
[0153] It was determined that rIFN-.alpha.10 was the most effective
of the interferons tested at promoting levels of IFN-gamma.
IFN-gamma has previously been suggested to be important in
providing an anti-cancer effect.
[0154] Similar results were achieved as shown in FIG. 18. Here the
effect of rIFN.alpha.14 and rIFN2 on IFN-gamma production by whole
blood incubated with phytohaemagglutinin (PHA) for 5 days was
tested using the same methodology. As indicated in FIG. 18 the
provision of rIFN-.alpha.10 caused a greater increase in IFN-gamma
than the provision of IFN-.alpha.2. rIFN-.alpha.10 is considered to
be the most potent interferon tested at increasing IFN-gamma
levels.
[0155] As shown in FIG. 19, the effect of the
IFN-.alpha.10-IFN-.alpha.14 hybrid of the present invention (SEQ ID
NO:1) on IL-17 production from whole human blood was compared to
the effect on IL-17 of the IFN-.alpha.10-IFN-.alpha.14 hybrid
disclosed in PCT/GB2015/050717.
[0156] Whole human blood was diluted 1/10 with RPMI 1640 culture
medium and was incubated with PHA (100 .mu.g/ml) in the presence of
a range of concentrations of either the IFN-.alpha.10-IFN-.alpha.14
hybrid of the present invention (IFN alpha-hybrid 1) or the
IFN-.alpha.10-IFN-.alpha.14 hybrid disclosed in PCT/GB2015/050717
(IFN alpha-hybrid 2) for 5 days at 37.degree. C. in an atmosphere
of 5% C02 in air in a humidified incubator. At the end of this
period supernatants were collected and levels of IL-17 measured by
ELISA. Values represent mean .+-. sem, for n=3 incubations.
P<0.05 for all points of the data between the two hybrids except
100 IU/ml, which was non significant The Red diamonds indicate
IFN-.alpha.10-IFN-.alpha.14 hybrid 1 and the blue circles indicate
IFN-.alpha.10-IFN-.alpha.14 hybrid 2.
[0157] It was determined that the IFN-.alpha.10-IFN-.alpha.14
hybrid of the present invention demonstrates a greater reduction in
IL-17.
EXAMPLE 6
Effects of Human Interferon Alpha-14 and Alpha-10 on Unstimulated
and Activated Human Mononuclear Leukocytes from Normal Subjects
TABLE-US-00002 [0158] TABLE 1 Synopsis of 400 interleukins,
chemokines, and protein markers estimations* following
IFN-.alpha.10/14 treatment of human mononuclear cells FOLD NUMBER
OF PHA- STIMULATED/PHA- FOLD NUMBER OF UNSTIMULATED/ STIMULATED
ALPHA-IFN ANALYTE ALPHA-IFN TREATED CELLS TREATED CELLS CYTOKINES
Alpha-14 Alpha-10 Alpha 14 Alpha 10 IL-1a 0 +23 1 +2 IL-1b 0 +70 -2
1 IL-1(F5 to F10) 0 0 0 0 IL-2 0 0 +7 +4 IL-3 0 0 -11 x IL-4 0 0 1
-3 IL-5 0 0 -420 1 IL-6 -19 +1000 1 1 IL-7 0 0 0 0 IL-8 1 +100 1 1
IL-9 0 0 0 0 IL-10 0 +5 +2 +2 IL-11 0 0 0 0 IL-12 p40 0 +350 0 +1
IL-12 p70 0 0 +11 0 IL-13 0 0 -5 1 IL-15 0 0 0 0 IL-16 1 1 1 1
IL-17 0 0 -43 -5 IL-18 0 0 0 0 IL-20 0 0 0 0 IL-21 0 x 0 x IL-23 +4
+3 +6 1 IL-24 0 0 0 0 IL-27 0 1 0 1 IL-28 0 0 0 0 IL-29 0 0 0 0
IL-31 0 0 0 0 IL-33 0 0 0 0 IL-34 0 0 0 0 IFN-gamma 0 0 +600 +3000
G-CSF -1500 +20 1 1 GM-CSF 0 0 0 1 CD MARKERS CD14 +2 +2 +2 +2 CD23
-22 1 -850 -3 CD30 0 0 0 0 CD40 +2 +2 1 1 CD97 -2 1 -5 -5 CD152
(CTLA-4) 1 0 -2 0 CD154 1 x +2 x CD163 -2 1 -2 1 CD200 1 1 -1 1
CD223 (LAG3) 0 0 -3 +3 SELECTED CHEMOKINES AND PROTEINS CXCL1
(GROa) -7600 -12 -3400 1 CXCL5 (ENA-78) -6 +16 -32 -3 CXCL10 (IP10)
+460 +10 +1 1 CCL1 (1-309) 0 1 -24 1 CCL7 (MCP-3) -2 -200 -149 1
CCL16 (HCC-4) 0 1 -100 1 CCL20 (MIP-3a) -69 +40 -2 1 MMP-2 +600
+200 +450 +500 (collagenase) MMP-10 -121 1 -2 -2 (proteoglycanase)
ACE-2 +12 0 +6 0 PDGF Ralpha -4 -7 -1 -3 Tie-1 -170 -200 -8 -280
ICAM-1 -1 +2 -3 1 TREM-1 +5 -2 +2 -5 E-SELECTIN 1 -7 1 -2 0 = no
analyte detected 1 = analyte present but no effect of alpha-10/14 x
= not determined The positive effect of alpha-10/14 is denoted by a
+ The negative effect of alpha-10/14 interferon is denoted by a -
*The assay system used was the RayBio Quantibody Human Cytokine
Array 9000 (QAH-CAA-9000 provided by Insight Bio Ltd.). This a
multiplex ELISA, measuring the concentrations of 400 proteins in a
single assay process, including pro- and anti-inflammatory markers,
interleukins, cancer markers, chemokines, growth factors and
related molecules. Human peripheral blood mononuclear leukocytes
(normal blood donors) were treated with 10 ng/ml IFN-.alpha.14/10
for 4 hours prior to assay. Tests were performed on 2 groups of
cells - a) unactivated and b) activated with PHA
(phytohaemagglutinin) to induce a high level of stimulation.
Effects of Alpha-14 on Activated Immune Cells
[0159] More than 30 interleukins were quantitated but only 6 showed
significant changes in the activated cells, indicating the targeted
and very specific nature of the interaction of the alpha-14 with
the human immune response.
[0160] Interleukin 2 increased by 7-fold, IL-12p70 +11 fold and
interferon-.gamma.+600 fold, indicating a strong proliferation of
the Th1 (cell-mediated) response while a 6-fold increase in IL-23
is in keeping with its role in cell-mediated immunity and its
association with IL-12.
[0161] Very large decreases were observed with IL-3 and IL-5 of 11
and 420-fold respectively. These molecules are associated with the
production of myeloid cells and immunoglobulin production (humoral
immunity). IL13 also decreased by 5-fold, which is important as
this interleukin is implicated in the secretion of IgE, the allergy
antibody. Also crucial was the 43-fold decrease in IL-17. This
regulatory cytokine is increased in autoimmune diseases, humoral
(antibody-mediated) immunity and stimulation of inflammation
through attraction of neutrophils.
[0162] CD23 or Fc.epsilon.RII is a receptor for the allergy
antibody, IgE, and is displayed widely on different types of
leukocytes. CD23 activation controls IgE production and significant
increases are seen in patients with allergic disorders. This
important marker was decreased by 850-fold, in the activated cells,
by alpha-14.
Effects of IFN Alpha-14 on Non-Activated Immune Cells
[0163] IL-6 decreased 19-fold. This cytokine stimulates liver
protein synthesis in responses to traumas, causes increases in body
temperature and is involved in muscle contraction. However, it is
its essential role in antibody-mediated immunity that is important
in allergy.
[0164] G-CSF was also decreased by more than 1000-fold. This
molecule can stimulate the bone marrow to make Increased numbers of
neutrophils that could be involved in inflammation. At the same
time the secretion of the chemokine CXCL1 was suppressed by
7,500-fold--this prevents it attracting neutrophils to the site of
a response and causing inflammation. Also the concentration of the
chemokine, CXCL10 was enhanced by 460-fold--its role is to attract
T-lymphocytes to an ongoing immune response.
Effects of IFN Alpha-10 on Activated Immune Cells
[0165] As with alpha-14, alpha-10 only regulated a small number of
cytokines out of the numbers assessed. Of particular note were the
increases in IL-2 and interferon-.gamma. of 4 and 3000 fold
respectively indicating a switch to cell-mediated immunity. IL-17
levels fell by 5 fold, confirming this change in balance.
[0166] The large reduction in CD23 was not evident with alpha-10
and its major effects on chemokines were on Tie-1 (tyrosine kinase
crucial in the process of lymphatic remodelling) and TREM-1
(neutrophil activation) where it caused reductions of 280 and 5
fold respectively.
Effects of Alpha-10 on Non-Activated Immune Cells
[0167] Alpha-10 showed significant activity in this context
enhancing IL-1.alpha./.beta. by up to 70fold and IL-6,8,10,12 (p40)
by 1000,100, 5 and 350 fold in keeping with a strong support for
cell-mediated over humoral immunity. G-CSF was also enhanced by 20
fold in total contrast to alpha-14.
[0168] Few changes were recorded with the CD markers but CXCL1 was
reduced by 12 fold while CXCL5 and 10 increased by 16 and 10 fold
and CCL20 rose by 40 fold. However, CCL7 and Tie-1 fell by 200 fold
each. These results are in keeping with a significant movement
towards cell-mediated immunity.
RESULT
[0169] The low doses of interferon-alpha 14 and 10 have modified
cytokine synthesis in order to enhance cell-mediated immunity at
the expense of antibody-mediated immunity. This would be invaluable
in enhancing the activities of certain vaccines where a humoral
immune response can be detrimental e.g. viral and cancer
vaccines.
[0170] In addition the results are totally in keeping with the
general understanding that allergy can be alleviated by changing
the immune response to an allergen by shifting an antibody response
to a cellular response. Such a change would be part of acquired
immunity and hence, potentially, a long-term solution by developing
tolerance.
[0171] In addition, the alpha-14 significantly suppressed the
capacity of leukocytes to make/utilise IgE and hence it inhibited
the immediate effects of an allergic reaction, together with
reducing inflammatory elements of immunity while enhancing the
involvement of more control elements.
[0172] All documents referred to in this specification are herein
incorporated by reference.
[0173] Various modifications and variations to the described
embodiments of the inventions will be apparent to those skilled in
the art without departing from the scope of the invention. Although
the 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
of carrying out the invention which are obvious to those skilled in
the art are intended to be covered by the present invention.
Sequence CWU 1
1
51166PRTHomo sapiens 1Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn
Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu Gly Gln Met Gly Arg Ile Ser
Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Ile Pro
Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala Gln Ala
Ile Ser Val Leu His Glu Met Met Gln Gln Thr 50 55 60 Phe Asn Leu
Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 Leu
Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu 85 90
95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met
100 105 110 Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg
Ile Thr 115 120 125 Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Leu Ser Phe
Ser Thr Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Asp 165
2507PRTHomo sapiens 2Ala Thr Gly Thr Gly Thr Gly Ala Thr Cys Thr
Gly Cys Cys Gly Cys 1 5 10 15 Ala Gly Ala Cys Cys Cys Ala Thr Ala
Gly Cys Cys Thr Gly Gly Gly 20 25 30 Thr Ala Ala Thr Cys Gly Thr
Cys Gly Thr Gly Cys Ala Cys Thr Gly 35 40 45 Ala Thr Thr Cys Thr
Gly Cys Thr Gly Gly Gly Thr Cys Ala Gly Ala 50 55 60 Thr Gly Gly
Gly Thr Cys Gly Thr Ala Thr Thr Ala Gly Cys Cys Cys 65 70 75 80 Gly
Thr Thr Thr Ala Gly Cys Thr Gly Thr Cys Thr Gly Ala Ala Ala 85 90
95 Gly Ala Thr Cys Gly Thr Cys Ala Thr Gly Ala Thr Thr Thr Thr Cys
100 105 110 Gly Thr Ala Thr Thr Cys Cys Gly Cys Ala Ala Gly Ala Gly
Gly Ala 115 120 125 Ala Thr Thr Thr Gly Ala Thr Gly Gly Cys Ala Ala
Cys Cys Ala Gly 130 135 140 Thr Thr Thr Cys Ala Gly Ala Ala Ala Gly
Cys Ala Cys Ala Gly Gly 145 150 155 160 Cys Ala Ala Thr Thr Ala Gly
Cys Gly Thr Thr Cys Thr Gly Cys Ala 165 170 175 Thr Gly Ala Ala Ala
Thr Gly Ala Thr Gly Cys Ala Gly Cys Ala Gly 180 185 190 Ala Cys Cys
Thr Thr Thr Ala Ala Cys Cys Thr Gly Thr Thr Thr Ala 195 200 205 Gly
Cys Ala Cys Cys Ala Ala Ala Ala Ala Thr Ala Gly Cys Ala Gly 210 215
220 Cys Gly Cys Ala Gly Cys Ala Thr Gly Gly Gly Ala Thr Gly Ala Ala
225 230 235 240 Ala Cys Cys Cys Thr Gly Cys Thr Gly Gly Ala Ala Ala
Ala Ala Thr 245 250 255 Thr Cys Thr Ala Thr Ala Thr Cys Gly Ala Ala
Cys Thr Gly Thr Thr 260 265 270 Thr Cys Ala Gly Cys Ala Gly Ala Thr
Gly Ala Ala Cys Gly Ala Thr 275 280 285 Cys Thr Gly Gly Ala Ala Gly
Cys Ala Thr Gly Thr Gly Thr Thr Ala 290 295 300 Thr Thr Cys Ala Ala
Gly Ala Ala Gly Thr Thr Gly Gly Cys Gly Thr 305 310 315 320 Thr Gly
Ala Ala Gly Ala Ala Ala Cys Ala Cys Cys Gly Cys Thr Gly 325 330 335
Ala Thr Gly Ala Ala Thr Gly Ala Ala Gly Ala Thr Ala Gly Cys Ala 340
345 350 Thr Thr Cys Thr Gly Gly Cys Ala Gly Thr Gly Ala Ala Ala Ala
Ala 355 360 365 Ala Thr Ala Cys Thr Thr Thr Cys Ala Gly Cys Gly Cys
Ala Thr Thr 370 375 380 Ala Cys Cys Cys Thr Gly Thr Ala Thr Cys Thr
Gly Ala Thr Cys Gly 385 390 395 400 Ala Ala Cys Gly Thr Ala Ala Ala
Thr Ala Thr Ala Gly Cys Cys Cys 405 410 415 Gly Thr Gly Thr Gly Cys
Ala Thr Gly Gly Gly Ala Ala Gly Thr Thr 420 425 430 Gly Thr Thr Cys
Gly Thr Gly Cys Ala Gly Ala Ala Ala Thr Thr Ala 435 440 445 Thr Gly
Cys Gly Thr Ala Gly Cys Cys Thr Gly Ala Gly Cys Thr Thr 450 455 460
Thr Ala Gly Cys Ala Cys Cys Ala Ala Thr Cys Thr Gly Cys Ala Ala 465
470 475 480 Ala Ala Ala Cys Gly Thr Cys Thr Gly Cys Gly Thr Cys Gly
Cys Ala 485 490 495 Ala Ala Gly Ala Thr Thr Ala Ala Thr Ala Ala 500
505 3498PRTHomo sapiens 3Thr Gly Tyr Gly Ala Tyr Tyr Thr Asn Cys
Cys Asn Cys Ala Arg Ala 1 5 10 15 Cys Asn Cys Ala Tyr Trp Ser Asn
Tyr Thr Asn Gly Gly Asn Ala Ala 20 25 30 Tyr Met Gly Asn Met Gly
Asn Gly Cys Asn Tyr Thr Asn Ala Thr His 35 40 45 Tyr Thr Asn Tyr
Thr Asn Gly Gly Asn Cys Ala Arg Ala Thr Gly Gly 50 55 60 Gly Asn
Met Gly Asn Ala Thr His Trp Ser Asn Cys Cys Asn Thr Thr 65 70 75 80
Tyr Trp Ser Asn Thr Gly Tyr Tyr Thr Asn Ala Ala Arg Gly Ala Tyr 85
90 95 Met Gly Asn Cys Ala Tyr Gly Ala Tyr Thr Thr Tyr Met Gly Asn
Ala 100 105 110 Thr His Cys Cys Asn Cys Ala Arg Gly Ala Arg Gly Ala
Arg Thr Thr 115 120 125 Tyr Gly Ala Tyr Gly Gly Asn Ala Ala Tyr Cys
Ala Arg Thr Thr Tyr 130 135 140 Cys Ala Arg Ala Ala Arg Gly Cys Asn
Cys Ala Arg Gly Cys Asn Ala 145 150 155 160 Thr His Trp Ser Asn Gly
Thr Asn Tyr Thr Asn Cys Ala Tyr Gly Ala 165 170 175 Arg Ala Thr Gly
Ala Thr Gly Cys Ala Arg Cys Ala Arg Ala Cys Asn 180 185 190 Thr Thr
Tyr Ala Ala Tyr Tyr Thr Asn Thr Thr Tyr Trp Ser Asn Ala 195 200 205
Cys Asn Gly Ala Arg Ala Ala Tyr Trp Ser Asn Trp Ser Asn Gly Cys 210
215 220 Asn Gly Cys Asn Thr Gly Gly Gly Ala Arg Cys Ala Arg Ala Cys
Asn 225 230 235 240 Tyr Thr Asn Tyr Thr Asn Gly Ala Arg Ala Ala Arg
Thr Thr Tyr Trp 245 250 255 Ser Asn Ala Thr His Gly Ala Arg Tyr Thr
Asn Thr Thr Tyr Cys Ala 260 265 270 Arg Cys Ala Arg Ala Thr Gly Ala
Ala Tyr Gly Ala Tyr Tyr Thr Asn 275 280 285 Gly Ala Arg Gly Cys Asn
Thr Gly Tyr Gly Thr Asn Ala Thr His Cys 290 295 300 Ala Arg Gly Ala
Arg Gly Thr Asn Gly Gly Asn Gly Thr Asn Gly Ala 305 310 315 320 Arg
Gly Ala Arg Ala Cys Asn Cys Cys Asn Tyr Thr Asn Ala Thr Gly 325 330
335 Ala Ala Tyr Gly Ala Arg Gly Ala Tyr Trp Ser Asn Ala Thr His Tyr
340 345 350 Thr Asn Gly Cys Asn Gly Thr Asn Met Gly Asn Ala Ala Arg
Thr Ala 355 360 365 Tyr Thr Thr Tyr Cys Ala Arg Met Gly Asn Ala Thr
His Ala Cys Asn 370 375 380 Tyr Thr Asn Thr Ala Tyr Tyr Thr Asn Ala
Thr His Gly Ala Arg Met 385 390 395 400 Gly Asn Ala Ala Arg Thr Ala
Tyr Trp Ser Asn Cys Cys Asn Thr Gly 405 410 415 Tyr Gly Cys Asn Thr
Gly Gly Gly Ala Arg Gly Thr Asn Gly Thr Asn 420 425 430 Met Gly Asn
Gly Cys Asn Gly Ala Arg Ala Thr His Ala Thr Gly Met 435 440 445 Gly
Asn Trp Ser Asn Tyr Thr Asn Trp Ser Asn Thr Thr Tyr Trp Ser 450 455
460 Asn Ala Cys Asn Ala Ala Tyr Tyr Thr Asn Cys Ala Arg Ala Ala Arg
465 470 475 480 Met Gly Asn Tyr Thr Asn Met Gly Asn Met Gly Asn Ala
Ala Arg Gly 485 490 495 Ala Tyr 4189PRTHomo sapiens 4Met Ala Leu
Ser Phe Ser Leu Leu Met Ala Val Leu Val Leu Ser Tyr 1 5 10 15 Lys
Ser Ile Cys Ser Leu Gly Cys Asp Leu Pro Gln Thr His Ser Leu 20 25
30 Gly Asn Arg Arg Ala Leu Ile Leu Leu Gly Gln Met Gly Arg Ile Ser
35 40 45 Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Arg Ile Pro
Gln Glu 50 55 60 Glu Phe Asp Gly Asn Gln Phe Gln Lys Ala Gln Ala
Ile Ser Val Leu 65 70 75 80 His Glu Met Ile Gln Gln Thr Phe Asn Leu
Phe Ser Thr Glu Asp Ser 85 90 95 Ser Ala Ala Trp Glu Gln Ser Leu
Leu Glu Lys Phe Ser Thr Glu Leu 100 105 110 Tyr Gln Gln Leu Asn Asp
Leu Glu Ala Cys Val Ile Gln Glu Val Gly 115 120 125 Val Glu Glu Thr
Pro Leu Met Asn Glu Asp Ser Ile Leu Ala Val Arg 130 135 140 Lys Tyr
Phe Gln Arg Ile Thr Leu Tyr Leu Ile Glu Arg Lys Tyr Ser 145 150 155
160 Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser
165 170 175 Phe Ser Thr Asn Leu Gln Lys Arg Leu Arg Arg Lys Asp 180
185 5189PRTHomo sapiens 5Met Ala Leu Pro Phe Ala Leu Met Met Ala
Leu Val Val Leu Ser Cys 1 5 10 15 Lys Ser Ser Cys Ser Leu Gly Cys
Asn Leu Ser Gln Thr His Ser Leu 20 25 30 Asn Asn Arg Arg Thr Leu
Met Leu Met Ala Gln Met Arg Arg Ile Ser 35 40 45 Pro Phe Ser Cys
Leu Lys Asp Arg His Asp Phe Glu Phe Pro Gln Glu 50 55 60 Glu Phe
Asp Gly Asn Gln Phe Gln Lys Ala Gln Ala Ile Ser Val Leu 65 70 75 80
His Glu Met Met Gln Gln Thr Phe Asn Leu Phe Ser Thr Lys Asn Ser 85
90 95 Ser Ala Ala Trp Asp Glu Thr Leu Leu Glu Lys Phe Tyr Ile Glu
Leu 100 105 110 Phe Gln Gln Met Asn Asp Leu Glu Ala Cys Val Ile Gln
Glu Val Gly 115 120 125 Val Glu Glu Thr Pro Leu Met Asn Glu Asp Ser
Ile Leu Ala Val Lys 130 135 140 Lys Tyr Phe Gln Arg Ile Thr Leu Tyr
Leu Met Glu Lys Lys Tyr Ser 145 150 155 160 Pro Cys Ala Trp Glu Val
Val Arg Ala Glu Ile Met Arg Ser Leu Ser 165 170 175 Phe Ser Thr Asn
Leu Gln Lys Arg Leu Arg Arg Lys Asp 180 185
* * * * *
References