U.S. patent application number 10/768744 was filed with the patent office on 2004-09-23 for methods for modulating an inflammatory response.
Invention is credited to Hunter, Christopher, Villarino, Alejandro.
Application Number | 20040185049 10/768744 |
Document ID | / |
Family ID | 32853378 |
Filed Date | 2004-09-23 |
United States Patent
Application |
20040185049 |
Kind Code |
A1 |
Hunter, Christopher ; et
al. |
September 23, 2004 |
Methods for modulating an inflammatory response
Abstract
The inventive subject matter relates to novel methods for
modulating an immune response in an animal, which comprises
administering to said animal an effective amount of an agent that
increases IL-27R/WSX-1 activity. Further, the inventive subject
matter relates to pharmaceutical compositions comprising an
effective amount of an agent that increases IL-27R/WSX-1
activity.
Inventors: |
Hunter, Christopher;
(Philadelphia, PA) ; Villarino, Alejandro;
(Philadelphia, PA) |
Correspondence
Address: |
NATH & ASSOCIATES, PLLC
Sixth Floor
1030 15th Street, N.W.
Washington
DC
20005
US
|
Family ID: |
32853378 |
Appl. No.: |
10/768744 |
Filed: |
February 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60444494 |
Jan 31, 2003 |
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60519074 |
Nov 10, 2003 |
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Current U.S.
Class: |
424/145.1 ;
514/1.7; 514/12.2; 514/13.2; 514/15.1; 514/16.6; 514/16.8;
514/17.1; 514/17.9; 514/19.8; 514/20.8; 514/7.7 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 13/08 20180101; A61P 35/04 20180101; A61P 43/00 20180101; A61P
17/00 20180101; A61P 21/00 20180101; A61P 3/10 20180101; A61P 17/06
20180101; A61P 21/04 20180101; A61P 27/02 20180101; A61P 31/18
20180101; A61P 9/10 20180101; C07K 16/2866 20130101; A61P 1/16
20180101; A61P 35/02 20180101; A61P 13/12 20180101; A61P 31/04
20180101; A61P 37/08 20180101; C07K 2317/76 20130101; A61P 37/00
20180101; A61K 38/208 20130101; A61P 25/00 20180101; A61P 29/00
20180101; A61P 7/00 20180101; A61P 1/04 20180101; A61P 9/08
20180101; A61P 19/02 20180101; A61K 38/20 20130101; A61P 7/06
20180101; A61P 19/10 20180101; A61P 1/18 20180101; A61P 13/00
20180101; A61P 1/02 20180101; A61P 37/02 20180101; Y02A 50/30
20180101; A61P 37/06 20180101; A61K 2039/505 20130101; A61P 5/14
20180101; A61P 11/06 20180101; A61P 25/16 20180101; A61P 1/14
20180101 |
Class at
Publication: |
424/145.1 ;
514/012 |
International
Class: |
A61K 039/395; A61K
038/17 |
Goverment Interests
[0002] This work was supported in part by National Institutes of
Health Grant Nos. AI41158, AI42334, and AI35914. The United States
government may have rights in this invention by virtue of this
support.
Claims
We claim:
1. A method for modulating an immune response in an animal in need
thereof, which comprises administering to said animal an effective
amount of an IL-27R/WSX-1 ligand.
2. The method of claim 1, wherein said modulation is suppression
and said ligand is an IL-27R/WSX-1 agonist.
3. The method of claim 2, wherein said agonist is selected from the
group consisting of IL-27, an active fragment of IL-27, and an
agonistic antibody to IL-27R/WSX-1 which enhances IL-27R/WSX-1
activity.
4. The method of claim 1, wherein said modulation is activation and
said ligand is an IL-27R/WSX-1 antagonist.
5. The method of claim 4, wherein said antagonist is an inactive
IL-27 fragment which retains IL-27R/WSX-1 binding affinity, or an
antagonist antibody to IL-27R/WSX-1 which suppresses IL-27R/WSX-1
activity.
6. A method for modulating a T-helper cell mediated immune response
in an animal in need thereof, which comprises administering to said
animal an effective amount of an IL-27R/WSX-1 ligand.
7. The method of claim 6, wherein said modulation is suppression
and said ligand is an IL-27R/WSX-1 agonist.
8. The method of claim 7, wherein said agonist is selected from the
group consisting of IL-27, an active fragment of IL-27, and an
agonistic antibody to IL-27R/WSX-1 which enhances IL-27R/WSX-1
activity.
9. The method of claim 6, wherein said modulation is activation and
said ligand is an IL-27R/WSX-1 antagonist.
10. The method of claim 9, wherein said antagonist is an inactive
IL-27 fragment which retains IL-27R/WSX-1 binding affinity, or an
antagonist antibody to IL-27R/WSX-1 which suppresses IL-27R/WSX-1
activity.
11. The method of claim 6, wherein said T-helper cell is Th1.
12. The method of claim 6, wherein said T-helper cell is Th2.
13. A method for modulating an interferon-.gamma.mediated immune
response in an animal in need thereof, which comprises
administering to said animal an effective amount of an IL-27R/WSX-1
ligand.
14. The method of claim 13, wherein said modulation is suppression
and said ligand is an IL-27R/WSX-1 agonist.
15. The method of claim 14, wherein said agonist is selected from
the group consisting of IL-27, an active fragment of IL-27, and an
agonistic antibody to IL-27R/WSX-1 which enhances IL-27R/WSX-1
activity.
16. The method of claim 13, wherein said modulation is activation
and said ligand is an IL-27R/WSX-1 antagonist.
17. The method of claim 16, wherein said antagonist is an inactive
IL-27 fragment which retains IL-27R/WSX-1 binding affinity, or an
antagonist antibody to IL-27R/WSX-1 which suppresses IL-27R/WSX-1
activity.
18. A method for treating immune hyperactivity in an animal in need
thereof, which comprises administering to said animal an effective
amount of an IL-27R/WSX-1 ligand.
19. A method for treating an immune hyperactivity disorder in an
animal in need thereof, which comprises administering to said
animal an effective amount of an IL-27R/WSX-1 ligand.
20. The method of claim 19, wherein said immune disorder is
selected from the group consisting of autoimmune disorders,
hypersensitivity disorders, allergies, and asthma.
21. The method of claim 20, wherein said immune disorder is
selected from the group consisting of Acquired Immune Deficiency
Syndrome; acute pancreatitis; Addison's disease; alcohol-induced
liver injury including alcoholic cirrhosis; Alzheimer's disease;
amyelolateroschlerosis; asthma and other pulmonary diseases;
atherosclerosis; autoimmune vasculitis; autoimmune
hepatitis-induced hepatic injury; biliary cirrhosis;
cachexia/anorexia, including AIDS-induced cachexia; cancer, such as
multiple myeloma and myelogenous and other leukemias, as well as
tumor metastasis; chronic fatigue syndrome; Clostridium associated
illnesses, including Clostridium-associated diarrhea; coronary
conditions and indications, including congestive heart failure,
coronary restenosis, myocardial infarction, myocardial dysfunction,
and coronary artery bypass graft; diabetes, including juvenile
onset Type 1, diabetes mellitus, and insulin resistance;
endometriosis, endometritis, and related conditions; epididymitis;
erythropoietin resistance; fever; fibromyalgia or analgesia;
glomerulonephritis; graft versus host disease/transplant rejection;
Graves' disease; Guillain-Barre syndrome; Hashimoto's disease;
hemolytic anemia; hemorrhagic shock; hyperalgesia; inflammatory
bowel diseases including ulcerative colitis and Crohn's disease;
inflammatory conditions of a joint and rheumatic diseases
including, osteoarthritis, rheumatoid arthritis, juvenile
(rheumatoid) arthritis, seronegative polyarthritis, ankylosing
spondylitis, Reiter's syndrome and reactive arthritis, Still's
disease, psoriatic arthritis, enteropathic arthritis, polymyositis,
dermatomyositis, scleroderma, systemic sclerosis, vasculitis (e.g.,
Kawasaki's disease), cerebral vasculitis, Lyme disease,
staphylococcal-inducedarthritis, Sjogren's syndrome, rheumatic
fever, polychondritis and polymyalgia rheumatica and giant cell
arteritis; inflammatory eye disease, as may be associated with, for
example, corneal transplant; inflammatory eye disease, as may be
associated with, e.g., corneal transplant; inflammatory bowel
disease; ischemia, including cerebral ischemia; Kawasaki's disease;
learning impairment; lung diseases; lupus nephritis; multiple
sclerosis; myasthenia gravis; myopathiesneuroinflammatory diseases;
neurotoxicity; ocular diseases and conditions, including ocular
degeneration and uveitis; osteoporosis; pain, including
cancer-related pain; Parkinson's disease; pemphigus; periodontal
disease; Pityriasis rubra pilaris; pre-term labor; prostatitis and
related conditions; psoriasis and related conditions; psoriatic
arthritis; pulmonary fibrosis; reperfusion injury; rheumatic fever;
rheumatoid arthritis; sarcoidosis; scleroderma; septic shock; side
effects from radiation therapy; Sjogren's syndrome; sleep
disturbance; spondyloarthropathies; systemic lupus erythematosus;
temporal mandibular joint disease; thyroiditis; tissue
transplantation or an inflammatory condition resulting from strain,
sprain, cartilage damage, trauma, and orthopedic surgery;
transplant rejection; uveitis; vasculitis; or an inflammatory
condition resulting from strain, sprain, cartilage damage, trauma,
orthopedic surgery, infection or other disease processes.
22. A method for treating a T-helper cell mediated disorder in an
animal in need thereof, which comprises administering to said
animal an effective amount of an IL-27R/WSX-1 ligand.
23. The method of claim 22, wherein said T-helper cell mediated
disorder is selected from the group consisting of Acquired Immune
Deficiency Syndrome; acute pancreatitis; Addison's disease;
alcohol-induced liver injury including alcoholic cirrhosis;
Alzheimer's disease; amyelolateroschlerosis; asthma and other
pulmonary diseases; atherosclerosis; autoimmune vasculitis;
autoimmune hepatitis-induced hepatic injury; biliary cirrhosis;
cachexia/anorexia, including AIDS-induced cachexia; cancer, such as
multiple myeloma and myelogenous and other leukemias, as well as
tumor metastasis; chronic fatigue syndrome; Clostridium associated
illnesses, including Clostridium-associated diarrhea; coronary
conditions and indications, including congestive heart failure,
coronary restenosis, myocardial infarction, myocardial dysfunction,
and coronary artery bypass graft; diabetes, including juvenile
onset Type 1, diabetes mellitus, and insulin resistance;
endometriosis, endometritis, and related conditions; epididymitis;
erythropoietin resistance; fever; fibromyalgia or analgesia;
glomerulonephritis; graft versus host disease/transplant rejection;
Graves' disease; Guillain-Barre syndrome; Hashimoto's disease;
hemolytic anemia; hemorrhagic shock; hyperalgesia; inflammatory
bowel diseases including ulcerative colitis and Crohn's disease;
inflammatory conditions of a joint and rheumatic diseases
including, osteoarthritis, rheumatoid arthritis, juvenile
(rheumatoid) arthritis, seronegative polyarthritis, ankylosing
spondylitis, Reiter's syndrome and reactive arthritis, Still's
disease, psoriatic arthritis, enteropathic arthritis, polymyositis,
dermatomyositis, scleroderma, systemic sclerosis, vasculitis (e.g.,
Kawasaki's disease), cerebral vasculitis, Lyme disease,
staphylococcal-inducedarthritis, Sjbgren's syndrome, rheumatic
fever, polychondritis and polymyalgia rheumatica and giant cell
arteritis; inflammatory eye disease, as may be associated with, for
example, corneal transplant; inflammatory eye disease, as may be
associated with, e.g., corneal transplant; inflammatory bowel
disease; ischemia, including cerebral ischemia; Kawasaki's disease;
learning impairment; lung diseases; lupus nephritis; multiple
sclerosis; myasthenia gravis; myopathiesneuroinflammatory diseases;
neurotoxicity; ocular diseases and conditions, including ocular
degeneration and uveitis; osteoporosis; pain, including
cancer-related pain; Parkinson's disease; pemphigus; periodontal
disease; Pityriasis rubra pilaris; pre-term labor; prostatitis and
related conditions; psoriasis and related conditions; psoriatic
arthritis; pulmonary fibrosis; reperfusion injury; rheumatic fever;
rheumatoid arthritis; sarcoidosis; scleroderma; septic shock; side
effects from radiation therapy; Sjogren's syndrome; sleep
disturbance; spondyloarthropathies; systemic lupus erythematosus;
temporal mandibular joint disease; thyroiditis; tissue
transplantation or an inflammatory condition resulting from strain,
sprain, cartilage damage, trauma, and orthopedic surgery;
transplant rejection; uveitis; vasculitis; or an inflammatory
condition resulting from strain, sprain, cartilage damage, trauma,
orthopedic surgery, infection or other disease processes.
24. A method for modulating a T-helper cell mediated immune
response in an animal in need thereof, which comprises
administering to said animal an effective amount of an IL-27R/WSX-1
ligand.
25. The method of claim 24, wherein said T-helper cell is Th1.
26. The method of claim 24, wherein said T-helper cell is Th2.
27. A pharmaceutical composition comprising: (i) an effective
amount of an IL-27R/WSX-1 ligand; and (ii) a pharmaceutically
acceptable carrier.
28. The pharmaceutical composition of claim 27, wherein said
IL-27R/WSX-1 ligand is an agent that increases WSX-1 activity.
29. The pharmaceutical composition of claim 28, wherein said agent
comprises IL-27 or an active fragment thereof.
30. The pharmaceutical composition of claim 28, wherein said agent
comprises an agonistic antibody that binds to an epitope on
WSX-1.
31. The pharmaceutical composition of claim 28, wherein said agent
comprises an agonistic antibody that binds to an epitope on
IL-27R.
32. The pharmaceutical composition of claim 28, wherein said agent
comprises an agonistic antibody that binds to an epitope on
IL-27RPP.
33. A method of treating immune hyperreactivity, which comprises
administering an effective amount of an agent that increases WSX-1
activity.
34. The method of claim 33, wherein the agent comprises IL-27 or an
active fragment thereof.
35. The method of claim 33, wherein the agent comprises an
agonistic antibody that binds to an epitope on WSX-1.
36. The method of claim 33, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27R.
37. The method of claim 33, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27RPP.
38. A method of suppressing polarized T cells, which comprises
administering an effective amount of an agent that increases WSX-1
activity.
39. The method of claim 38, wherein the agent comprises IL-27 or an
active fragment thereof.
40. The method of claim 38, wherein the agent comprises an
agonistic antibody that binds to an epitope on WSX-1.
41. The method of claim 38, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27R.
42. The method of claim 38, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27RPP.
43. A method of treating Th1-mediated disease, which comprises
administering an effective amount of an agent that increases WSX-1
activity.
44. The method of claim 43, wherein the agent comprises IL-27 or an
active fragment thereof.
45. The method of claim 43, wherein the agent comprises an
agonistic antibody that binds to an epitope on WSX-1.
46. The method of claim 43, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27R.
47. The method of claim 43, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27RPP.
48. A method of treating Th2-mediated disease, which comprises
administering an effective amount of an agent that increases WSX-1
activity.
49. The method of claim 48, wherein the agent comprises IL-27 or an
active fragment thereof.
50. The method of claim 48, wherein the agent comprises an
agonistic antibody that binds to an epitope on WSX-1.
51. The method of claim 48, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27R.
52. The method of claim 48, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27RPP.
53. A method of treating IFN-g mediated disease, which comprises
administering an effective amount of an agent that increases WSX-1
activity.
54. The method of claim 53, wherein the agent comprises IL-27 or an
active fragment thereof.
55. The method of claim 53, wherein the agent comprises an
agonistic antibody that binds to an epitope on WSX-1.
56. The method of claim 53, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27R.
57. The method of claim 53, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27RPP.
58. A method of treating IgE-mediated disease, which comprises
administering an effective amount of an agent that increases WSX-1
activity.
59. The method of claim 58, wherein the agent comprises IL-27 or an
active fragment thereof.
60. The method of claim 58, wherein the agent comprises an
agonistic antibody that binds to an epitope on WSX-1.
61. The method of claim 58, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27R.
62. The method of claim 58, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27RPP.
63. A method of treating asthma, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
64. The method of claim 64, wherein the agent comprises IL-27 or an
active fragment thereof.
65. The method of claim 64, wherein the agent comprises an
agonistic antibody that binds to an epitope on WSX-1.
66. The method of claim 64, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27R.
67. The method of claim 64, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27RPP.
68. A method of treating allergy, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
69. The method of claim 68, wherein the agent comprises IL-27 or an
active fragment thereof.
70. The method of claim 68, wherein the agent comprises an
agonistic antibody that binds to an epitope on WSX-1.
71. The method of claim 68, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27R.
72. The method of claim 68, wherein the agent comprises an
agonistic antibody that binds to an epitope on IL-27RPP.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/444,494, filed Jan. 31, 2003, and U.S.
Provisional Patent Application No. 60/519,074, filed Nov. 10, 2003,
the contents of which are hereby incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION 1. Field of Invention
[0003] The inventive subject matter relates to novel methods for
modulating an immune response in an animal, which comprises
administering to said animal an effective amount of an agent that
increases IL-27R/WSX-1 activity. Further, the inventive subject
matter relates to pharmaceutical compositions comprising an
effective amount of an agent that increases IL-27R/WSX-1
activity.
[0004] 2. Background
[0005] WSX-1 is a class I cytokine receptor that is homologous to
the P2 chain of the IL-12R in both sequence and structure. This
receptor is highly expressed by resting/naive CD4.sup.+ T cells and
CD8.sup.+ T cells. Recent studies have identified IL-27,
heterodimeric cytokine composed of the subunits EBI3 and IL-27p28,
as the ligand for WSX-1. EBI3, a member of the class I cytokine
receptor family, shares significant structural homology to
IL-12p40, and IL-27p28 is closely related to IL-12p35. In addition
to the structural similarity between the IL-12/IL-12R and
IL-27/WSX-1 ligand/receptor pairs, there are also reports that show
functional similarity. While the IL-12R plays a critical role in
the development of Th1 type responses, it has been reported that
WSX-1 deficient cells have impaired IFN-.gamma. production during
early Th1 differentiation. Moreover, recombinant IL-27, like IL-12,
can enhance Th1 differentiation in highly purified naive helper T
cells. As a consequence of these studies, an early consensus
emerged that IL-27/WSX-1 was, like the IL-12/IL-12R interaction, an
important factor in the initial differentiation of Th1
responses.
[0006] Although recent studies have described IL-27 and its
receptor, WSX-1, as promoters of Th1 differentiation in naive
CD4.sup.+ T cells, Applicants have determined that signaling
through this receptor is involved in limiting the intensity and
duration of T cell activity. When WSX-1-deficient mice are infected
with the intracellular pathogen Toxoplasma gondii, they establish
protective T cell responses, characterized by production of
inflammatory cytokines and control of parasite replication.
However, infected WSX-1.sup.-/- mice are unable to downregulate
these protective responses, and develop a lethal, T cell-mediated
inflammatory disease.
[0007] Contrary to the previous consensus understanding, we have
demonstrated that WSX-1-deficient mice infected with T. gondii are
able to develop a strong Th1 type response and control parasite
replication, but are unable to downregulate this protective
response and develop a lethal, T cell-mediated inflammatory
disease. This pathology was characterized by the excessive
production of IFN-.gamma., persistence of highly activated T cells,
and enhanced T cell proliferation in vivo. The phenotype could be
recapitulated in vitro as Th1 polarization of WSX-1.sup.-/-
CD4.sup.+ T cells led to increased proliferation and IFN-.gamma.
secretion. However, these studies also confirmed that, under
nonpolarizing conditions, WSX-1 is required for optimal IFN-.gamma.
production. Further analysis revealed that exogenous IL-27 can
activate STAT1, STAT3, and STAT5: STAT family members that have
traditionally been associated with cellular activation but have
also recently been linked with the inhibition of immune functions.
Together, these findings demonstrate that WSX-1 is not required for
the generation of IFN-.gamma.-mediated immunity to T. gondii and
identify a novel function for WSX-1 as a potent antagonist of T
cell-mediated immune hyperactivity.
[0008] Thus, we have found a method for treating immune
hyperreactivity, wherein the method comprises administering an
effective amount of an agent that increases WSX-1 activity. Such
agents include IL-27, an active fragment thereof, an agonistic
antibody that binds to an epitope on WSX-1 and, as WSX-1 is part of
the heterodimeric receptor IL-27R along with IL-27RPP, an agonistic
antibody that binds to an epitope on dimeric IL-27R, and an
agonistic antibody that binds to an epitope on IL-27RPP. Such
agents may also be used to suppress the function of polarized T
cells, to treat Th1-mediated disease, to treat Th2-mediated
disease, to treat IFN-.gamma. mediated disease, to modulate the
response of non-lymphoid cells (e.g., MAST cells) to Immunoglobulin
E (IgE), and to treat IgE-mediated disease (e.g., asthma, allergy,
and the like). Such agents may be used to treat a number of
autoimmune diseases, as further disclosed in detail below.
[0009] We have also found a method for increasing immune response,
which is useful in treating patients with suppressed immune
systems. Such immunosuppression can result, for example, from
disease or from chemotherapy. This method comprises administering
an effective amount of an agent that binds to IL-27 and/or
IL-27R/WQSX-1 and prevents activation of IL-27R by IL-27. Such
agents include an antibody that binds to IL-27, an antibody that
binds to EB13 (a subunit of IL-27), an antibody that binds to
IL27p28 (also a subunit of IL-27), a soluble form of WSX-1, a
soluble form of dimeric IL-27R, a soluble form of IL-27RPP, and a
non-activating IL-27R/WSX-1 ligand. Such agents may be used to
treat a number of diseases and conditions related to
immunosuppression, as further detailed hereinbelow.
[0010] Further in accordance with the present invention, also
provided is a method for screening for molecules useful in the
methods described hereinabove, which comprises (a) treating cells
comprising IL-27 receptor with a test agent; (b) determining any
effect of the test agent on IL-27 receptor activity.
SUMMARY OF THE INVENTION
[0011] The inventive subject matter relates to a method for
modulating an immune response in an animal in need thereof, which
comprises administering to said animal an effective amount of an
IL-27R/WSX-1 ligand.
[0012] The inventive subject matter further relates to a method for
modulating a T-helper cell mediated immune response in an animal in
need thereof, which comprises administering to said animal an
effective amount of an IL-27R/WSX-1 ligand.
[0013] method for modulating an interferon-.gamma. mediated immune
response in an animal in need thereof, which comprises
administering to said animal an effective amount of an IL-27R/WSX-1
ligand.
[0014] The inventive subject matter further relates to a method for
treating immune hyperactivity in an animal in need thereof, which
comprises administering to said animal an effective amount of an
IL-27R/WSX-1 ligand.
[0015] The inventive subject matter further relates to a method for
treating a T-helper cell mediated disorder in an animal in need
thereof, which comprises administering to said animal an effective
amount of an IL-27R/WSX-1 ligand.
[0016] The inventive subject matter further relates to a method for
modulating a T-helper cell mediated immune response in an animal in
need thereof, which comprises administering to said animal an
effective amount of an IL-27R/WSX-1 ligand.
[0017] The inventive subject matter further relates to a
pharmaceutical composition comprising:
[0018] (i) an effective amount of an IL-27R/WSX-1 ligand; and
[0019] (ii) a pharmaceutically acceptable carrier.
[0020] The inventive subject matter further relates to a method of
treating immune hyperreactivity, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0021] The inventive subject matter further relates to a method of
suppressing polarized T cells, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0022] The inventive subject matter further relates to a method of
treating Th1-mediated disease, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0023] The inventive subject matter further relates to a method of
treating Th2-mediated disease, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0024] The inventive subject matter further relates to a method of
treating IFN-mediated disease, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0025] The inventive subject matter further relates to a method of
treating IgE-mediated disease, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0026] The inventive subject matter further relates to a method of
treating asthma, which comprises administering an effective amount
of an agent that increases WSX-1 activity.
[0027] The inventive subject matter further relates to a method of
treating allergy, which comprises administering an effective amount
of an agent that increases WSX-1 activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1(A) is a photograph which depicts a protein gel
showing expression levels of mRNA for IL-27p28, EBI3, WSX-1, and
.beta.-actin.
[0029] FIG. 1(B) is a graph which depicts survival times of wild
type, WSX-1.sup.-/-, and IL-12p40.sup.-/- mice infected with T.
gondii.
[0030] FIG. 1(C) is a graph which depicts percentage of cells
infected with T. gondii.
[0031] FIG. 1(D) is a graph which depicts survival of WSX-1.sup.-/-
mice infected with T. gondii.
[0032] FIGS. 1(E-H) are histological slides which depict liver
tissue of wild type and WSX-1.sup.-/- animals before infection and
after being infected for 12 days.
[0033] FIG. 2(A) is a graph which depicts IL-12p40 circulating
level at 0, 7, and 11 days postinfection in wild type and
WSX-1.sup.-/- mice.
[0034] FIG. 2(B) is a graph which depicts IL-12p40 circulating
level in splenocytes cultured with soluble Toxoplasma antigen in
wild type and WSX-1.sup.-/- mice.
[0035] FIG. 2(C) is a graph which depicts IL-12p40 circulating
level in splenocytes cultured with plate bound .alpha.CD3 antibody
in wild type and WSX-1.sup.-/- mice.
[0036] FIG. 2(D) is a graph which depicts IFN-.gamma. circulating
level at 0, 7, and 11 days postinfection in wild type and
WSX-1.sup.-/- mice.
[0037] FIG. 2(E) is a graph which depicts IFN-.gamma. circulating
level in splenocytes cultured with soluble Toxoplasma antigen in
wild type and WSX-1.sup.-/- mice.
[0038] FIG. 2(F) is a graph which depicts IFN-.gamma. circulating
level in splenocytes cultured with plate bound .alpha.CD3 antibody
in wild type and WSX-1.sup.-/- mice.
[0039] FIG. 2(G) is a graph which depicts IL-10 circulating level
in splenocytes cultured with soluble Toxoplasma antigen in wild
type and WSX-1.sup.-/- mice.
[0040] FIG. 2(H) is a graph which depicts IL-10 circulating level
in splenocytes cultured with plate bound .alpha.CD3 antibody in
wild type and WSX-1.sup.-/- mice.
[0041] FIG. 2(I) is a graph which depicts IL-2 production in
splenocytes cultured with plate bound .alpha.CD3 antibody in wild
type and WSX-1.sup.-/- mice.
[0042] FIG. 2(J)is a graph which depicts survival of wild type,
WSX-1.sup.-/-, and IL-10.sup.-/- mice infected with T. gondii.
[0043] FIG. 2(K) is a graph which depicts IL-12p40 concentration in
splenocytes from wild type, WSX-1.sup.-/-, and IL-10.sup.-/- mice
prior to infection and 10 days postinfection.
[0044] FIG. 2(L) is a graph which depicts IFN-.gamma. concentration
in splenocytes from wild type, WSX-1.sup.-/-, and IL-10.sup.-/-
mice prior to infection and 10 days postinfection.
[0045] FIG. 3(A) is a series of flow cytometry photographs which
depict splenocytes from wild type and WSX-1.sup.-/- mice infected
for 0, 7, and 10 days, and stained for CD4 and intracellular
IFN-.gamma..
[0046] FIG. 3(B) is a series of flow cytometry photographs which
depict splenocytes from wild type and WSX-1.sup.-/- mice infected
for 11 days, and stained for CD4 and intracellular IFN-.gamma..
[0047] FIG. 4(A) is a series of flow cytometry photographs which
depict splenocytes from wild type and WSX-1.sup.-/- mice infected
for 0, 7, and 10 days, stained for expression of CD4, CD25, and
CD62L.
[0048] FIG. 4(B) is a series of flow cytometry photographs which
depict splenocytes from wild type and WSX-1.sup.-/- mice infected
and treated for 3 days prior to sacrifice at days 0, 10, and 14
postinfection, stained for CD4 expression and incorporation of
BrdU.
[0049] FIG. 5(A) is a pair of graphs which depict IFN-.gamma.
production in wild type or WSX-1.sup.-/- cells adoptively
transferred into RAG-2.sup.-/- mice.
[0050] FIG. 5(B) is a pair of flow cytometry photographs which
depict splenocytes isolated at 11 days postinfection, and stained
for CD4 and intracellular IFN-.gamma..
[0051] FIG. 5(C) is a pair of flow cytometry photographs which
depict splenocytes isolated at 11 days postinfection, and stained
for expression of CD4, CD25, and CD62L.
[0052] FIG. 6(A) is a flow cytometry photograph and two graphs
which depict naive CD4.sup.+ T cells purified from uninfected wild
type or WSX-1.sup.-/- spleens, stained with CFSE, and activated
with soluble .alpha.CD3 under nonpolarizing conditions.
[0053] FIG. 6(B) is a flow cytometry photograph and two graphs
which depict naive CD4.sup.+ T cells purified from uninfected wild
type or WSX-1.sup.-/- spleens, stained with CFSE, and activated
with Th1 soluble .alpha.CD3 under polarizing conditions.
[0054] FIG. 7 is a photograph of a Western blot which depicts total
and tyrosine phosphorylated STAT-1, STAT-3, and STAT-5 detected
following stimulation of naive CD4.sup.+CD45RB.sup.hi T cells with
rIFN-.gamma., IFN-.gamma., or rIL-27.
[0055] FIG. 8 is a photograph that depicts primary bone
marrow-derived mouse mast cells, a spontaneously immortalized
BM-derived mouse mast cell line (T*) or CD4+ splenocytes
(9.times.10e6) which were immunoprecepitated with antibody against
WSX-1. The blot was probed with the same anti-WSX-1 Ab.
[0056] FIGS. 9-13 are graphs which depict a Flow Cytometry analysis
of whole blood from wild type and WSX-1 knockout mice stimulated
with conditioned medium from either mock-transfected or
IL-27-transfected 293 cells. The cells are stained for
intracellular phospho-STAT1 as a measure of IL-27 signaling and a
surface maker to identify the cell lineage of the responding cells.
The data show that, in whole blood, two major cell types respond;
CD4 T cells and a cell type that is currently characterized by its
scatter properties only, but appears to be of non-lymphoid nature:
mast cells or possibly basophils. This data shows that these two
major cell types not only express IL-27 receptor, but that IL-27R
is functionally competent on both cell types.
DETAILED DESCRIPTION OF THE INVENTION
[0057] Definitions
[0058] Unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the
singular.
[0059] Throughout this document, reference is made to the terms
"WSX-1", "IL-27R", and "IL-27R/WSX-1". It is to be understood that
these terms are used interchangeably to refer to the receptor for
IL-27.
[0060] The term "ligand" as used herein refers to a molecule, or a
domain of a molecule, which is bound or able to bind selectivley
and stoichiometrically, either covalently or not, to one or more
specific sites on another molecule. Non-limiting examples of
ligands include an antibody and its antigen, a hormone and its
receptor, and an enzyme and its substrate.
[0061] Terms Related to Treatment of Disease
[0062] The term "patient" includes human and animal subjects. The
term "modulating" refers to regulating or adjusting the degree of
activity of a process or the degree of an effect. "Modulating"
includes activation, amplification, attenuation, and
suppression.
[0063] The term "effecting" refers to the process of producing an
effect on biological activity, function, health, or condition of an
organism in which such biological activity, function, health, or
condition is maintained, enhanced, diminished, or treated in a
manner which is consistent with the general health and well-being
of the organism.
[0064] The term "enhancing" the biological activity, function,
health, or condition of an organism refers to the process of
augmenting, fortifying, strengthening, or improving.
[0065] A "treatment" or "treating" of a disorder, condition, or
disease (including an autoimmune disease), encompasses alleviation
of at least one symptom thereof, a reduction in the severity
thereof, or the delay or prevention of progression to a more
serious disease that occurs with some frequency following the
treated disease or disorder. Treatment need not mean that the
disease is totally cured. A useful therapeutic agent needs only to
reduce the severity of a disease, reduce the severity of a symptom
or symptoms associated with the disease or its treatment, or
provide improvement to a patient's quality of life, or delay the
onset of a more serious disease that can occur with some frequency
following the treated disease, disorder, or condition. For example,
if the disease is rheumatoid arthritis, a therapeutic agent may
decrease swelling of joints, reduce the number of joints affected,
or delay or inhibit bone loss. An SLE patient can have symptoms
such as skin lesions, fever, weakness, arthritis, lymphadenopathy,
pleurisy, pericarditis, and/or anemia, among others. Such symptoms
can be assessed by any of a number of conventional techniques
including, for example, visual observation, photography,
measurement of temperature, grip strength, or joint size, and/or
microscopic examination of blood to determine the concentration of
red blood cells. The invention encompasses a method for treatment
comprising administering to a patient an agent in an amount and for
a time sufficient to induce a sustained improvement over baseline
of an indicator that reflects the severity of a particular disease,
disorder, or condition or the severity of symptoms caused thereby
or to delay or prevent the onset of a more serious disease that
follows the treated disease, disorder, or condition in some or all
cases. The invention does not exclude possible treatment with other
therapeutic agents before, after, and/or during treatment with the
agents described herein.
[0066] A disease or medical condition is considered to be a
"Th1-mediated disease" or "Th1-mediated disorder" if the
naturally-occurring or experimentally-induced disease or medical
condition is associated with proliferation or increased
differentiation of Th1 cells. Th1-mediated disease can be
identified by (1) levels of Th1 cells that exceed those normally
found in a human, animal, or cell culture; (2) pathological
findings associated with the disease or medical condition that can
be mimicked experimentally in animals by administration of agents
that upregulate proliferation or differentiation of Th1 cells; or
(3) a pathology induced in experimental animal models of the
disease or medical condition can be inhibited or abolished by
treatment with agents that inhibit the proliferation or
differentiation of Th1 cells. In most Th1-mediated diseases, at
least two of the three conditions are met.
[0067] A non-exclusive list of acute and chronic Th1-mediated
diseases includes but is not limited to the following: acute
pancreatitis; amyelolateroschlerosis (ALS); Alzheimer's disease;
cachexia/anorexia, including AIDS-induced cachexia; asthma and
other pulmonary diseases; atherosclerosis; autoimmune vasculitis;
chronic fatigue syndrome; Clostridium associated illnesses,
including Clostridium-associated diarrhea; coronary conditions and
indications, including congestive heart failure, coronary
restenosis, myocardial infarction, myocardial dysfunction (e.g.,
related to sepsis), and coronary artery bypass graft; cancer, such
as multiple myeloma and myelogenous (e.g., AML or CML) and other
leukemias, as well as tumor metastasis; diabetes (e.g.,
insulin-dependent diabetes); endometriosis; fever; fibromyalgia;
glomerulonephritis; graft versus host disease/transplant rejection;
hemorrhagic shock; hyperalgesia; inflammatory bowel disease;
inflammatory conditions of a joint, including osteoarthritis,
psoriatic arthritis and rheumatoid arthritis; inflammatory eye
disease, as may be associated with, e.g., corneal transplant;
ischemia, including cerebral ischemia (e.g., brain injury as a
result of trauma, epilepsy, hemorrhage or stroke, each of which may
lead to neurodegeneration); Kawasaki's disease; learning
impairment; lung diseases (e.g., ARDS); multiple sclerosis;
myopathies (e.g., muscle protein metabolism, especially in sepsis);
neurotoxicity (e.g., as induced by HIV); osteoporosis; pain,
including cancer-related pain; Parkinson's disease; periodontal
disease; pre-term labor; psoriasis; reperfusion injury; septic
shock; side effects from radiation therapy; temporal mandibular
joint disease; sleep disturbance; uveitis; or an inflammatory
condition resulting from strain, sprain, cartilage damage, trauma,
orthopedic surgery, infection or other disease processes.
[0068] A disease or medical condition is considered to be a
"Th2-mediated disease" if the naturally-occurring or
experimentally-induced disease or medical condition is associated
with proliferation or increased differentiation of Th2 cells.
Th2-mediated disease can be identified by (1) levels of Th2 cells
that exceed those normally found in a human, animal, or cell
culture; (2) pathological findings associated with the disease or
medical condition that can be mimicked experimentally in animals by
administration of agents that upregulate proliferation or
differentiation of Th2 cells; or (3) a pathology induced in
experimental animal models of the disease or medical condition can
be inhibited or abolished by treatment with agents that inhibit the
proliferation or differentiation of Th2 cells. In most Th2-mediated
diseases, at least two of the three conditions are met.
[0069] A non-exclusive list of acute and chronic Th2-mediated
diseases includes but is not limited to the following: acute
pancreatitis; amyelolateroschlerosis (ALS); Alzheimer's disease;
cachexia/anorexia, including AIDS-induced cachexia; asthma and
other pulmonary diseases; atherosclerosis; autoimmune vasculitis;
chronic fatigue syndrome; Clostridium associated illnesses,
including Clostridium-associated diarrhea; coronary conditions and
indications, including congestive heart failure, coronary
restenosis, myocardial infarction, myocardial dysfunction (e.g.,
related to sepsis), and coronary artery bypass graft; cancer, such
as multiple myeloma and myelogenous (e.g., AML or CML) and other
leukemias, as well as tumor metastasis; diabetes (e.g.,
insulin-dependent diabetes); endometriosis; fever; fibromyalgia;
glomerulonephritis; graft versus host disease/transplant rejection;
hemorrhagic shock; hyperalgesia; inflammatory bowel disease;
inflammatory conditions of a joint, including osteoarthritis,
psoriatic arthritis and rheumatoid arthritis; inflammatory eye
disease, as may be associated with, e.g., corneal transplant;
ischemia, including cerebral ischemia (e.g., brain injury as a
result of trauma, epilepsy, hemorrhage or stroke, each of which may
lead to neurodegeneration); Kawasaki's disease; learning
impairment; lung diseases (e.g., ARDS); multiple sclerosis;
myopathies (e.g., muscle protein metabolism, especially in sepsis);
neurotoxicity (e.g., as induced by HIV); osteoporosis; pain,
including cancer-related pain; Parkinson's disease; periodontal
disease; pre-term labor; psoriasis; reperfusion injury; septic
shock; side effects from radiation therapy; temporal mandibular
joint disease; sleep disturbance; uveitis; or an inflammatory
condition resulting from strain, sprain, cartilage damage, trauma,
orthopedic surgery, infection or other disease processes.
[0070] The term "IFN-.gamma. mediated disease" includes, but is not
limited to, inflammatory, infectious, and autoimmune diseases. An
"autoimmune disease" as used herein refers to disease states and
conditions wherein a patient's immune response is directed toward
the patient's own constituents. For example, IFN-.gamma. mediated
diseases include, but are not limited to, Acquired Immune
Deficiency Syndrome (AIDS), rheumatoid arthritis, inflammatory
bowel diseases including ulcerative colitis and Crohn's disease,
multiple sclerosis, Addison's disease, diabetes (type I),
epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre
syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus
erythematosus (SLE), lupus nephritis, myasthenia gravis, pemphigus,
psoriasis, psoriatic arthritis, atherosclerosis, erythropoietin
resistance, graft versus host disease, transplant rejection,
autoimmune hepatitis-induced hepatic injury, biliary cirrhosis,
alcohol-induced liver injury including alcoholic cirrhosis,
rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome,
spondyloarthropathies, thyroiditis, and vasculitis. Because
IFN-.gamma. is a cytokine with multiple functions, including
protecting the body from viral infection and regulating several
aspects of the immune response, increased IFN-.gamma. activity can
contribute to several pathological conditions. The term "IFN-gamma
mediated disease" also encompasses any medical condition associated
with increased levels of IFN-.gamma. or increased sensitivity to
IFN-.gamma.. Additional IFN-.gamma. mediated diseases include:
acute pancreatitis; ALS; Alzheimer's disease; cachexia/anorexia,
including AIDS-induced cachexia; asthma and other pulmonary
diseases; atherosclerosis; chronic fatigue syndrome; Clostridium
associated illnesses, including Clostridium-associated diarrhea;
coronary conditions and indications, including congestive heart
failure, coronary restenosis, myocardial infarction, myocardial
dysfunction (e.g., related to sepsis), and coronary artery bypass
graft; cancer, such as multiple myeloma and myelogenous (e.g., AML
and CML) and other leukemias, as well as tumor metastasis; fever;
glomerulonephritis; graft versus host disease/transplant rejection;
hemohorragic shock; inflammatory eye disease, as may be associated
with, for example, corneal transplant; ischemia, including cerebral
ischemia (e.g., brain injury as a result of trauma, epilepsy,
hemorrhage or stroke, each of which may lead to neurodegeneration);
learning impairment; multiple sclerosis; myopathies (e.g., muscle
protein metabolism, esp. in sepsis); neurotoxicity (e.g., as
induced by HIV); osteoporosis; pain, including cancer-related pain;
Parkinson's disease; periodontal disease; neurotoxicity; pre-term
labor; psoriasis; reperfusion injury; septic shock; side effects
from radiation therapy; temporal mandibular joint disease; sleep
disturbance; uveitis; or an inflammatory condition resulting from
strain, sprain, cartilage damage, trauma, orthopedic surgery,
infection or other disease processes; diabetes, including juvenile
onset Type 1, diabetes mellitus, and insulin resistance (e.g., as
associated with obesity); endometriosis, endometritis, and related
conditions; fibromyalgia or analgesia; hyperalgesia; lung diseases
(e.g., adult respiratory distress syndrome, and pulmonary
fibrosis); neuroinflammatory diseases; ocular diseases and
conditions, including ocular degeneration and uveitis; Pityriasis
rubra pilaris (PRP); prostatitis (bacterial or non-bacterial) and
related conditions; psoriasis and related conditions; pulmonary
fibrosis; reperfusion injury; inflammatory conditions of a joint
and rheumatic diseases including, osteoarthritis, rheumatoid
arthritis, juvenile (rheumatoid) arthritis, seronegative
polyarthritis, ankylosing spondylitis, Reiter's syndrome and
reactive arthritis, Still's disease, psoriatic arthritis,
enteropathic arthritis, polymyositis, dermatomyositis, scleroderma,
systemic sclerosis, vasculitis (e.g., Kawasaki's disease), cerebral
vasculitis, Lyme disease, staphylococcal-induced ("septic")
arthritis, Sjogren's syndrome, rheumatic fever, polychondritis and
polymyalgia rheumatica and giant cell arteritis; septic shock; side
effects from radiation therapy; temporal mandibular joint disease;
thyroiditis; tissue transplantation or an inflammatory condition
resulting from strain, sprain, cartilage damage, trauma, and
orthopedic surgery.
[0071] The term "IgE-related diseases" refers to diseases,
disorders, and conditions associated with increased production of
Immunoglobulin E. Such diseases, disorders, and conditions comprise
asthma, allergy, and the like.
[0072] The terms "WSX-1 activity" and "IL-27R activity" refer to
any biological activity heretofore or hereafter found to be
associated with interaction of IL-27 and its receptor, known
variously as IL-27R or WSX-1. By way of example, WSX-1 activity
includes, but is not limited to, resistance to infection,
modulation of infection-induced cytokine production (including
IFN-.gamma.), and modulation of levels of CD4+ and CD8+ T cells as
shown in the accompanying figures and the disclosed materials and
methods.
[0073] The term "selective binding agent" refers to a molecule
which preferentially binds a protein of interest. A selective
binding agent may include a protein, peptide, nucleic acid,
carbohydrate, lipid, or small molecular weight compound. Examples
of proteins that are selective binding agents of the inventive
subject matter include soluble receptors (i.e., proteins having all
or part of the extracellular domain of a naturally occurring
membrane-bound protein but not the transmembrane domain or
intracellular domain); antibodies and fragments thereof; variants,
derivatives and fusion proteins of antibodies and soluble
receptors; peptidomimetic compounds; and organo-mimetic compounds.
In a preferred embodiment, a selective binding agent is an
antibody, such as polyclonal antibodies, monoclonal antibodies
(mAbs), chimeric antibodies, CDR-grafted antibodies, anti-idiotypic
(anti-Id) antibodies to antibodies that can be labeled in soluble
or bound form, as well as fragments, regions or derivatives
thereof, provided by known techniques, including, but not limited
to enzymatic cleavage, peptide synthesis or recombinant techniques.
The selective binding agents of the present inventive subject
matter are capable of binding portions of their respective protein
of interest that inhibit the binding of the protein of interest to
its cognate receptor or ligand.
[0074] Assays for Selective Binding Agents
[0075] Screening methods for identifying selective binding agents
which partially or completely mimic or inhibit at least one
biological activity of a protein of interest (e.g., mimicking the
activity of IL-27) are provided by the inventive subject matter.
Inhibiting the biological activity of a protein of interest
includes, but is not limited to, inhibiting binding of the protein
to its cognate receptor, inhibiting the activity thereof as
measured by in vitro or in vivo assays. Mimicking the biological
activity of a protein of interest includes, but is not limited to,
binding to the protein's cognate receptor and causing biological
activity similar to the protein of interest as measured by in vitro
or in vivo assays. In vitro assays include those that detect
binding of the protein to its cognate receptor or ligand and may be
used to screen selective binding agents for their ability to
increase or decrease the rate or extent of such binding. In one
type of assay, a polypeptide, such as a soluble receptor, is
immobilized on a solid support (e.g., agarose or acrylic beads) and
its cognate ligand is added either in the presence or absence of a
selective binding agent. The extent of binding of the soluble
receptor and its cognate ligand in the presence or absence of a
selective binding agent present is measured. Binding can be
detected by for example radioactive labeling, fluorescent labeling
or enzymatic reaction.
[0076] Alternatively, the binding reaction may be carried out using
a surface plasmon resonance detector system such as the BIAcore
assay system (Pharmacia, Piscataway, N.J.). Binding reactions may
be carried out according to the manufacturer's protocol.
[0077] In vitro assays such as those described above may be used
advantageously to screen rapidly large numbers of selective binding
agents. The assays may be automated to screen compounds generated
in phage display, synthetic peptide and chemical synthesis
libraries.
[0078] Selective binding agents may also be screened in cell
culture using cells and cell lines expressing either polypeptide.
Cells and cell lines may be obtained from any mammal, but
preferably will be from human or other primate, canine, or rodent
sources. As an example, the binding of receptor and cognate ligand
on the cell surface is evaluated in the presence or absence of
selective binding agents, with the extent of binding determined by
flow cytometry using a biotinylated antibody to the ligand.
[0079] The selective binding agents of the inventive subject matter
may be employed in any known assay method, such as
radioimmunoassays, competitive binding assays, direct and indirect
sandwich assays (ELISAs), and immunoprecipitation assays (Sola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, 1987)).
[0080] Terms Related to Soluble Protein Agents
[0081] The term "half-life extender" refers to a molecule that
prevents degradation and/or increases half-life, reduces toxicity,
reduces immunogenicity, or increases biological activity of a
therapeutic protein. Exemplary vehicles include an Fc domain (which
is preferred) as well as a linear polymer (e.g., polyethylene
glycol (PEG), polylysine, dextran, etc.); a branched-chain polymer
(see, for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al.,
issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul.
20, 1993; WO 93/21259 by Frechet et al., published 28 Oct. 1993); a
lipid; a cholesterol group (such as a steroid); a carbohydrate or
oligosaccharide (e.g., dextran); any natural or synthetic protein,
polypeptide or peptide that binds to a salvage receptor; albumin,
including human serum albumin (HSA), leucine zipper domain, and
other such proteins and protein fragments. Vehicles are further
described hereinafter.
[0082] The term "native Fc" refers to molecule or sequence
comprising the sequence of a non-antigen-binding fragment resulting
from digestion of whole antibody, whether in monomeric or
multimeric form. The original immunoglobulin source of the native
Fc is preferably of human origin and may be any of the
immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc's
are made up of monomeric polypeptides that may be linked into
dimeric or multimeric forms by covalent (i.e., disulfide bonds) and
non-covalent association. The number of intermolecular disulfide
bonds between monomeric subunits of native Fc molecules ranges from
1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g.,
IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a
disulfide-bonded dimer resulting from papain digestion of an IgG
(see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The
term "native Fc" as used herein is generic to the monomeric,
dimeric, and multimeric forms.
[0083] The term "Fc variant" refers to a molecule or sequence that
is modified from a native Fc but still comprises a binding site for
the salvage receptor, FcRn. International applications WO 97/34631
(published 25 Sep. 1997) and WO 96/32478 describe exemplary Fc
variants, as well as interaction with the salvage receptor, and are
hereby incorporated by reference in their entirety. Thus, the term
"Fc variant" comprises a molecule or sequence that is humanized
from a non-human native Fc. Furthermore, a native Fc comprises
sites that may be removed because they provide structural features
or biological activity that are not required for the fusion
molecules of the present invention. Thus, the term "Fc variant"
comprises a molecule or sequence that lacks one or more native Fc
sites or residues that affect or are involved in (1) disulfide bond
formation, (2) incompatibility with a selected host cell (3)
N-terminal heterogeneity upon expression in a selected host cell,
(4) glycosylation, (5) interaction with complement, (6) binding to
an Fc receptor other than a salvage receptor, or (7)
antibody-dependent cellular cytotoxicity (ADCC). Fc variants are
described in further detail hereinafter.
[0084] The term "Fc domain" encompasses native Fc and Fc variant
molecules and sequences as defined above. As with Fc variants and
native Fc's, the term "Fc domain" includes molecules in monomeric
or multimeric form, whether digested from whole antibody or
produced by other means.
[0085] Antibody-Related Terms
[0086] The term "antigen" refers to a molecule or a portion of a
molecule capable of being bound by a selective binding agent, such
as an antibody, and additionally capable of being used in an animal
to produce antibodies capable of binding to an epitope of that
antigen. An antigen may have one or more epitopes.
[0087] The term "epitope" includes any determinant, preferably a
polypeptide determinant, capable of specific binding to an
immunoglobulin or T-cell receptor. In certain embodiments, epitope
determinants include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific
three-dimensional structural characteristics, and/or specific
charge characteristics. An epitope is a region of an antigen that
is bound by an antibody. In certain embodiments, an antibody is
said to specifically bind an antigen when it preferentially
recognizes its target antigen in a complex mixture of proteins
and/or macromolecules. In preferred embodiments, an antibody is
said to specifically bind an antigen when the dissociation constant
is less than or equal to about 10 nM, more preferably when the
dissociation constant is less than or equal to about 100 pM, and
most preferably when the dissociation constant is less than or
equal to about 10 pM. "Antibody" or "antibody peptide(s)" refer to
an intact antibody, or a binding fragment thereof that competes
with the intact antibody for specific binding and includes
chimeric, humanized, fully human, and bispecific antibodies. In
certain embodiments, binding fragments are produced by recombinant
DNA techniques. In additional embodiments, binding fragments are
produced by enzymatic or chemical cleavage of intact antibodies.
Binding fragments include, but are not limited to, Fab, Fab',
F(ab').sub.2, Fv, immunologically functional immunoglobulin
fragments, heavy chain, light chain, and single-chain
antibodies.
[0088] The term "heavy chain" includes a full-length heavy chain
and fragments thereof having sufficient variable region sequence to
confer binding specificity. A full-length heavy chain includes a
variable region domain, V.sub.H, and three constant region domains,
C.sub.H1, C.sub.H2, and C.sub.H3. The V.sub.H domain is at the
amino-terminus of the polypeptide, and the CH.sup.3 domain is at
the carboxyl-terminus.
[0089] The term "light chain" includes a full-length light chain
and fragments thereof having sufficient variable region sequence to
confer binding specificity. A full-length light chain includes a
variable region domain, V.sub.L, and a constant region domain,
C.sub.L. Like the heavy chain, the variable region domain of the
light chain is at the amino-terminus of the polypeptide.
[0090] A "Fab fragment" is comprised of one light chain and the
C.sub.H1 and variable regions of one heavy chain. The heavy chain
of a Fab molecule cannot form a disulfide bond with another heavy
chain molecule.
[0091] A "Fab' fragment" contains one light chain and one heavy
chain that contains more of the constant region, between the
C.sub.H1 and C.sub.H2 domains, such that an interchain disulfide
bond can be formed between two heavy chains to form a F(ab').sub.2
molecule.
[0092] A "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond is formed between two heavy chains.
[0093] The "Fv region" comprises the variable regions from both the
heavy and light chains, but lacks the constant regions.
[0094] "Single-chain antibodies" are Fv molecules in which the
heavy and light chain variable regions have been connected by a
flexible linker to form a single polypeptide chain, which forms an
antigen-binding region. Single chain antibodies are discussed in
detail in International Patent Application Publication No. WO
88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the
disclosures of which are incorporated by reference for any
purpose.
[0095] A "bivalent antibody" other than a "multispecific" or
"multifunctional" antibody, in certain embodiments, is understood
to comprise binding sites having identical antigenic
specificity.
[0096] A "bispecific" or "bifunctional" antibody is a hybrid
antibody having two different heavy/light chain pairs and two
different binding sites. Bispecific antibodies may be produced by a
variety of methods including, but not limited to, fusion of
hybridomas or linking of Fab' fragments. See, e.g., Songsivilai
& Lachmann (1990), Clin. Exp. Immunol. 79:315-321; Kostelny et
al. (1992), J. Immunol. 148:1547-1553.
[0097] In assessing antibody binding and specificity according to
the invention, an antibody "substantially inhibits" adhesion of an
antigen to a binding partner therefor when an excess of antibody
reduces the quantity of antigen bound to binding partner by at
least about 20%, 40%, 60%, 80%, 85%, or more (as measured in an in
vitro competitive binding assay).
[0098] The term "immunologically functional immunoglobulin
fragment" as used herein refers to a polypeptide fragment that
contains at least the variable domains of the immunoglobulin heavy
and light chains. An immunologically functional immunoglobulin
fragment of the invention is capable of binding to an antigen,
preventing binding of the antigen to a binding partner therefor,
interrupting the biological response resulting from binding of the
antigen and binding partner, or any combination thereof.
[0099] The term "agent" means a chemical compound, a mixture of
chemical compounds, a biological macromolecule, or an extract made
from biological materials.
[0100] DNA and Protein Preparation Terms
[0101] Conventional techniques may be used for preparing
recombinant DNA, performing oligonucleotide synthesis, and
practicing tissue culture and transformation (e.g.,
electroporation, transfection or lipofection). Enzymatic reactions
and purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See, e.g., Sambrook et al., 2001, Molecular
Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated
herein by reference for any purpose. Unless specific definitions
are provided, the nomenclature utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques may be used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0102] The term "isolated polynucleotide" means that the subject
polynucleotide, (1) is not associated (covalently or noncovalently)
with all or a portion of other polynucleotides with which the
subject polynucleotide is associated in nature, (2) is associated
with a molecule with which it is not associated in nature, or (3)
does not occur in nature associated with any other polynucleotides.
Such an isolated polynucleotide may be genomic DNA, cDNA, mRNA or
other RNA, of synthetic origin, or any combination thereof.
[0103] The term "isolated protein" referred to herein means that a
subject protein (1) is free of at least some other proteins with
which it would normally be found, (2) is essentially free of other
proteins from the same source, e.g., from the same species, (3) is
expressed by a cell from a different species, (4) has been
separated from at least about 50 percent of polynucleotides,
lipids, carbohydrates, or other materials with which it is
associated in nature, (5) is not associated (by covalent or
noncovalent interaction) with portions of a protein with which the
"isolated protein" is associated in nature, (6) is operably
associated (by covalent or noncovalent interaction) with a
polypeptide with which it is not associated in nature, or (7) does
not occur in nature. Genomic DNA, cDNA, mRNA or other RNA, of
synthetic origin, or any combination thereof may encode such an
isolated protein. Preferably, the isolated protein is substantially
free from proteins or polypeptides or other contaminants that are
found in its natural environment that would interfere with its
therapeutic, diagnostic, prophylactic, research or other use.
[0104] The terms "polypeptide" or "protein" means molecules having
the sequence of native proteins, that is, proteins produced by
naturally-occurring and specifically non-recombinant cells, or
genetically-engineered or recombinant cells, and comprise molecules
having the amino acid sequence of the native protein, or molecules
having deletions from, additions to, and/or substitutions of one or
more amino acids of the native sequence. The terms "polypeptide"
and "protein" specifically encompass antibodies, or sequences that
have deletions from, additions to, and/or substitutions of one or
more amino acid of such antibody. The term "polypeptide fragment"
refers to a polypeptide that has an amino-terminal deletion, a
carboxyl-terminal deletion, and/or an internal deletion. In certain
embodiments, fragments are at least 5 to about 500 amino acids
long. It will be appreciated that in certain embodiments, fragments
are at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250,
300, 350, 400, or 450 amino acids long. Particularly useful
polypeptide fragments include functional domains, including binding
domains. In the case of an antibody, useful fragments include but
are not limited to a CDR region, a variable domain of a heavy or
light chain, a portion of an antibody chain or just its variable
region including two CDRs, and the like.
[0105] The terms "naturally occurring" and "native" mean that the
biological materials (molecules, sequences, protein complexes,
cells, and the like) to which the terms are applied can be found in
nature and are not manipulated by man. For example, a polypeptide
or polynucleotide sequence that is present in an organism
(including viruses) that can be isolated from a source in nature
and that has not been intentionally modified by man is naturally
occurring. Likewise, the terms "non-naturally occurring" or
"non-native" refer to a material that is not found in nature or
that has been structurally modified or synthesized by man.
[0106] The term "operably linked" means that the components to
which the term is applied are in a relationship that allows them to
carry out their inherent functions under suitable conditions. For
example, a control sequence "operably linked" to a protein coding
sequence is ligated thereto so that expression of the protein
coding sequence is achieved under conditions compatible with the
transcriptional activity of the control sequences.
[0107] The term "control sequence" means that the subject
polynucleotide sequence can effect expression and processing of
coding sequences to which it is ligated. The nature of such control
sequences may depend upon the host organism. In particular
embodiments, control sequences for prokaryotes may include a
promoter, ribosomal binding site, and transcription termination
sequence. In other particular embodiments, control sequences for
eukaryotes may include promoters comprising one or a plurality of
recognition sites for transcription factors, transcription enhancer
sequences, and transcription termination sequence. In certain
embodiments, "control sequences" can include leader sequences
and/or fusion partner sequences.
[0108] The term "polynucleotide" means single-stranded or
double-stranded nucleic acid polymers of at least 10 bases in
length. In certain embodiments, the nucleotides comprising the
polynucleotide can be ribonucleotides or deoxyribonucleotides or a
modified form of either type of nucleotide. Said modifications
include base modifications such as bromouridine and inosine
derivatives, ribose modifications such as 2',3'-dideoxyribose, and
internucleotide linkage modifications such as phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The
term includes single and double stranded forms of DNA.
[0109] The term "oligonucleotide" means a polynucleotide comprising
a length of 200 bases or fewer. In preferred embodiments,
oligonucleotides are 10 to 60 bases in length. In more preferred
embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19,
or 20 to 40 bases in length. Oligonucleotides may be single
stranded or double stranded, e.g., for use in the construction of a
mutant gene. Oligonucleotides of the invention may be sense or
antisense oligonucleotides.
[0110] The term "naturally occurring nucleotides" includes
deoxyribonucleotides and ribonucleotides. The term "modified
nucleotides" includes nucleotides with modified or substituted
sugar groups or modified or substituted bases. The term
"oligonucleotide linkages" includes linkages such as
phosphorothioate, phosphorodithioate, phosphoroselenoate,
phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,
phosphoroamidate, and the like. See, e.g., LaPlanche et al. (1986),
Nucl. Acids Res. 14:9081; Stec et al. (1984), J. Am. Chem. Soc.
106:6077; Stein et al. (1988), Nucl. Acids Res. 16:3209; Zon et al.
(1991), Anti-Cancer Drug Design 6:539; Zon et al. (1991),
Oligonucleotides and Analogues: A Practical Approach, pp. 87-108
(F. Eckstein, ed.), Oxford University Press, Oxford England; Stec
et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman (1990),
Chemical Reviews 90:543, the disclosures of which are hereby
incorporated by reference for any purpose. An oligonucleotide of
the invention can include a label, including a radiolabel, a
fluorescent label, a hapten or an antigenic label, for detection
assays.
[0111] The term "vector" means any molecule (e.g., nucleic acid,
plasmid, or virus) used to transfer coding information to a host
cell.
[0112] The term "expression vector" or "expression construct"
refers to a vector that is suitable for transformation of a host
cell and contains nucleic acid sequences that direct and/or control
(in conjunction with the host cell) expression of one or more
heterologous coding regions operatively linked thereto. An
expression construct may include, but is not limited to, sequences
that affect or control transcription, translation, and RNA
splicing, if introns are present, of a coding region operably
linked thereto.
[0113] The term "host cell" means a cell that has been transformed,
or is capable of being transformed, with a nucleic acid sequence
and thereby expresses a selected gene of interest. The term
includes the progeny of the parent cell, whether or not the progeny
is identical in morphology or in genetic make-up to the original
parent cell, so long as the selected gene is present.
[0114] The term "transduction" means the transfer of genes from one
bacterium to another, usually by phage. "Transduction" also refers
to the acquisition and transfer of eukaryotic cellular sequences by
retroviruses.
[0115] The term "transfection" means the uptake of foreign or
exogenous DNA by a cell, and a cell has been "transfected" when the
exogenous DNA has been introduced inside the cell membrane. A
number of transfection techniques are well known in the art and are
disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456;
Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Id.;
Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier;
and Chu et al., 1981, Gene 13:197. Such techniques can be used to
introduce one or more exogenous DNA moieties into suitable host
cells.
[0116] The term "transformation" refers to a change in a cell's
genetic characteristics, and a cell has been transformed when it
has been modified to contain new DNA. For example, a cell is
transformed where it is genetically modified from its native state
by transfection, transduction, or other techniques. Following
transfection or transduction, the transforming DNA may recombine
with that of the cell by physically integrating into a chromosome
of the cell, or may be maintained transiently as an episomal
element without being replicated, or may replicate independently as
a plasmid. A cell is considered to have been "stably transformed"
when the transforming DNA is replicated with the division of the
cell.
[0117] The term "identity" refers to a relationship between the
sequences of two or more polypeptide molecules or two or more
nucleic acid molecules, as determined by comparing the sequences
thereof. In the art, "identity" also means the degree of sequence
relatedness between nucleic acid molecules or polypeptides, as the
case may be, as determined by the match between sequences of two or
more nucleotides or two or more amino acids. "Identity" measures
the percentage of identical matches between the smaller of two or
more sequences with gap alignments (if any) addressed by a
particular mathematical model or computer program (i.e.,
"algorithms").
[0118] The term "similarity" is used in the art with regard to a
related concept; in contrast to "identity," however, "similarity"
refers to a measure of relatedness that includes both identical
matches and conservative substitution matches. If two polypeptide
sequences have, for example, {fraction (10/20)} identical amino
acids, and the remainder are all non-conservative substitutions,
then the percentage identity and similarity would both be 50%. If
in the same example, there are five more positions where there are
conservative substitutions, then the percentage identity remains
50%, but the percentage similarity would be 75% ({fraction
(15/20)}). Therefore, in cases where there are conservative
substitutions, the percentage similarity between two polypeptides
will be higher than the percentage identity between those two
polypeptides.
[0119] Identity and similarity of related nucleic acids and
polypeptides can be readily calculated by known methods. Such
methods include, but are not limited to, those described in
Computational Molecular Biology, (Lesk, A. M., ed.), 1988, Oxford
University Press, New York; Biocomputing: Informatics and Genome
Projects, (Smith, D. W., ed.), 1993, Academic Press, New York;
Computer Analysis of Sequence Data, Part 1, (Griffin, A. M., and
Griffin, H. G., eds.), 1994, Humana Press, New Jersey; von Heinje,
G., Sequence Analysis in Molecular Biology, 1987, Academic Press;
Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.),
1991, M. Stockton Press, New York; Carillo et al., 1988, SIAM J.
Applied Math. 48:1073; and Durbin et al., 1998, Biological Sequence
Analysis, Cambridge University Press.
[0120] Preferred methods to determine identity are designed to give
the largest match between the sequences tested. Methods to
determine identity are described in publicly available computer
programs. Preferred computer program methods to determine identity
between two sequences include, but are not limited to, the GCG
program package, including GAP (Devereux et al., 1984, Nucl. Acid.
Res. 12:387; Genetics Computer Group, University of Wisconsin,
Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., 1990,
J. Mol. Biol. 215:403-410). The BLASTX program is publicly
available from the National Center for Biotechnology Information
(NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH
Bethesda, Md. 20894; Altschul et al., 1990, supra). The well-known
Smith Waterman algorithm may also be used to determine
identity.
[0121] Certain alignment schemes for aligning two amino acid
sequences may result in matching of only a short region of the two
sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, in certain
embodiments, the selected alignment method (GAP program) will
result in an alignment that spans at least 50 contiguous amino
acids of the target polypeptide.
[0122] For example, using the computer algorithm GAP (Genetics
Computer Group, University of Wisconsin, Madison, Wis.), two
polypeptides-for which the percentage sequence identity is to be
determined are aligned for optimal matching of their respective
amino acids (the "matched span", as determined by the algorithm).
In certain embodiments, a gap opening penalty (which is calculated
as three-times the average diagonal; where the "average diagonal"
is the average of the diagonal of the comparison matrix being used;
the "diagonal" is the score or number assigned to each perfect
amino acid match by the particular comparison matrix) and a gap
extension penalty (which is usually one-tenth of the gap opening
penalty), as well as a comparison matrix such as PAM250 or BLOSUM
62 are used in conjunction with the algorithm. In certain
embodiments, a standard comparison matrix (see Dayhoff et al.,
1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM
250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci
USA 89:10915-10919 for the BLOSUM 62 comparison matrix) is also
used by the algorithm.
[0123] In certain embodiments, the parameters for a polypeptide
sequence comparison include the following:
[0124] Algorithm: Needleman et al. (1970), J. Mol. Biol.
48:443-453;
[0125] Comparison matrix: BLOSUM 62 from Henikoff et al. (1992),
supra;
[0126] Gap Penalty: 12
[0127] Gap Length Penalty: 4
[0128] Threshold of Similarity: 0
[0129] The GAP program may be useful with the above parameters. In
certain embodiments, the aforementioned parameters are the default
parameters for polypeptide comparisons (along with no penalty for
end gaps) using the GAP algorithm.
[0130] The term "naturally occurring," as used to refer to amino
acids, refers to the twenty conventional amino acids. See
Immunology--A Synthesis, 2nd Edition, (E. S. Golub and D. R. Gren,
eds.), Sinauer Associates: Sunderland, Mass. (1991), incorporated
herein by reference for any purpose. Peptide analogs are commonly
used in the pharmaceutical industry as non-peptide drugs with
properties analogous to those of the template peptide. These types
of non-peptide compounds are termed "peptide mimetics" or
"peptidomimetics". See Fauchere (1986), Adv. Drug Res. 15:29; Veber
& Freidinger, 1985, TINS p.392; and Evans et al. (1987), J.
Med. Chem. 30:1229, which are incorporated herein by reference for
any purpose. Such compounds are often developed with the aid of
computerized molecular modeling. Peptide mimetics that are
structurally similar to therapeutically useful peptides may be used
to produce a similar therapeutic or prophylactic effect. Generally,
peptidomimetics are structurally similar to a paradigm peptide or
polypeptide (i.e., a peptide or polypeptide that has a biochemical
property or pharmacological activity), such as human antibody, but
have one or more peptide linkages optionally replaced by a linkage
selected from: --CH.sub.2--NH--, --CH.sub.2--S--,
--CH.sub.2--CH.sub.2--, --CH.dbd.CH-- (cis and trans) ,
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--, by methods
well known in the art. Systematic substitution of one or more amino
acids of a consensus sequence with a D-amino acid of the same type
(e.g., D-lysine in place of L-lysine) may be used in certain
embodiments to generate more stable peptides. In addition,
constrained peptides comprising a consensus sequence or a
substantially identical consensus sequence variation may be
generated by methods known in the art (Rizo & Gierasch, 1992,
Ann. Rev. Biochem. 61:387, incorporated herein by reference for any
purpose); for example, by adding internal cysteine residues capable
of forming intramolecular disulfide bridges which cyclize the
peptide.
[0131] The terms "label" or "labeled" refers to incorporation of a
detectable marker, e.g., by incorporation of a radiolabeled amino
acid or attachment to a polypeptide of biotin moieties that can be
detected by marked avidin (e.g., streptavidin preferably comprising
a detectable marker such as a fluorescent marker, a
chemiluminescent marker or an enzymatic activity that can be
detected by optical or colorimetric methods). In certain
embodiments, the label can also be therapeutic. Various methods of
labeling polypeptides and glycoproteins are known in the art and
may be used advantageously in the methods disclosed herein.
Examples of labels for polypeptides include, but are not limited
to, the following: radioisotopes or radionuclides (e.g., .sup.3H,
.sup.14C .sup.15N, .sup.35S, .sup.90Y, .sup.99mTc, .sup.111In,
.sup.125I, .sup.131I), fluorescent labels (e.g., fluorescein
isothiocyanate (FITC), rhodamine, or lanthanide phosphors),
enzymatic labels (e.g., horseradish peroxidase,
.beta.-galactosidase, luciferase, alkaline phosphatase),
chemiluminescent labels, hapten labels such as biotinyl groups, and
predetermined polypeptide epitopes recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, epitope tags). In
certain embodiments, labels are attached by spacer arms (such as
(CH.sub.2).sub.n, where n<about 20) of various lengths to reduce
potential steric hindrance.
[0132] The term "biological sample" includes, but is not limited
to, any quantity of a substance from a living thing or formerly
living thing. Such living things include, but are not limited to,
humans, mice, monkeys, rats, rabbits, and other animals. Such
substances include, but are not limited to, blood, serum, urine,
cells, organs, tissues, bone, bone marrow, lymph, lymph nodes,
synovial tissue, chondrocytes, synovial macrophages, endothelial
cells, vascular tissue (particularly inflamed vascular tissue), and
skin. The terms "pharmaceutical agent" and "drug" refer to a
chemical compound or composition capable of inducing a desired
therapeutic effect when properly administered to a patient.
[0133] The terms "substantially pure" and "substantially purified"
mean a compound or species that is the predominant species present
(i.e., on a molar basis it is more abundant than any other
individual species in the composition). In certain embodiments, a
substantially purified fraction is a composition wherein the
species comprises at least about 50 percent (on a molar basis) of
all macromolecular species present. In certain embodiments, a
substantially pure composition will comprise more than about 80%,
85%, 90%, 95%, or 99% of all macromolar species present in the
composition. In certain embodiments, the species is purified to
essential homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
[0134] Amino Acids
[0135] The twenty naturally-occurring amino acids and their
abbreviations follow conventional usage. See Immunology-A
Synthesis, 2nd Edition, (E. S. Golub and D. R. Gren, eds.), Sinauer
Associates: Sunderland, Mass. (1991), incorporated herein by
reference for any purpose. Stereoisomers (e.g., D-amino acids) of
the twenty conventional amino acids, unnatural amino acids such as
-, -disubstituted amino acids, N-alkyl amino acids, and other
unconventional amino acids may also be suitable components for
polypeptides of the invention. Examples of unconventional amino
acids include: 4-hydroxyproline, -carboxyglutamate,
--N,N,N-trimethyllysine, --N--acetyllysine, O-phosphoserine,
N-acetylserine, N--formylmethionine, 3-methylhistidine,
5-hydroxylysine, --N-methylarginine, and other similar amino acids
and imino acids (e.g., 4-hydroxyproline). In the polypeptide
notation used herein, the left-hand direction is the amino terminal
direction and the right-hand direction is the carboxyl-terminal
direction, in accordance with standard usage and convention.
[0136] Similarly, unless specified otherwise, the left-hand end of
single-stranded polynucleotide sequences is the 5' end; the
left-hand direction of double-stranded polynucleotide sequences is
referred to as the 5' direction. The direction of 5' to 3' addition
of nascent RNA transcripts is referred to as the transcription
direction; sequence regions on the DNA strand having the same
sequence as the RNA transcript that are 5' to the 5' end of the RNA
transcript are referred to as "upstream sequences"; sequence
regions on the DNA strand having the same sequence as the RNA
transcript that are 3' to the 3' end of the RNA transcript are
referred to as "downstream sequences".
[0137] Naturally occurring amino acid residues may be divided into
classes based on common side chain properties:
[0138] 1) hydrophobic: norleucine (Nor or Nle), Met, Ala, Val, Leu,
Ile;
[0139] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0140] 3) acidic: Asp, Glu;
[0141] 4) basic: His, Lys, Arg;
[0142] 5) residues that influence chain orientation: Gly, Pro;
and
[0143] 6) aromatic: Trp, Tyr, Phe.
[0144] Conservative amino acid substitutions may involve exchange
of a member of one of these classes with another member of the same
class. Conservative amino acid substitutions may encompass
non-naturally occurring amino acid residues, which are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics and other
reversed or inverted forms of amino acid moieties.
[0145] Non-conservative substitutions may involve the exchange of a
member of one of these classes for a member from another class.
Such substituted residues may be introduced, for example, into
regions of a human antibody that are homologous with non-human
antibodies, or into the non-homologous regions of the molecule.
[0146] In making such changes, according to certain embodiments,
the hydropathic index of amino acids may be considered. Each amino
acid has been assigned a hydropathic index on the basis of its
hydrophobicity and charge characteristics. They are: isoleucine
(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine
(-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9);
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate
(-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5);
lysine (-3.9); and arginine (-4.5).
[0147] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art (see, for example, Kyte et al., 1982, J. Mol.
Biol. 157:105-131). It is known that certain amino acids may be
substituted for other amino acids having a similar hydropathic
index or score and still retain a similar biological activity. In
making changes based upon the hydropathic index, in certain
embodiments, the substitution of amino acids whose hydropathic
indices are within .+-.2 is included. In certain embodiments, those
that are within .+-.1 are included, and in certain embodiments,
those within .+-.0.5 are included.
[0148] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functional
protein or peptide thereby created is intended for use in
immunological embodiments, as disclosed herein. In certain
embodiments, the greatest local average hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.,
with a biological property of the protein.
[0149] The following hydrophilicity values have been assigned to
these amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and
tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, in certain embodiments, the substitution of
amino acids whose hydrophilicity values are within .+-.2 is
included, in certain embodiments, those that are within .+-.1 are
included, and in certain embodiments, those within .+-.0.5 are
included. One may also identify epitopes from primary amino acid
sequences on the basis of hydrophilicity. These regions are also
referred to as "epitopic core regions."
[0150] Exemplary amino acid substitutions are set forth in Table
1.
1TABLE 1 Amino Acid Substitutions Original Preferred Residues
Exemplary Substitutions Substitutions Ala Val, Leu, Ile Val Arg
Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn
Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile
Leu, Val, Met, Ala, Phe, Leu Norleucine Leu Norleucine, Ile, Val,
Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyricAcid, Arg Gln, Asn
Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly
Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe,
Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu
[0151] A skilled artisan will be able to determine suitable
variants of polypeptides as set forth herein using well-known
techniques. In certain embodiments, one skilled in the art may
identify suitable areas of the molecule that may be changed without
destroying activity by targeting regions not believed to be
important for activity. In other embodiments, the skilled artisan
can identify residues and portions of the molecules that are
conserved among similar polypeptides. In further embodiments, even
areas that may be important for biological activity or for
structure may be subject to conservative amino acid substitutions
without destroying the biological activity or without adversely
affecting the polypeptide structure.
[0152] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, the skilled artisan can predict the
importance of amino acid residues in a protein that correspond to
amino acid residues important for activity or structure in similar
proteins. One skilled in the art may opt for chemically similar
amino acid substitutions for such predicted important amino acid
residues.
[0153] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of such
information, one skilled in the art may predict the alignment of
amino acid residues of an antibody with respect to its
three-dimensional structure. In certain embodiments, one skilled in
the art may choose to not make radical changes to amino acid
residues predicted to be on the surface of the protein, since such
residues may be involved in important interactions with other
molecules. Moreover, one skilled in the art may generate test
variants containing a single amino acid substitution at each
desired amino acid residue. The variants can then be screened using
activity assays known to those skilled in the art. Such variants
could be used to gather information about suitable variants. For
example, if one discovered that a change to a particular amino acid
residue resulted in destroyed, undesirably reduced, or unsuitable
activity, variants with such a change can be avoided. In other
words, based on information gathered from such routine experiments,
one skilled in the art can readily determine the amino acids where
further substitutions should be avoided either alone or in
combination with other mutations.
[0154] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult, 1996, Curr. Op. in
Biotech. 7:422-427; Chou et al., 1974, Biochemistry 13:222-245;
Chou et al., 1974, Biochemistry 113:211-222; Chou et al., 1978,
Adv.. Enzymol. Relat. Areas Mol. Biol. 47:45-148; Chou et al.,
1979, Ann. Rev. Biochem. 47:251-276; and Chou et al., 1979,
Biophys. J. 26:367-384. Moreover, computer programs are currently
available to assist with predicting secondary structure. One method
for predicting secondary structure is based upon homology modeling.
For example, two polypeptides or proteins that have a sequence
identity of greater than 30%, or similarity greater than 40% often
have similar structural topologies. The recent growth of the
protein structural database (PDB) has provided enhanced
predictability of secondary structure, including the potential
number of folds within a polypeptide's or protein's structure. See
Holm et al., 1999, Nucl. Acid. Res. 27:244-247. It has been
suggested (Brenner et al., 1997, Curr. Op. Struct. Biol. 7:369-376)
that there are a limited number of folds in a given polypeptide or
protein and that once a critical number of structures have been
resolved, structural prediction will become dramatically more
accurate.
[0155] Additional methods of predicting secondary structure include
"threading" (Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl
et al., 1996, Structure 4:15-19), "profile analysis" (Bowie et al.,
1991, Science 253:164-170; Gribskov et al., 1990, Meth. Enzym.
183:146-159; Gribskov et al., 1987, Proc. Nat. Acad. Sci.
84:4355-4358), and "evolutionary linkage" (See Holm, 1999, supra;
and Brenner, 1997, supra).
[0156] In certain embodiments, antibody variants include
glycosylation variants wherein the number and/or type of
glycosylation site has been altered compared to the amino acid
sequences of the parent polypeptide. In certain embodiments,
protein variants comprise a greater or a lesser number of N-linked
glycosylation sites than the native protein. An N-linked
glycosylation site is characterized by the sequence: Asn-X-Ser or
Asn-X-Thr, wherein the amino acid residue designated as X may be
any amino acid residue except proline. The substitution of amino
acid residues to create this sequence provides a potential new site
for the addition of an N-linked carbohydrate chain. Alternatively,
substitutions that eliminate this sequence will remove an existing
N-linked carbohydrate chain. Also provided is a rearrangement of
N-linked carbohydrate chains wherein one or more N-linked
glycosylation sites (typically those that are naturally occurring)
are eliminated and one or more new N-linked sites are created.
Additional preferred antibody variants include cysteine variants
wherein one or more cysteine residues are deleted from or
substituted for another amino acid (e.g., serine) compared to the
parent amino acid sequence. Cysteine variants may be useful when
antibodies must be refolded into a biologically active conformation
such as after the isolation of insoluble inclusion bodies. Cysteine
variants generally have fewer cysteine residues than the native
protein, and typically have an even number to minimize interactions
resulting from unpaired cysteines.
[0157] According to certain embodiments, amino acid substitutions
are those that: (1) reduce susceptibility to proteolysis, (2)
reduce susceptibility to oxidation, (3) alter binding affinity for
forming protein complexes, (4) alter binding affinities, and/or (5)
confer or modify other physicochemical or functional properties on
such polypeptides. According to certain embodiments, single or
multiple amino acid substitutions (in certain embodiments,
conservative amino acid substitutions) may be made in the naturally
occurring sequence (in certain embodiments, in the portion of the
polypeptide outside the domain(s) forming intermolecular contacts).
In preferred embodiments, a conservative amino acid substitution
typically does not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence). Examples of art-recognized
polypeptide secondary and tertiary structures are described in
Proteins, Structures and Molecular Principles, (Creighton, ed.),
1984, W. H. Freeman and Company, New York; Introduction to Protein
Structure (C. Branden and J. Tooze, eds.), 1991, Garland
Publishing, New York, N.Y.; and Thornton et al. (1991), Nature
354:105, each of which are incorporated herein by reference.
[0158] Preparation of Antibodies
[0159] Naturally occurring antibody structural units typically
comprise a tetramer. Each such tetramer typically is composed of
two identical pairs of polypeptide chains, each pair having one
full-length "light" chain (typically having a molecular weight of
about 25 kDa) and one full-length "heavy" chain (typically having a
molecular weight of about 50-70 kDa). The amino-terminal portion of
each chain typically includes a variable region of about 100 to 110
or more amino acids that typically is responsible for antigen
recognition. The carboxy-terminal portion of each chain typically
defines a constant region responsible for effector function. Human
light chains are typically classified as kappa and lambda light
chains. Heavy chains are typically classified as mu, delta, gamma,
alpha, or epsilon, and define the antibody's isotype as IgM, IgD,
IgG, IgA, and IgE, respectively. IgG has several subclasses,
including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has
subclasses including, but not limited to, IgM1 and IgM2. IgA is
similarly subdivided into subclasses including, but not limited to,
IgA1 and IgA2. Within full-length light and heavy chains,
typically, a "J" region of about 12 or more amino acids joins the
variable region and constant regions, with the heavy chain also
including a "D" region of about 10 more amino acids. See, e.g.,
Fundamental Immunology, Ch. 7, 2.sup.nd ed., (Paul, W., ed.), 1989,
Raven Press, N.Y. (incorporated by reference in its entirety for
all purposes). The combination of the variable regions of each
light chain/heavy chain pair typically forms the antigen-binding
site.
[0160] The variable regions of each of the heavy chains and light
chains typically exhibit the same general structure comprising four
relatively conserved framework regions (FR) joined by three hyper
variable regions, also called complementarity determining regions
or CDRs. The CDRs from the two chains of each pair typically are
aligned by the framework regions, which alignment may enable
binding to a specific epitope. From N-terminal to C-terminal, both
light and heavy chain variable regions typically comprise the
domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of
amino acids to each domain is typically in accordance with the
definitions of Kabat Sequences of Proteins of Immunological
Interest (1987 and 1991, National Institutes of Health, Bethesda,
Md.), Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917, or
Chothia et al., 1989, Nature 342:878-883).
[0161] Antibodies became useful and of interest as pharmaceutical
agents with the development of monoclonal antibodies. Monoclonal
antibodies are produced using any method that produces antibody
molecules by continuous cell lines in culture. Examples of suitable
methods for preparing monoclonal antibodies include the hybridoma
methods of Kohler et al. (1975, Nature 256:495-497) and the human
B-cell hybridoma method (Kozbor, 1984, J. Immunol. 133:3001; and
Brodeur et al., 1987, Monoclonal Antibody Production Techniques and
Applications, (Marcel Dekker, Inc., New York), pp. 51-63).
[0162] Monoclonal antibodies may be modified for use as
therapeutics. One example is a "chimeric" antibody in which a
portion of the heavy chain and/or light chain is identical with or
homologous to a corresponding sequence in antibodies derived from a
particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is/are identical with
or homologous to a corresponding sequence in antibodies derived
from another species or belonging to another antibody class or
subclass. Other examples are fragments of such antibodies, so long
as they exhibit the desired biological activity. See, U.S. Pat. No.
4,816,567; and Morrison et al. (1985), Proc. Natl. Acad. Sci. USA
81:6851-6855. A related development is the "CDR-grafted" antibody,
in which the antibody comprises one or more complementarity
determining regions (CDRs) from a particular species or belonging
to a particular antibody class or subclass, while the remainder of
the antibody chain(s) is/are identical with or homologous to a
corresponding sequence in antibodies derived from another species
or belonging to another antibody class or subclass.
[0163] Another development is the "humanized" antibody. Methods for
humanizing non-human antibodies are well known in the art. (See
U.S. Pat. Nos. 5,585,089, and 5,693,762). Generally, a humanized
antibody is produced by a non-human animal, and then certain amino
acid residues, typically from non-antigen recognizing portions of
the antibody, are modified to be homologous to said residues in a
human antibody of corresponding isotype. Humanization can be
performed, for example, using methods described in the art (Jones
et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature
332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536), by
substituting at least a portion of a rodent variable region for the
corresponding regions of a human antibody.
[0164] More recent and more promising is the development of human
antibodies without exposure of antigen to human beings ("fully
human antibodies"). Using transgenic animals (e.g., mice) that are
capable of producing a repertoire of human antibodies in the
absence of endogenous mouse immunoglobulin production, such
antibodies are produced by immunization with an antigen (typically
having at least 6 contiguous amino acids), optionally conjugated to
a carrier. See, for example, Jakobovits et al., 1993, Proc. Natl.
Acad. Sci. USA 90:2551-2555; Jakobovits et al., 1993, Nature
362:255-258; and Bruggermann et al., 1993, Year in Immunol. 7:33.
In one example of these methods, transgenic animals are produced by
incapacitating the endogenous mouse immunoglobulin loci encoding
the mouse heavy and light immunoglobulin chains therein, and
inserting loci encoding human heavy and light chain proteins into
the genome thereof. Partially modified animals, which have less
than the full complement of modifications, are then cross-bred to
obtain an animal having all of the desired immune system
modifications. When administered an immunogen, these transgenic
animals produce antibodies that are immunospecific for these
antigens having human (rather than murine) amino acid sequences,
including variable regions. See PCT Publication Nos. WO96/33735 and
WO94/02602, incorporated by reference. Additional methods are
described in U.S. Pat. No. 5,545,807, PCT Publication Nos.
WO91/10741, WO90/04036, and in EP 546073B1 and EP 546073A1,
incorporated by reference. Human antibodies may also be produced by
the expression of recombinant DNA in host cells or by expression in
hybridoma cells as described herein.
[0165] Fully human antibodies can also be produced from
phage-display libraries (as disclosed in Hoogenboom et al., 1991,
J. Mol. Biol. 227:381; and Marks et al., 1991, J. Mol. Biol.
222:581). These processes mimic immune selection through the
display of antibody repertoires on the surface of filamentous
bacteriophage, and subsequent selection of phage by their binding
to an antigen of choice. One such technique is described in PCT
Publication No. WO99/10494, incorporated by reference, which
describes the isolation of high affinity and functional agonistic
antibodies for MPL- and msk-receptors using such an approach.
[0166] Once the nucleotide sequences encoding such antibodies have
been determined, chimeric, CDR-grafted, humanized, and fully human
antibodies also may be produced by recombinant methods. Nucleic
acids encoding the antibodies are introduced into host cells and
expressed using materials and procedures generally known in the
art.
[0167] The invention provides for use of one or a plurality of
monoclonal antibodies. In preferred embodiments, the invention
provides nucleotide sequences encoding, and amino acid sequences
comprising, heavy and light chain immunoglobulin molecules,
particularly sequences corresponding to the variable regions
thereof. In preferred embodiments, sequences corresponding to
complementarity determining regions (CDR's), specifically from CDR1
through CDR3, are provided. In additional preferred embodiments,
the invention provides hybridoma cell lines expressing such
immunoglobulin molecules and monoclonal antibodies produced
therefrom.
[0168] The ability to clone and reconstruct megabase-sized human
loci in yeast artificial chromosomes (YACs) and to introduce them
into the mouse germline provides an advantageous approach to
elucidating the functional components of very large or crudely
mapped loci as well as generating useful models of human disease.
Furthermore, the utilization of such technology for substitution of
mouse loci with their human equivalents provides unique insights
into expression and regulation of human gene products during
development, their communication with other systems, and their
involvement in disease induction and progression.
[0169] An important practical application of such a strategy is the
"humanization" of the mouse humoral immune system. Introduction of
human immunoglobulin (Ig) loci into mice in which the endogenous Ig
genes have been inactivated offers the opportunity to study the
mechanisms underlying programmed expression and assembly of
antibodies as well as their role in B-cell development.
Furthermore, such a strategy provides a source for production of
fully human monoclonal antibodies (MAbs), particularly for use as
therapeutic agents. Fully human antibodies are expected to minimize
the immunogenic and allergic responses intrinsic to mouse or
mouse-derivatized Mabs, and to thereby increase the efficacy and
safety of administered antibodies in therapeutic applications.
Fully human antibodies can be used in the treatment of chronic and
recurring human diseases, such as osteoarthritis, rheumatoid
arthritis, and other inflammatory conditions, the treatment thereof
requiring repeated antibody administration.
[0170] One skilled in the art can engineer mouse strains deficient
in mouse antibody production with large fragments of the human Ig
loci so that such mice produce human antibodies in the absence of
mouse antibodies. Large human Ig fragments may preserve the large
variable gene diversity as well as the proper regulation of
antibody production and expression. By exploiting the mouse
machinery for antibody diversification and selection and the lack
of immunological tolerance to human proteins, the reproduced human
antibody repertoire in these mouse strains yields high affinity
antibodies against any antigen of interest, including human
antigens. Using the hybridoma technology, antigen-specific human
MAbs with the desired specificity may be produced and selected.
[0171] In certain embodiments, the skilled artisan can use constant
regions from species other than human along with the human variable
region(s) in such mice to produce chimeric antibodies. The
antibodies of the invention can be produced by immunizing such
animals with full-length antigen or a fragment thereof. See, for
example, International Patent Application, Publication WO
93/12227).
[0172] The CDRs of the light and heavy chain variable regions of
antibodies of the invention can be grafted to framework regions
(FRs) from the same, or another, species. In certain embodiments,
the CDRs of the light and heavy chain variable regions of antibody
may be grafted to consensus human FRs. To create consensus human
FRs, FRs from several human heavy chain or light chain amino acid
sequences are aligned to identify a consensus amino acid sequence.
The FRs of the antibody heavy chain or light chain can be replaced
with the FRs from a different heavy chain or light chain. Rare
amino acids in the FRs of the heavy and light chains of anti-IL-1R1
antibody typically are not replaced, while the rest of the FR amino
acids can be replaced. Rare amino acids are specific amino acids
that are in positions in which they are not usually found in FRs.
The grafted variable regions from antibodies of the invention can
be used with a constant region that is different from the constant
region of an antibody of this invention. Alternatively, the grafted
variable regions are part of a single chain Fv antibody. CDR
grafting is described, e.g., in U.S. Pat. Nos. 6,180,370,
5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are hereby
incorporated by reference for any purpose.
[0173] Antibodies of the invention are preferably prepared using
transgenic mice that have a substantial portion of the human
antibody-producing locus inserted in antibody-producing cells of
the mice, and that are further engineered to be deficient in
producing endogenous, murine, antibodies. Such mice are capable of
producing human immunoglobulin molecules and antibodies and do not
produce or produce substantially reduced amounts of murine
immunoglobulin molecules and antibodies. Technologies utilized for
achieving this result are disclosed in the patents, applications,
and references disclosed in the specification herein. In preferred
embodiments, the skilled worker may employ methods as disclosed in
International Patent Application Publication No. WO 98/24893, which
is hereby incorporated by reference for any purpose. See also
Mendez et al., 1997, Nature Genetics 15:146-156, which is hereby
incorporated by reference for any purpose.
[0174] The monoclonal antibodies (MAbs) and other agents of the
invention can be produced by a variety of techniques, including
conventional monoclonal antibody methodology, e.g., the standard
somatic cell hybridization technique of Kohler and Milstein, 1975,
Nature 256:495. Although somatic cell hybridization procedures are
preferred, in principle, other techniques for producing monoclonal
antibodies can be employed, e.g., viral or oncogenic transformation
of B-lymphocytes.
[0175] In a preferred embodiment, human monoclonal antibodies can
be generated using mice referred to as "HuMab" mice, contain a
human immunoglobulin gene minilocus that encodes unrearranged human
heavy (.mu.and .gamma.) and k light chain immunoglobulin sequences,
together with targeted mutations that inactivate the endogenous
.mu.and k chain loci. Lonberg et al., 1994, Nature 368:856-859.
Accordingly, the mice exhibit reduced expression of mouse IgM or k
and in response to immunization, the introduced human heavy and
light chain transgenes undergo class switching and somatic mutation
to generate high affinity human IgG k monoclonal antibodies.
Lonberg et al., supra; Lonberg and Huszar, 1995, Intern. Rev.
Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci.
764:536-546. The preparation of HuMab mice is described in detail
in Taylor et al., 1992, Nucleic Acids Res. 20:6287-6295; Chen et
al., 1993, International Immunology 5:647-656; Tuaillon et al.,
1994, J. Immunol. 152:2912-2920; Lonberg et al., 1994, Nature
368:856-859; Lonberg, 1994, Handbook of Exp. Pharmacology
113:49-101; Taylor et al., 1994, International Immunology
6:579-591; Lonberg & Huszar, 1995, Intern. Rev. Immunol.
13:65-93; Harding & Lonberg, 1995, Ann. N.Y. Acad. Sci
764:536-546; Fishwild et al., 1996, Nature Biotechnology
14:845-851, the contents of all of which are hereby incorporated by
reference in their entirety. See further U.S. Pat. Nos. 5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;
5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, as
well as U.S. Pat. No. 5,545,807 to Surani et al.; International
Patent Application Publication Nos. WO 93/1227, published Jun. 24,
1993; WO 92/22646, published Dec. 23, 1992; and WO 92/03918,
published Mar. 19, 1992, the disclosures of all of which are hereby
incorporated by reference in their entirety.
[0176] Advantageously, fully human monoclonal antibodies are
produced as follows. Transgenic mice containing human
immunoglobulin genes are immunized with the antigen of interest.
Lymphatic cells (such as B-cells) from the mice that express
antibodies are obtained. Such recovered cells are fused with a
myeloid-type cell line to prepare immortal hybridoma cell lines,
and such hybridoma cell lines are screened and selected to identify
hybridoma cell lines that produce antibodies specific to the
antigen of interest. In certain embodiments, the production of a
hybridoma cell line that produces antibodies specific to the
antigen of interest is provided.
[0177] In preferred embodiments, antibodies of the invention are
produced by hybridoma lines. In these embodiments, antibodies of
the invention would typically bind to their associated antigen with
a dissociation constant (K.sub.d) of between approximately 4 pM and
100 pM.
[0178] In preferred embodiments, the antibodies of the invention
are of the IgG1, IgG2, or IgG4 isotype, with the IgG2 isotype most
preferred. In preferred embodiments of the invention, the
antibodies comprise a human kappa light chain and a human IgG1,
IgG2, or IgG4 heavy chain. In particular embodiments, the variable
regions of the antibodies are ligated to a constant region other
than the constant region for the IgG1, IgG2, or IgG4 isotype. In
certain embodiments, the antibodies of the invention have been
cloned for expression in mammalian cells.
[0179] In certain embodiments, conservative amino acid
substitutions to the heavy and light chains of the antibody (and
corresponding modifications to the encoding nucleotides) will
produce antibodies having functional and chemical characteristics
similar to those of the unsubstituted antibody. In contrast,
substantial modifications in the functional and/or chemical
characteristics of the antibody may be accomplished by selecting
substitutions in the amino acid sequence of the heavy and light
chains that differ significantly in their effect on maintaining (a)
the structure of the molecular backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain.
[0180] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
nonnative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described for
"alanine scanning mutagenesis" (Wells, 1991, Methods Enzymol.
202:390 (ed. J. J. Langone), Academic Press, London).
[0181] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. In certain embodiments,
amino acid substitutions can be used to identify important residues
of the antibody, or to increase or decrease the affinity of the
antibodies described herein.
[0182] In alternative embodiments, antibodies of the invention can
be expressed in cell lines other than hybridoma cell lines. In
these embodiments, sequences encoding particular antibodies can be
used for transformation of a suitable mammalian host cell.
According to these embodiments, transformation can be achieved
using any known method for introducing polynucleotides into a host
cell, including, for example packaging the polynucleotide in a
virus (or into a viral vector) and transducing a host cell with the
virus (or vector) or by transfection procedures known in the art.
Such procedures are exemplified by U.S. Pat. Nos. 4,399,216,
4,912,040, 4,740,461, and 4,959,455 (all of which are hereby
incorporated herein by reference for any purpose). Generally, the
transformation procedure used may depend upon the host to be
transformed. Methods for introducing heterologous polynucleotides
into mammalian cells are well known in the art and include, but are
not limited to, dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0183] According to certain embodiments of the methods of the
invention, a nucleic acid molecule encoding the amino acid sequence
of a heavy chain constant region, a heavy chain variable region, a
light chain constant region, or a light chain variable region of an
antibody of the invention is inserted into an appropriate
expression vector using standard ligation techniques. In a
preferred embodiment, the heavy or light chain constant region is
appended to the C-terminus of the appropriate variable region and
is ligated into an expression vector. The vector is typically
selected to be functional in the particular host cell employed
(i.e., the vector is compatible with the host cell machinery such
that amplification of the gene and/or expression of the gene can
occur). For a review of expression vectors, see, Goeddel (ed.),
1990, Meth. Enzymol. Vol. 185, Academic Press. N.Y.
[0184] Typically, expression vectors used in any of the host cells
will contain sequences for plasmid maintenance and for cloning and
expression of exogenous nucleotide sequences. Such sequences,
collectively referred to as "flanking sequences" in certain
embodiments will typically include one or more of the following
nucleotide sequences: a promoter, one or more enhancer sequences,
an origin of replication, a transcriptional termination sequence, a
complete intron sequence containing a donor and acceptor splice
site, a sequence encoding a leader sequence for polypeptide
secretion, a ribosome binding site, a polyadenylation sequence, a
polylinker region for inserting the nucleic acid encoding the
polypeptide to be expressed, and a selectable marker element. Each
of these sequences is discussed below.
[0185] Optionally, the vector may contain a "tag"-encoding
sequence, i.e., an oligonucleotide molecule located at the 5' or 3'
end of the polypeptide coding sequence; the oligonucleotide
sequence encodes polyHis (such as hexaHis), or another "tag" such
as FLAG, HA (hemaglutinin influenza virus), or myc for which
commercially available antibodies exist. This tag is typically
fused to the polypeptide upon expression of the polypeptide, and
can serve as a means for affinity purification or detection of the
antibody from the host cell. Affinity purification can be
accomplished, for example, by column chromatography using
antibodies against the tag as an affinity matrix. Optionally, the
tag can subsequently be removed from the purified polypeptide by
various means such as using certain peptidases for cleavage.
[0186] Flanking sequences may be homologous (i.e., from the same
species and/or strain as the host cell), heterologous (i.e., from a
species other than the host cell species or strain), hybrid (i.e.,
a combination of flanking sequences from more than one source),
synthetic or native. As such, the source of a flanking sequence may
be any prokaryotic or eukaryotic organism, any vertebrate or
invertebrate organism, or any plant, provided that the flanking
sequence is functional in, and can be activated by, the host cell
machinery.
[0187] Flanking sequences useful in the vectors of this invention
may be obtained by any of several methods well known in the art.
Typically, flanking sequences useful herein will have been
previously identified by mapping and/or by restriction endonuclease
digestion and can thus be isolated from the proper tissue source
using the appropriate restriction endonucleases. In some cases, the
full nucleotide sequence of a flanking sequence may be known. Here,
the flanking sequence may be synthesized using the methods
described herein for nucleic acid synthesis or cloning.
[0188] Where all or only a portion of the flanking sequence is
known, it may be obtained using polymerase chain reaction (PCR)
and/or by screening a genomic library with a suitable probe such as
an oligonucleotide and/or flanking sequence fragment from the same
or another species. Where the flanking sequence is not known, a
fragment of DNA containing a flanking sequence may be isolated from
a larger piece of DNA that may contain, for example, a coding
sequence or even another gene or genes. Isolation may be
accomplished by restriction endonuclease digestion to produce the
proper DNA fragment followed by isolation using agarose gel
purification, Qiagen.RTM. column chromatography (Chatsworth,
Calif.), or other methods known to the skilled artisan. The
selection of suitable enzymes to accomplish this purpose will be
readily apparent to one of ordinary skill in the art.
[0189] An origin of replication is typically a part of those
prokaryotic expression vectors purchased commercially, and the
origin aids in the amplification of the vector in a host cell. If
the vector of choice does not contain an origin of replication
site, one may be chemically synthesized based on a known sequence,
and ligated into the vector. For example, the origin of replication
from the plasmid pBR322 (New England Biolabs, Beverly, Mass.) is
suitable for most gram-negative bacteria and various viral origins
(e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV),
or papillomaviruses such as HPV or BPV) are useful for cloning
vectors in mammalian cells. Generally, the origin of replication
component is not needed for mammalian expression vectors (for
example, the SV40 origin is often used only because it also
contains the virus early promoter).
[0190] A transcription termination sequence is typically located 3'
to the end of a polypeptide coding region and serves to terminate
transcription. Usually, a transcription termination sequence in
prokaryotic cells is a G-C rich fragment followed by a poly-T
sequence. While the sequence is easily cloned from a library or
even purchased commercially as part of a vector, it can also be
readily synthesized using methods for nucleic acid synthesis such
as those described herein.
[0191] A selectable marker gene encodes a protein necessary for the
survival and growth of a host cell grown in a selective culture
medium. Typical selection marker genes encode proteins that (a)
confer resistance to antibiotics or other toxins, e.g., ampicillin,
tetracycline, or kanamycin for prokaryotic host cells; (b)
complement auxotrophic deficiencies of the cell; or (c) supply
critical nutrients not available from complex or defined media.
Preferred selectable markers are the kanamycin resistance gene, the
ampicillin resistance gene, and the tetracycline resistance gene. A
neomycin resistance gene may also be used for selection in both
prokaryotic and eukaryotic host cells.
[0192] Other selectable genes may be used to amplify the gene that
will be expressed. Amplification is the process wherein genes that
are in greater demand for the production of a protein critical for
growth or cell survival are reiterated generally in tandem within
the chromosomes of successive generations of recombinant cells.
Examples of suitable selectable markers for mammalian cells include
dihydrofolate reductase (DHFR) and promoterless thymidine kinase.
Mammalian cell transformants are placed under selection pressure
wherein only the transformants are uniquely adapted to survive by
virtue of the selectable gene present in the vector. Selection
pressure is imposed by culturing the transformed cells under
conditions in which the concentration of selection agent in the
medium is successively increased, thereby leading to the
amplification of both the selectable gene and the DNA that encodes
another gene. As a result, increased quantities of a polypeptide
can be synthesized from the amplified DNA.
[0193] A ribosome-binding site is usually necessary for translation
initiation of mRNA and is characterized by a Shine-Dalgarno
sequence (prokaryotes) or a Kozak sequence (eukaryotes). The
element is typically located 3' to the promoter and 5' to the
coding sequence of the polypeptide to be expressed.
[0194] In some cases, such as where glycosylation is desired in a
eukaryotic host cell expression system, one may manipulate the
various pre- or prosequences to improve glycosylation or yield. For
example, one may alter the peptidase cleavage site of a particular
signal peptide, or add pro-sequences, which also may affect
glycosylation. The final protein product may have, in the -1
position (relative to the first amino acid of the mature protein)
one or more additional amino acids incident to expression, which
may not have been totally removed. For example, the final protein
product may have one or two amino acid residues found in the
peptidase cleavage site, attached to the amino-terminus.
Alternatively, use of some enzyme cleavage sites may result in a
slightly truncated form of the desired polypeptide, if the enzyme
cuts at such area within the mature polypeptide.
[0195] The expression and cloning vectors of the invention will
typically contain a promoter that is recognized by the host
organism and operably linked to the molecule encoding the antibody.
Promoters are untranscribed sequences located upstream (i.e., 5')
to the start codon of a structural gene (generally within about 100
to 1000 bp) that control the transcription of the structural gene.
Promoters are conventionally grouped into one of two classes:
inducible promoters and constitutive promoters. Inducible promoters
initiate increased levels of transcription from DNA under their
control in response to some change in culture conditions, such as
the presence or absence of a nutrient or a change in temperature.
Constitutive promoters, on the other hand, initiate continual gene
product production; that is, there is little or no control over
gene expression. A large number of promoters, recognized by a
variety of potential host cells, are well known. A suitable
promoter is operably linked to the DNA encoding heavy chain or
light chain comprising an antibody of the invention by removing the
promoter from the source DNA by restriction enzyme digestion and
inserting the desired promoter sequence into the vector.
[0196] Suitable promoters for use with yeast hosts are also well
known in the art. Yeast enhancers are advantageously used with
yeast promoters. Suitable promoters for use with mammalian host
cells are well known and include, but are not limited to, those
obtained from the genomes of viruses such as polyoma virus, fowlpox
virus, adenovirus (such as Adenovirus 2), bovine papilloma virus,
avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B
virus and most preferably Simian Virus 40 (SV40). Other suitable
mammalian promoters include heterologous mammalian promoters, for
example, heat-shock promoters and the actin promoter.
[0197] Additional promoters which may be of interest include, but
are not limited to: the SV40 early promoter region (Bernoist and
Chambon, 1981, Nature 290:304-10); the CMV promoter; the promoter
contained in the 3' long terminal repeat of Rous sarcoma virus
(Yamamoto et al., 1980, Cell 22:787-97); the herpes thymidine
kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA
78:1444-45); the regulatory sequences of the metallothionine gene
(Brinster et al., 1982, Nature 296:39-42); prokaryotic expression
vectors such as the beta-lactamase promoter (Villa-Kamaroff et al.,
1978, Proc. Natl. Acad. Sci. USA 75:3727-31); or the tac promoter
(DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Also of
interest are the following animal transcriptional control regions,
which exhibit tissue specificity and have been utilized in
transgenic animals: the elastase I gene control region that is
active in pancreatic acinar cells (Swift et al., 1984, Cell
38:639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.
Biol. 50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515);
the insulin gene control region that is active in pancreatic beta
cells (Hanahan, 1985, Nature 315:115-22); the immunoglobulin gene
control region that is active in lymphoid cells (Grosschedl et al.,
1984, Cell 38:647-58; Adames et al., 1985, Nature 318:533-38;
Alexander et al., 1987, Mol. Cell. Biol. 7:1436-44); the mouse
mammary tumor virus control region that is active in testicular,
breast, lymphoid and mast cells (Leder et al., 1986, Cell
45:485-95); the albumin gene control region that is active in liver
(Pinkert et al., 1987, Genes and Devel. 1:268-76); the
alpha-feto-protein gene control region that is active in liver
(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-48; Hammer et al.,
1987, Science 235:53-58); the alpha 1-antitrypsin gene control
region that is active in liver (Kelsey et al., 1987, Genes and
Devel. 1:161-71); the beta-globin gene control region that is
active in myeloid cells (Mogram et al., 1985, Nature 315:338-40;
Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene
control region that is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-12); the myosin light chain-2
gene control region that is active in skeletal muscle (Sani, 1985,
Nature 314:283-86); and the gonadotropic releasing hormone gene
control region that is active in the hypothalamus (Mason et al.,
1986, Science 234:1372-78).
[0198] An enhancer sequence may be inserted into the vector to
increase transcription of DNA encoding light chain or heavy chain
comprising an antibody of the invention by higher eukaryotes.
Enhancers are cis-acting elements of DNA, usually about 10-300 bp
in length, that act on the promoter to increase transcription.
Enhancers are relatively orientation- and position-independent.
They have been found 5' and 3' to the transcription unit. Several
enhancer sequences available from mammalian genes (e.g., globin,
elastase, albumin, alpha-feto-protein and insulin) are known.
Typically, however, an enhancer from a virus is used. The SV40
enhancer, the cytomegalovirus early promoter enhancer, the polyoma
enhancer, and adenovirus enhancers known in the art are exemplary
enhancing elements for the activation of eukaryotic promoters.
While an enhancer may be spliced into the vector at a position 5'
or 3' to a nucleic acid molecule, it is typically located at a site
5' from the promoter.
[0199] Expression vectors of the invention may be constructed from
a starting vector such as a commercially available vector. Such
vectors may or may not contain all of the desired flanking
sequences. Where one or more of the flanking sequences described
herein are not already present in the vector, they may be
individually obtained and ligated into the vector. Methods used for
obtaining each of the flanking sequences are well known to one
skilled in the art.
[0200] After the vector has been constructed and a nucleic acid
molecule encoding light chain or heavy chain or light chain and
heavy chain has been inserted into the proper site of the vector,
the completed vector may be inserted into a suitable host cell for
amplification and/or polypeptide expression. The transformation of
an expression vector for an antibody into a selected host cell may
be accomplished by well known methods including transfection,
infection, calcium phosphate co-precipitation, electroporation,
microinjection, lipofection, DEAE-dextran mediated transfection, or
other known techniques. The method selected will in part be a
function of the type of host cell to be used. These methods and
other suitable methods are well known to the skilled artisan, and
are set forth, for example, in Sambrook et al., supra.
[0201] The host cell, when cultured under appropriate conditions,
synthesizes an antibody that can subsequently be collected from the
culture medium (if the host cell secretes it into the medium) or
directly from the host cell producing it (if it is not secreted).
The selection of an appropriate host cell will depend upon various
factors, such as desired expression levels, polypeptide
modifications that are desirable or necessary for activity (such as
glycosylation or phosphorylation) and ease of folding into a
biologically active molecule.
[0202] Mammalian cell lines available as hosts for expression are
well known in the art and include, but are not limited to, many
immortalized cell lines available from the American Type Culture
Collection (A.T.C.C.), including but not limited to Chinese hamster
ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,
monkey kidney cells (COS), human hepatocellular carcinoma cells
(e.g., Hep G2), and a number of other cell lines. In certain
embodiments, one may select cell lines by determining which cell
lines have high expression levels and produce antibodies with
constitutive binding properties. In another embodiment, one may
select a cell line from the B cell lineage that does not make its
own antibody but has a capacity to make and secrete a heterologous
antibody (e.g., mouse myeloma cell lines NS0 and SP2/0).
[0203] Preparation of Other Agents
[0204] Persons of ordinary skill in the art are able to prepare
additional agents, such as soluble fragments of IL-27 receptor and
WSX-1, using the techniques described hereinabove. Further in
accordance with the present invention, such an agent may be linked
to a half-life extender such as a polymer (e.g., PEG or dextran),
human serum albumin (HSA), transthyretin (TTR), a leucine zipper
domain (LZ) or an Fc domain, which is preferred. The half-life
extender and the agent may be linked through the N- or C-terminus
of the agent. The preferred half-life extender is an Fc domain, and
the preferred Fc domain is an IgG Fc domain.
[0205] The term "pharmaceutically acceptable salt, ester, or
solvate" refers to a salt, ester, or solvate of a subject compound
which possesses the desired pharmacological activity and which is
neither biologically nor otherwise undesirable. A salt, ester, or
solvate can be formed with inorganic acids such as acetate,
adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, gluconate,
glycerophosphate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,
lactate, maleate, methanesulfonate, naphthylate,
2-naphthalenesulfonate, nicotinate, oxalate, sulfate, thiocyanate,
tosylate and undecanoate. Examples of base salts, esters, or
solvates include ammonium salts; alkali metal salts, such as sodium
and potassium salts; alkaline earth metal salts, such as calcium
and magnesium salts; salts with organic bases, such as
dicyclohexylamine salts; N-methyl-D-glucamine; and salts with amino
acids, such as arginine, lysine, and so forth. Also, the basic
nitrogen-containing groups can be quarternized with such agents as
lower alkyl halides, such as methyl, ethyl, propyl, and butyl
chlorides, bromides, and iodides; dialkyl sulfates, such as
dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain
halides, such as decyl, lauryl, myristyl, and stearyl chlorides,
bromides, and iodides; aralkyl halides, such as benzyl and
phenethyl bromides; and others. Water or oil-soluble or dispersible
products are thereby obtained.
The Inventive Subject Matter
[0206] The inventive subject matter relates to methods for
modulating an immune response, and pharmaceutical compositions
comprising an effective amount of an IL-27R/WSX-1 ligand.
[0207] Although recent work has described IL-27 and its receptor,
WSX-1, as promoters of Th1 differentiation in naive CD4.sup.+ T
cells, Applicants have determined that signaling through this
receptor is involved in limiting the intensity and duration of T
cell activity. When WSX-1-deficient mice are infected with the
intracellular pathogen Toxoplasma gondii, they establish protective
T cell responses, characterized by production of inflammatory
cytokines and control of parasite replication. However, infected
WSX-1.sup.-/- mice are unable to downregulate these protective
responses, and develop a lethal, T cell-mediated inflammatory
disease. This pathology was characterized by the excessive
production of IFN-_, persistence of highly activated T cells, and
enhanced T cell proliferation in vivo. Together, these findings
demonstrate that WSX-1 is not required for the generation of
IFN-.gamma.-mediated immunity to this parasitic infection and
identify a novel function for this receptor as a potent antagonist
of T cell-mediated, immune hyperactivity.
[0208] Contrary to the previous consensus understanding, we have
demonstrated that WSX-1-deficient mice infected with T. gondii are
able to develop a strong Th1 type response and control parasite
replication, but are unable to downregulate this protective
response and develop a lethal, T cell-mediated inflammatory
disease. This pathology was characterized by the excessive
production of IFN-.gamma., persistence of highly activated T cells,
and enhanced T cell proliferation in vivo. The phenotype could be
recapitulated in vitro as Th1 polarization of WSX-1.sup.-/-
CD4.sup.+ T cells led to increased proliferation and IFN-.gamma.
secretion. However, this work also confirmed that, under
nonpolarizing conditions, WSX-1 is required for optimal IFN-.gamma.
production. Further analysis revealed that exogenous IL-27 can
activate STAT1, STAT3, and STAT5: STAT family members that have
traditionally been associated with cellular activation but have
also recently been linked with the inhibition of immune functions.
Together, these findings demonstrate that WSX-1 is not required for
the generation of IFN-.gamma.-mediated immunity to T. gondii and
identify a novel function for WSX-1 as a potent antagonist of T
cell-mediated immune hyperactivity.
[0209] Given the importance of IL-12 in resistance to intracellular
pathogens, experiments were performed to determine the role of
IL-27/WSX-1 in immunity to Toxoplasma gondii, an obligate
intracellular parasite that is an important opportunistic pathogen
of prenatal infants and immunocompromised adults. Resistance to
this pathogen is characterized by the development of an
IL-12-dependent Th1 type response dominated by the production of
IFN-.gamma. by CD4+ and CD8.sup.+ T cells. A strong, protective
response leads to control of parasite replication but a failure to
appropriately regulate this response can lead to severe T cell
mediated immune-pathology characterized by the overproduction of
inflammatory cytokines.
[0210] Methods of the Inventive Subject Matter
[0211] Based primarily on in vitro experiments that showed the
IL-27/WSX-1 receptor-ligand interaction could enhance IFN-.gamma.
production, a consensus emerged that WSX-1 was an essential
receptor for the development of Th1 type responses. However, the
present disclosure demonstrates that under the strongly Th1
polarizing conditions that occur during toxoplasmosis or in vitro
culture, WSX-1 is not required for the development of Th1 effector
cells. Unexpectedly, through infection of WSX-1-deficient mice with
T. gondii, we have found a regulatory role for this receptor.
WSX-1.sup.-/- animals exhibit prolonged IFN-.gamma. responses and
an accumulation of highly activated T cells that is associated with
increased T cell proliferation.
[0212] It is well established that antigen dose and cytokine
environment are critical factors in the differentiation of naive
CD4.sup.+ T cells into effector Th1 and Th2 cells. Moreover, T
cells must proliferate, not only to acquire effector functions, but
also to curb activation and thereby limit the duration and
intensity of an immune response.
[0213] Because the role of IL-27/WSX-1 in T cell differentiation
was previously assessed using ConA in combination with polarizing
cytokines, it is possible that the enhanced proliferation of these
cells led to abbreviated cytokine production, which could then be
interpreted as a defect in Th1 differentiation. We show here that
when T cells are activated through TCR ligation and in highly
polarizing conditions, there is an important role for IL-27/WSX-1
as a negative regulator of Th1 type responses. These finding are
consistent with a recent report in which CD4.sup.+ T cells from
mice lacking the EBI3 component of IL-27 produced significantly
more IFN-.gamma. than their wild-type counterparts in vitro. While
it is clear that WSX-1.sup.-/- CD4.sup.+ T cells do not have an
intrinsic defect in their ability to become Th1 effector cells, it
has been reported that recombinant IL-27 can synergize with IL-12
to enhance IFN-.gamma. production by naive T cells and NK cells.
Therefore, the ability of IL-27 to activate STAT-1 and thereby
induce expression of T-bet, a key transcription factor in Th1
differentiation, may be crucial for maximal Th1 differentiation
when concentrations of polarizing cytokine are limiting (FIG. 6A).
However, in the context of high IL-12 concentrations, like those
induced by acute toxoplasmosis, there is no requirement for
IL-27-induced expression of T-bet, thus confirming a central role
for IL-12, and not IL-27, in the development of IFN-.gamma.
responses that are essential for control of T. gondii.
[0214] During infection, the ability to downregulate T cell
responses after pathogen control is a critical function of an
appropriate immune response, but little was known about the
mechanisms that mediate this process. In our work, STAT4
phosphorylation could not be detected after treatment of naive
cells with either IL-12 or IL-27 . Though the absence of functional
IL-12R on the surface of naive CD4.sup.+ T cells explains the
relative inactivity of IL-12 in this assay, a recent report on
WSX-1 signaling in naive human lymphocytes suggests that despite
functional receptor expression, IL-27 fails to activate STAT4.
Furthermore, this finding is consistent with previous work that
indicate that exogenous IL-27 does not lead to STAT4
phosphorylation in WSX-1 transfected murine cell lines.
Nevertheless, the recognition that WSX-1 can activate STAT1, STAT3,
and STAT5 does provide an insight into the pathways that are
involved in the negative regulation of T cell responses. While
tyrosine phosphorylation of these STAT family members has been
previously associated with the activation of immune cells, it is
becoming clear that they also have a crucial role in preventing
immune hyperactivity. Cytokines like IFN-.gamma.,
IFN-.alpha./.beta., and IL-6 activate similar STAT pathways and can
have profound suppressive effects on immune responses. Although
little is known about the molecular mechanisms governing these
inhibitory processes, several studies have shown that STAT1 is a
negative regulator of proliferation and IFN-.gamma. production by
Th1 cells. Additionally, ablation of STAT3 in bone marrow
haematopoetic progenitors led to the development of immune-mediated
colitis in mice, while germline deletion of the STAT3.beta. isoform
results in impaired recovery from LPS-induced shock. Thus, the
absence of IL-27-mediated STAT activation could provide a molecular
mechanism for the T cell hyperactivity observed in WSX-1.sup.-/-
mice that have been infected with T. gondii. However, it is still
uncertain whether WSX-1-dependent STAT activation can directly
inhibit T cell responses or operates through other trans factors,
such as SOCS family members to limit the ability of effector T
cells to respond to growth and survival stimuli. Although
SOCS1-deficient mice spontaneously develop severe liver pathology
similar that of WSX-1.sup.-/- mice infected with T. gondii, initial
work has not revealed decreased, in vitro expression of SOCS1 by
WSX-1.sup.-/- T cells.
[0215] A role for WSX-1 in the downregulation of T cell responses
does not appear to be restricted to toxoplasmosis as infection of
WSX-1.sup.-/- mice with Trypanosoma cruzi also resulted in the
development of immune pathology. Other work has also found that
when WSX-1.sup.-/- mice were infected with the intestinal helminth
Trichuris muris, they developed an exaggerated Th2 response that
was associated with enhanced expulsion of the parasite (unpublished
data). Consequently, the previous report of increased
susceptibility of WSX-1.sup.-/- mice to L. major cannot simply be
attributed to a failure in the generation of Th1 responses. Because
resistant mouse strains produce an acute Th2 response when
challenged with L. major, an inability to regulate this acute IL-4
production, in combination with the absence of WSX-1-dependent
STAT-1 activation, could inhibit initial generation of protective
Th1 type cells and thereby delay disease resolution in
WSX-1.sup.-/- mice. In support of this hypothesis, in vivo
neutralization of IL-4 was found to restore the ability of
WSX-1.sup.-/- mice to control L. major and, in these experiments,
WSX-1.sup.-/- T cells were found to produce more IFN-.gamma. than
those of similarly treated wild type mice (unpublished data).
[0216] Together, this work indicates that one role of WSX-1 is to
control the kinetics, but not polarity, of an immune response and
that signaling through this receptor may act as a general negative
regulator of infection-induced T cell effector functions.
Consequently, our determination of a role for WSX-1 in the
suppression of T cell hyperactivity has immediate clinical
implications for T cell-mediated inflammatory disorders and
represents a novel target for immune based therapies.
[0217] To further demonstrate the importance of IL-27/WSX-1
interactions in modulating immune responses, we have shown the
development of a T helper (Th) 2 type immunity response of
WSX-1.sup.-/- mice infected with the gut dwelling helminth
Trichuris muris. In contrast to wild type mice the WSX-1.sup.-/-
mice displayed increased production of Th2 cytokines, elevated
intestinal goblet cell and mast cell responses, and accelerated
expulsion of T. muris. In addition, mast cells were also shown to
express WSX-1 and in a model of IgE-mediated mast cell dependent
anaphylaxis WSX-1.sup.-/- mice displayed increased changes in
vascular permeability. Importantly, the accelerated
parasite-induced Th2 responses in WSX-1.sup.-/- mice did not appear
to be due to an intrinsic defect in IFN-.gamma. production since
the blockade of Th1 responses in wild type mice did not result in
enhanced Th2 responses or resistance to T. muris. Moreover, in
vitro assays revealed that naive WSX-1.sup.-/- CD4.sup.+ T cells
stimulated under Th2 polarizing conditions displayed enhanced
proliferation and secreted elevated levels of IL-5 and IL-13
compared to wild type cells and exogenous IL-27 was shown to
suppress the production of IL-4 by wild type CD4.sup.+ T cells.
[0218] As discussed above, the initial consensus that IL-27/WSX-1
was required for optimal Th1 type responses led to the hypothesis
that WSX-1.sup.-/- mice would be more resistant to T. muris as a
consequence of reduced IFN-.gamma. responses. Indeed, the finding
that WSX-1.sup.-/- mice displayed enhanced resistance to T. muris
supported this hypothesis. However, since WSX-1.sup.-/- mice did
not appear to have an early defect in IFN-.gamma. production, and
the finding that blockade of Th1 responses in wild type mice did
not result in enhanced resistance suggested IL-27/WSX-1 signaling
was involved in the inhibition of Th2 responses. Moreover, under
Th2 polarizing conditions in vitro, WSX-1.sup.-/- CD4.sup.+ T cells
displayed enhanced production of Th2 cytokines whereas IL-27 could
downregulate the production of IL-4 by wild type CD4.sup.+ T cells,
supporting a direct role for IL-27/WSX-1 as a negative regulator of
Th2 cell responses.
[0219] While previous work has shown that the IL-27/WSX-1
interaction can promote the production of IFN-.gamma. under
non-polarizing conditions, the results presented here identify a
role for IL-27/WSX-1 in regulating the development of Th2 responses
in vitro and in vivo. These data are in accord with studies in a
variety of experimental systems that demonstrated enhanced Th2 type
responses in the absence of IL-27/WSX-1 signaling. In addition,
recent studies have shown that IL-27 can down-regulate the levels
of mRNA for GATA3, a transcription factor critical for the
development of Th2 responses but whether this is a direct effect of
IL-27 or a secondary consequence of other inhibitory effects
remains unclear. In some of these experimental systems these
effects have previously been attributed to reduced IFN-.gamma.
responses, but the data presented here prompt a reappraisal of the
role of IL-27/WSX-1 in the regulation of type 2 immunity. For
instance, the enhanced susceptibility of WSX-1.sup.-/- mice to
Leishmania major infection may not be due solely to a defect in
IFN-.gamma. production as previously reported, but may also be the
result of enhanced Th2 cytokine responses in the absence of WSX-1.
Supporting this hypothesis, in vivo depletion of IL-4 in
WSX-1.sup.-/- mice infected with L. major restored IFN-.gamma.
production and protective immunity.
[0220] While most work to date on IL-27/WSX-1 have focused on the
role of this cytokine/receptor interaction in the regulation of
lymphocyte functions the finding that mast cells can express high
levels of WSX-1 and can respond to IL-27 was unexpected. However,
it is now becoming clear that other cell types express functional
IL-27R; it has been reported that macrophages, dendritic cells, as
well as mast cells express this receptor and that primary human
mast cells can activate and STAT3 in response to IL-27. The finding
that in the absence of WSX-1 mast cell responses are enhanced in a
model of passive cutaneous anaphlyaxis demonstrates that the
IL-27/WSX-1 interaction may provide a general mechanism to
downregulate immune activity in multiple cell types. While our data
indicates a direct role for IL-27/WSX-1 in the inhibition of Th2
responses, previous studies have demonstrated that the ability of
mast cells to produce IL-4 contributes to the development of
infection-induced Th2 responses.
[0221] The findings that WSX-1.sup.-/- mice develop immune-mediated
chronic inflammation when infected with T. gondii or T. cruzi, two
parasites that induce strong type 1 immunity, in combination with
other work disclosed herein, support the idea that IL-27/WSX-1 has
inhibitory effects on pathogen-induced Th1 and Th2 type responses.
Thus, it appears that IL-27/WSX-1 signaling can deliver a negative
regulatory signal to activated CD4.sup.+ T cells and thereby limit
T cell responses. Such enhanced proliferative responses of
WSX-1.sup.-/- T cells may be a function of enhanced cell survival,
accelerated cell cycle progression, or increased sensitivity to
growth factors like IL-2. However, our studies have found that
IL-27 does not inhibit T cell proliferation, and the basis for the
difference between the enhanced proliferative responses of
WSX-1.sup.-/- T cells and the effects of rIL-27 are unclear.
Without being bound to any particular theory of mode of activity, a
possible explanation may be that the different IL-27R components
mediate different functional activities or that there is an
additional ligand for WSX-1 that is involved in the regulation of T
cell proliferation. The identification of a role for the
IL-27/WSX-1 interaction in the suppression of in vivo Th2 responses
demonstrates that this pathway represents a viable therapeutic
target for the treatment of a number of inflammatory conditions
associated with aberrant Th2 cytokine production, including asthma,
allergy, and autoimmunity.
[0222] Taken together, these studies demonstrate a further, novel
role for IL-27/WSX-1, in limiting different elements associated
with innate and adaptive components of Th2 type responses. Thus, we
have shown that the IL-27/WSX-1 interaction is additionally a
target for the treatment of inflammatory conditions associated with
Th2 type responses.
[0223] Thus, the inventive subject matter relates to a method for
modulating an immune response in an animal in need thereof, which
comprises administering to said animal an effective amount of an
IL-27R/WSX-1 ligand.
[0224] In another aspect, said modulation is suppression and said
ligand is an IL-27R/WSX-1 agonist.
[0225] In another aspect, said agonist is selected from the group
consisting of IL-27, an active fragment of IL-27, and an agonistic
antibody to IL-27R/WSX-1 which enhances IL-27R/WSX-1 activity.
[0226] In another aspect, said modulation is activation and said
ligand is an IL-27R/WSX-1 antagonist.
[0227] In another aspect, wherein said antagonist is an inactive
IL-27 fragment which retains IL-27R/WSX-1 binding affinity, or an
antagonist antibody to IL-27R/WSX-1 which suppresses IL-27R/WSX-1
activity.
[0228] The inventive subject matter further relates to a method for
modulating a T-helper cell mediated immune response in an animal in
need thereof, which comprises administering to said animal an
effective amount of an IL-27R/WSX-1 ligand.
[0229] In another aspect, said modulation is suppression and said
ligand is an IL-27R/WSX-1 agonist.
[0230] In another aspect, said agonist is selected from the group
consisting of IL-27, an active fragment of IL-27, and an agonistic
antibody to IL-27R/WSX-1 which enhances IL-27R/WSX-1 activity.
[0231] In another aspect, said modulation is activation and said
ligand is an IL-27R/WSX-1 antagonist.
[0232] In another aspect, said antagonist is an inactive IL-27
fragment which retains IL-27R/WSX-1 binding affinity, or an
antagonist antibody to IL-27R/WSX-1 which suppresses IL-27R/WSX-1
activity.
[0233] In another aspect, said T-helper cell is Th1.
[0234] In another aspect, said T-helper cell is Th2.
[0235] The inventive subject matter further relates to a method for
modulating an interferon-.gamma. mediated immune response in an
animal in need thereof, which comprises administering to said
animal an effective amount of an IL-27R/WSX-1 ligand.
[0236] In another aspect, said modulation is suppression and said
ligand is an IL-27R/WSX-1 agonist.
[0237] In another aspect, said agonist is selected from the group
consisting of IL-27, an active fragment of IL-27, and an agonistic
antibody to IL-27R/WSX-1 which enhances IL-27R/WSX-1 activity.
[0238] In another aspect, said modulation is activation and said
ligand is an IL-27R/WSX-1 antagonist.
[0239] In another aspect, said antagonist is an inactive IL-27
fragment which retains IL-27R/WSX-1 binding affinity, or an
antagonist antibody to IL-27R/WSX-1 which suppresses IL-27R/WSX-1
activity.
[0240] The inventive subject matter further relates to a method for
treating immune hyperactivity in an animal in need thereof, which
comprises administering to said animal an effective amount of an
IL-27R/WSX-1 ligand.
[0241] The inventive subject matter further relates to a method for
treating an immune hyperactivity disorder in an animal in need
thereof, which comprises administering to said animal an effective
amount of an IL-27R/WSX-1 ligand.
[0242] In another aspect, said immune disorder is selected from the
group consisting of autoimmune disorders, hypersensitivity
disorders, allergies, and asthma.
[0243] In another aspect, said immune disorder is selected from the
group consisting of Acquired Immune Deficiency Syndrome; acute
pancreatitis; Addison's disease; alcohol-induced liver injury
including alcoholic cirrhosis; Alzheimer's disease;
amyelolateroschlerosis; asthma and other pulmonary diseases;
atherosclerosis; autoimmune vasculitis; autoimmune
hepatitis-induced hepatic injury; biliary cirrhosis;
cachexia/anorexia, including AIDS-induced cachexia; cancer, such as
multiple myeloma and myelogenous and other leukemias, as well as
tumor metastasis; chronic fatigue syndrome; Clostridium associated
illnesses, including Clostridium-associated diarrhea; coronary
conditions and indications, including congestive heart failure,
coronary restenosis, myocardial infarction, myocardial dysfunction,
and coronary artery bypass graft; diabetes, including juvenile
onset Type 1, diabetes mellitus, and insulin resistance;
endometriosis, endometritis, and related conditions; epididymitis;
erythropoietin resistance; fever; fibromyalgia or analgesia;
glomerulonephritis; graft versus host disease/transplant rejection;
Graves' disease; Guillain-Barre syndrome; Hashimoto's disease;
hemolytic anemia; hemorrhagic shock; hyperalgesia; inflammatory
bowel diseases including ulcerative colitis and Crohn's disease;
inflammatory conditions of a joint and rheumatic diseases
including, osteoarthritis, rheumatoid arthritis, juvenile
(rheumatoid) arthritis, seronegative polyarthritis, ankylosing
spondylitis, Reiter's syndrome and reactive arthritis, Still's
disease, psoriatic arthritis, enteropathic arthritis, polymyositis,
dermatomyositis, scleroderma, systemic sclerosis, vasculitis (e.g.,
Kawasaki's disease), cerebral vasculitis, Lyme disease,
staphylococcal-inducedarthritis, Sjbgren's syndrome, rheumatic
fever, polychondritis and polymyalgia rheumatica and giant cell
arteritis; inflammatory eye disease, as may be associated with, for
example, corneal transplant; inflammatory eye disease, as may be
associated with, e.g., corneal transplant; inflammatory bowel
disease; ischemia, including cerebral ischemia; Kawasaki's disease;
learning impairment; lung diseases; lupus nephritis; multiple
sclerosis; myasthenia gravis; myopathiesneuroinflammatory diseases;
neurotoxicity; ocular diseases and conditions, including ocular
degeneration and uveitis; osteoporosis; pain, including
cancer-related pain; Parkinson's disease; pemphigus; periodontal
disease; Pityriasis rubra pilaris; pre-term labor; prostatitis and
related conditions; psoriasis and related conditions; psoriatic
arthritis; pulmonary fibrosis; reperfusion injury; rheumatic fever;
rheumatoid arthritis; sarcoidosis; scleroderma; septic shock; side
effects from radiation therapy; Sjogren's syndrome; sleep
disturbance; spondyloarthropathies; systemic lupus erythematosus;
temporal mandibular joint disease; thyroiditis; tissue
transplantation or an inflammatory condition resulting from strain,
sprain, cartilage damage, trauma, and orthopedic surgery;
transplant rejection; uveitis; vasculitis; or an inflammatory
condition resulting from strain, sprain, cartilage damage, trauma,
orthopedic surgery, infection or other disease processes.
[0244] The inventive subject matter further relates to a method for
treating a T-helper cell mediated disorder in an animal in need
thereof, which comprises administering to said animal an effective
amount of an IL-27R/WSX-1 ligand.
[0245] In another aspect, said T-helper cell mediated disorder is
selected from the group consisting of Acquired Immune Deficiency
Syndrome; acute pancreatitis; Addison's disease; alcohol-induced
liver injury including alcoholic cirrhosis; Alzheimer's disease;
amyelolateroschlerosis; asthma and other pulmonary diseases;
atherosclerosis; autoimmune vasculitis; autoimmune
hepatitis-induced hepatic injury; biliary cirrhosis;
cachexia/anorexia, including AIDS-induced cachexia; cancer, such as
multiple myeloma and myelogenous and other leukemias, as well as
tumor metastasis; chronic fatigue syndrome; Clostridium associated
illnesses, including Clostridium-associated diarrhea; coronary
conditions and indications, including congestive heart failure,
coronary restenosis, myocardial infarction, myocardial dysfunction,
and coronary artery bypass graft; diabetes, including juvenile
onset Type 1, diabetes mellitus, and insulin resistance;
endometriosis, endometritis, and related conditions; epididymitis;
erythropoietin resistance; fever; fibromyalgia or analgesia;
glomerulonephritis; graft versus host disease/transplant rejection;
Graves' disease; Guillain-Barre syndrome; Hashimoto's disease;
hemolytic anemia; hemorrhagic shock; hyperalgesia; inflammatory
bowel diseases including ulcerative colitis and Crohn's disease;
inflammatory conditions of a joint and rheumatic diseases
including, osteoarthritis, rheumatoid arthritis, juvenile
(rheumatoid) arthritis, seronegative polyarthritis, ankylosing
spondylitis, Reiter's syndrome and reactive arthritis, Still's
disease, psoriatic arthritis, enteropathic arthritis, polymyositis,
dermatomyositis, scleroderma, systemic sclerosis, vasculitis (e.g.,
Kawasaki's disease), cerebral vasculitis, Lyme disease,
staphylococcal-inducedarthritis, Sjogren's syndrome, rheumatic
fever, polychondritis and polymyalgia rheumatica and giant cell
arteritis; inflammatory eye disease, as may be associated with, for
example, corneal transplant; inflammatory eye disease, as may be
associated with, e.g., corneal transplant; inflammatory bowel
disease; ischemia, including cerebral ischemia; Kawasaki's disease;
learning impairment; lung diseases; lupus nephritis; multiple
sclerosis; myasthenia gravis; myopathiesneuroinflammatory diseases;
neurotoxicity; ocular diseases and conditions, including ocular
degeneration and uveitis; osteoporosis; pain, including
cancer-related pain; Parkinson's disease; pemphigus; periodontal
disease; Pityriasis rubra pilaris; pre-term labor; prostatitis and
related conditions; psoriasis and related conditions; psoriatic
arthritis; pulmonary fibrosis; reperfusion injury; rheumatic fever;
rheumatoid arthritis; sarcoidosis; scleroderma; septic shock; side
effects from radiation therapy; Sjogren's syndrome; sleep
disturbance; spondyloarthropathies; systemic lupus erythematosus;
temporal mandibular joint disease; thyroiditis; tissue
transplantation or an inflammatory condition resulting from strain,
sprain, cartilage damage, trauma, and orthopedic surgery;
transplant rejection; uveitis; vasculitis; or an inflammatory
condition resulting from strain, sprain, cartilage damage, trauma,
orthopedic surgery, infection or other disease processes.
[0246] The inventive subject matter further relates to a method for
modulating a T-helper cell mediated immune response in an animal in
need thereof, which comprises administering to said animal an
effective amount of an IL-27R/WSX-1 ligand.
[0247] In another aspect, said T-helper cell is Th1.
[0248] In another aspect, said T-helper cell is Th2.
[0249] The inventive subject matter further relates to a method of
treating immune hyperreactivity, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0250] In another aspect, the agent comprises IL-27 or an active
fragment thereof.
[0251] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on WSX-1.
[0252] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27R.
[0253] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27RPP.
[0254] The inventive subject matter further relates to a method of
suppressing polarized T cells, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0255] In another aspect, the agent comprises IL-27 or an active
fragment thereof.
[0256] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on WSX-1.
[0257] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27R.
[0258] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27RPP.
[0259] The inventive subject matter further relates to a method of
treating Th1-mediated disease, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0260] In another aspect, the agent comprises IL-27 or an active
fragment thereof.
[0261] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on WSX-1.
[0262] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27R.
[0263] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27RPP.
[0264] The inventive subject matter further relates to a method of
treating Th2-mediated disease, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0265] In another aspect, the agent comprises IL-27 or an active
fragment thereof.
[0266] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on WSX-1.
[0267] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27R.
[0268] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27RPP.
[0269] The inventive subject matter further relates to a method of
treating IFN-mediated disease, which comprises administering an
effective-amount of an agent that increases WSX-1 activity.
[0270] In another aspect, the agent comprises IL-27 or an active
fragment thereof.
[0271] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on WSX-1.
[0272] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27R.
[0273] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27RPP.
[0274] The inventive subject matter further relates to a method of
treating IgE-mediated disease, which comprises administering an
effective amount of an agent that increases WSX-1 activity.
[0275] In another aspect, the agent comprises IL-27 or an active
fragment thereof.
[0276] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on WSX-1.
[0277] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27R.
[0278] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27RPP.
[0279] The inventive subject matter further relates to a method of
treating asthma, which comprises administering an effective amount
of an agent that increases WSX-1 activity.
[0280] In another aspect, the agent comprises IL-27 or an active
fragment thereof.
[0281] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on WSX-1.
[0282] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27R.
[0283] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27RPP.
[0284] The inventive subject matter further relates to a method of
treating allergy, which comprises administering an effective amount
of an agent that increases WSX-1 activity.
[0285] In another aspect, the agent comprises IL-27 or an active
fragment thereof.
[0286] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on WSX-1.
[0287] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27R.
[0288] In another aspect, the agent comprises an agonistic antibody
that binds to an epitope on IL-27RPP.
Inventive Pharmaceutical Compositions
[0289] The inventive subject matter also relates to a
pharmaceutical composition comprising:
[0290] (i) an effective amount of a compound of formula I . . . ;
and
[0291] (ii) a pharmaceutically acceptable carrier.
[0292] In another aspect, A pharmaceutical composition
comprising:
[0293] (i) an effective amount of an IL-27R/WSX-1 ligand; and
[0294] (ii) a pharmaceutically acceptable carrier.
[0295] In another aspect, The pharmaceutical composition of claim
28, said
[0296] IL-27R/WSX-1 ligand is an agent that increases WSX-1
activity.
[0297] In another aspect, The pharmaceutical composition of claim
29, said agent comprises IL-27 or an active fragment thereof.
[0298] In another aspect, The pharmaceutical composition of claim
29, said agent comprises an agonistic antibody that binds to an
epitope on WSX-1.
[0299] In another aspect, The pharmaceutical composition of claim
28, said agent comprises an agonistic antibody that binds to an
epitope on IL-27R.
[0300] In another aspect, The pharmaceutical composition of claim
28, said agent comprises an agonistic antibody that binds to an
epitope on IL-27RPP.
[0301] The novel pharmaceutical compositions of the invention
include a therapeutically effective amount of the active agent
indicated above. This effective amount will generally comprise from
about 0.1 mg to about 100 mg of the active agent per kilogram of
patient body weight per day. This effective amount can vary
depending upon the physical status of the patient and other factors
well known in the art. Moreover, it will be understood that this
dosage of active agent can be administered in a single or multiple
dosage units to provide the desired therapeutic effect. If desired,
other therapeutic agents can be employed in conjunction with those
provided by the inventive subject matter.
[0302] The compounds of the invention are preferably delivered to
the patient by means of a pharmaceutically acceptable carrier. Such
carriers are well known in the art and generally will be in either
solid or liquid form. Solid form pharmaceutical preparations which
may be prepared according to the inventive subject matter include
powders, tablets, dispersible granules, capsules, cachets and
suppositories. In general, solid form preparations will comprise
from about 5% to about 90% by weight of the active agent.
[0303] In preferred embodiments, the invention also provides
pharmaceutical compositions comprising a therapeutically effective
amount of one or a plurality of the antibodies or other agents of
the invention together with a pharmaceutically acceptable diluent,
carrier, solubilizer, emulsifier, preservative and/or adjuvant.
Preferably, acceptable formulation materials are nontoxic to
recipients at the dosages and concentrations employed. In preferred
embodiments, pharmaceutical compositions comprising a
therapeutically effective amount of anti-IL-1R1 antibodies are
provided.
[0304] In certain embodiments, the pharmaceutical composition may
contain formulation materials for modifying, maintaining or
preserving, for example, the pH, osmolarity, viscosity, clarity,
color, isotonicity, odor, sterility, stability, rate of dissolution
or release, adsorption or penetration of the composition. In such
embodiments, suitable formulation materials include, but are not
limited to, amino acids (such as glycine, glutamine, asparagine,
arginine or lysine); antimicrobials; antioxidants (such as ascorbic
acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as
borate, bicarbonate, Tris-HCl, citrates, phosphates or other
organic acids); bulking agents (such as mannitol or glycine);
chelating agents (such as ethylenediamine tetraacetic acid (EDTA));
complexing agents (such as caffeine, polyvinylpyrrolidone,
beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;
monosaccharides; disaccharides; and other carbohydrates (such as
glucose, mannose or dextrins); proteins (such as serum albumin,
gelatin or immunoglobulins); coloring, flavoring and diluting
agents; emulsifying agents; hydrophilic polymers (such as
polyvinylpyrrolidone); low molecular weight polypeptides;
salt-forming counterions (such as sodium); preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin,
propylene glycol or polyethylene glycol); sugar alcohols (such as
mannitol or sorbitol); suspending agents; surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate 80, triton, trimethamine, lecithin,
cholesterol, tyloxapal); stability enhancing agents (such as
sucrose or sorbitol); tonicity enhancing agents (such as alkali
metal halides, preferably sodium or potassium chloride, mannitol
sorbitol); delivery vehicles; diluents; excipients and/or
pharmaceutical adjuvants. See, Remington's Pharmaceutical Sciences,
18.sup.th Edition, (A. R. Gennaro, ed.), 1990, Mack Publishing
Company.
[0305] A solid carrier can be one or more substances which may also
act as diluents, flavoring agents, solubilizers, lubricants,
suspending agents, binders or tablet disintegrating agents; it can
also be encapsulating material. In powders, the carrier is a finely
divided solid which is in admixture with the viscous active
compound. In tablets, the active compound is mixed with a carrier
having the necessary binding properties in suitable proportions and
compacted to the shape and size desired. Suitable solid carriers
include magnesium carbonate, magnesium stearate, talc, sugar,
lactose, pectin, dextrin, starch, gelatin, tragacanth,
methylcellulose, sodium carboxymethylcellulose, a low melting wax,
cocoa butter, and the like. The term "preparation is intended to
include the formulation of the active compound with encapsulating
materials as a carrier which may provide a capsule in which the
active component (with or without other carriers) is surrounded by
carrier, which is thus in association with it. Similarly, cachets
are included. Tablets, powders, cachets, and capsules can be used
as solid dosage forms suitable for oral administration. If desired
for reasons of convenience or patient acceptance, pharmaceutical
tablets prepared according to the invention may be provided in
chewable form, using techniques well known in the art.
[0306] For preparing suppositories, a low melting wax such as a
mixture of fatty acid glycerides or cocoa butter is first melted,
and the active ingredient is dispersed homogeneously therein as by
stirring. The molten homogeneous mixture is then poured into
convenient sized molds, allowed to cool and thereby to
solidify.
[0307] Liquid form preparations include solutions, suspensions, and
emulsions. As an example may be mentioned water or water/propylene
glycol solutions for parenteral injection. Liquid preparations can
also be formulated in solution in aqueous polyethylene glycol
solution. Aqueous solutions suitable for oral use can be prepared
by dissolving the active component in water and adding suitable
colorants, flavors, stabilizers and thickening agents as desired.
Aqueous suspensions suitable for oral use can be made my dispersing
the finely divided active component in water with a viscous
material, i.e., natural or synthetic gums, resins, methylcellulose,
sodium carboxymethylcellulose, and other well known suspending
agents. Liquid pharmaceutical preparations may comprise up to 100%
by weight of the subject active agent.
[0308] In certain embodiments, the primary vehicle or carrier in a
pharmaceutical composition may be either aqueous or non-aqueous in
nature. For example, a suitable vehicle or carrier may be water for
injection, physiological saline solution or artificial
cerebrospinal fluid, possibly supplemented with other materials
common in compositions for parenteral administration. Neutral
buffered saline or saline mixed with serum albumin are further
exemplary vehicles. In preferred embodiments, pharmaceutical
compositions comprise Tris buffer of about pH 7.0-8.5, or acetate
buffer of about pH 4.0-5.5, and may further include sorbitol or a
suitable substitute therefor. In certain embodiments of the
invention, compositions may be prepared for storage by mixing the
selected composition having the desired degree of purity with
optional formulation agents ("Remington's Pharmaceutical Sciences",
18th ed. (1990, Mack Publishing Co., Easton, Pa. 18042)) in the
form of a lyophilized cake or an aqueous solution. Further, in
certain embodiments, the product may be formulated as a
lyophilizate using appropriate excipients such as sucrose.
[0309] Also contemplated as suitable carriers are solid form
preparations which are intended to be converted, shortly before
use, to liquid form preparations for either oral or parenteral
administration. Such liquid forms include solutions, suspensions,
and emulsions. These particular solid form preparations are most
conveniently provided in unit dose form and as such are used to
provide a single liquid dosage unit. Alternately, sufficient solid
may be provided so that after conversion to liquid form, multiple
individual liquid doses may be obtained by measuring predetermined
volumes of the liquid form preparation as with a syringe, teaspoon,
or other volumetric container. When multiple liquid doses are so
prepared, it is preferred to maintain the unused portion of said
liquid doses at low temperature (i.e., under refrigeration) in
order to retard possible decomposition. The solid form preparations
intended to be converted to liquid form may contain, in addition to
the active material, flavorants, colorants, stabilizers, buffers,
artificial and natural sweeteners, dispersants, thickeners,
solubilizing agents, and the like. The liquid utilized for
preparing useful liquid form preparations may be water, isotonic
water, ethanol, glycerine, propylene glycol, and the like as well
as mixtures thereof. Naturally, the liquid utilized will be chosen
with regard to the route of administration. For example, liquid
preparations containing large amounts of ethanol are not suitable
for parenteral use.
[0310] The pharmaceutical preparation may also be in a unit dosage
form. In such form, the preparation is subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of preparation, for example, packeted tablets,
capsules, and powders in vials or ampoules. The unit dosage form
can also be a capsule, cachet, or tablet itself or it can be the
appropriate number of any of these in packaged form.
[0311] The pharmaceutical preparations of the invention may include
one or more preservatives well known in the art, such as benzoic
acid, sorbic acid, methylparaben, propylparaben and
ethylenediaminetetraacetic acid (EDTA). Preservatives are generally
present in amounts up to about 1% and preferably from about 0.05 to
about 0.5% by weight of the pharmaceutical composition.
[0312] Useful buffers for purposes of the invention include citric
acid-sodium citrate, phosphoric acid-sodium phosphate, and acetic
acid-sodium acetate in amounts up to about 1% and preferably from
about 0.05 to about 0.5% by weight of the pharmaceutical
composition. Useful suspending agents or thickeners include
cellulosics like methylcellulose, carageenans like alginic acid and
its derivatives, xanthan gums, gelatin, acacia, and
microcrystalline cellulose in amounts up to about 20% and
preferably from about 1% to about 15% by weight of the
pharmaceutical composition.
[0313] Sweeteners which may be employed include those sweeteners,
both natural and artificial, well known in the art. Sweetening
agents such as monosaccharides, disaccharides and polysaccharides
such as xylose, ribose, glucose, mannose, galactose, fructose,
dextrose, sucrose, maltose, partially hydrolyzed starch or corn
syrup solids and sugar alcohols such as sorbitol, xylitol, mannitol
and mixtures thereof may be utilized in amounts from about 10% to
about 60% and preferably from about 20% to about 50% by weight of
the pharmaceutical composition. Water soluble artificial sweeteners
such as saccharin and saccharin salts such as sodium or calcium,
cyclamate salts, acesulfame-K, aspartame and the like and mixtures
thereof may be utilized in amounts from about 0.001% to about 5% by
weight of the composition.
[0314] Flavorants which may be employed in the pharmaceutical
products of the invention include both natural and artificial
flavors, and mints such as peppermint, menthol, vanilla, artificial
vanilla, chocolate, artificial chocolate, cinnamon, various fruit
flavors, both individually and mixed, in amounts from about 0.5% to
about 5% by weight of the pharmaceutical composition.
[0315] Colorants useful in the inventive subject matter include
pigments which may be incorporated in amounts of up to about 6% by
weight of the composition. A preferred pigment, titanium dioxide,
may be incorporated in amounts up to about 1%. Also, the colorants
may include other dyes suitable for food, drug and cosmetic
applications, known as F.D.&C. dyes and the like. Such dyes are
generally present in amounts up to about 0.25% and preferably from
about 0.05% to about 0.2% by weight of the pharmaceutical
composition. A full recitation of all F.D.&C. and D.&C.
dyes and their corresponding chemical structures may be found in
the Kirk-Othmer Encyclopedia of Chemical Technology, in Volume 5,
at pages 857-884, which text is accordingly incorporated herein by
reference.
[0316] Useful solubilizers include alcohol, propylene glycol,
polyethylene glycol and the like and may be used to solubilize the
flavors. Solubilizing agents are generally present in amounts up to
about 10%; preferably from about 2% to about 5% by weight of the
pharmaceutical composition.
[0317] Lubricating agents which may be used when desired in the
instant compositions include silicone oils or fluids such as
substituted and unsubstituted polysiloxanes, e.g., dimethyl
polysiloxane, also known as dimethicone. Other well known
lubricating agents may be employed.
[0318] Combination Therapy
[0319] It is not expected that compounds of the inventive subject
matter will display significant adverse interactions with other
synthetic or naturally occurring substances. Thus, a compound of
the inventive subject matter may be administered in combination
with other compounds and compositions useful for modulating an
immune response. In particular the compounds of the inventive
subject matter may be administered in combination with other
compounds of the inventive subject matter; other immunomodulating
substances; etc.
[0320] Therapeutic agents of the inventive subject matter can be
administered alone or in combination with other therapeutic agents
to prevent or to treat various diseases, disorders, and conditions,
such as inflammatory or autoimmune diseases. Depending on the
disease, disorder or condition and the desired level of treatment,
two, three, or more agents may be administered. These agents may be
provided together by inclusion in the same formulation or inclusion
in a treatment kit, or they may be provided separately. When
administered by gene therapy, the genes encoding the protein agents
may be included in the same vector, optionally under the control of
the same promoter region, or in separate vectors. Particularly
preferred molecules in the aforementioned classes are as
follows:
[0321] IL-1 inhibitors: IL-1ra proteins and soluble IL-1 receptors.
The most preferred IL-1 inhibitor is anakinra.
[0322] TNF-.alpha. inhibitors: soluble tumor necrosis factor
receptor type I (sTNF-RI; -RI is also called the p55 receptor);
soluble tumor necrosis factor receptor type II (also called the p75
receptor); and monoclonal antibodies that bind the TNF receptor.
Most preferred is sTNF-RI as described in WO 98/24463, etanercept
(Enbrel.RTM.), and Avakine.RTM.. Exemplary TNF-inhibitors are
described in EP 422 339, EP 308 378, EP 393 438, EP 398 327, and EP
418 014.
[0323] Serine protease inhibitors: SLPI, ALP, MPI, HUSI-I, BMI, and
CUSI. These inhibitors also may be viewed as exemplary LPS
modulators, as SLPI has been shown to inhibit LPS responses.
[0324] Jin et al. (1997), Cell 88(3): 417-26 (incorporated by
reference).
[0325] In certain embodiments, the optimal pharmaceutical
formulations will be determined by one skilled in the art depending
upon considerations such as, for example, the intended route of
administration, delivery format, and desired dosage. See, for
example, Remington's Pharmaceutical Sciences, supra, pp. 1435-1712,
the disclosure of which is hereby incorporated by reference. In
certain embodiments, such compositions may influence the physical
state, stability, rate of in vivo release, and rate of in vivo
clearance of the inventive therapeutic agents.
SYNTHESIS OF COMPOUNDS OF THE INVENTION
[0326] The inventive compositions may be readily prepared by
standard techniques of molecular biology, utilizing techniques
known to those of ordinary skill in the art and as described in
greater detail herein.
[0327] The products and intermediates may be isolated or purified
using one or more standard purification techniques known to one of
ordinary skill in the art, including, for example, one or more of
simple solvent evaporation, recrystallization, distillation,
sublimation, filtration, polymerase chain reaction, Southern
blotting, Northern blotting, Western blotting, chromatography,
including thin-layer chromatography, affinity chromatography, gel
filtration chromatography, ion exchange chromatography, FPLC, HPLC
(e.g. reverse phase HPLC), column chromatography, flash
chromatography, radial chromatography, trituration, salt
precipitation, two-phase separation, polymer precipitation, heat
denaturation, isoelectric separation, dialysis, and the like.
[0328] It is contemplated that suitable IL-27R/WSX-1 ligands may
optionally be synthesized as small molecule chemical compounds.
Such inventive compounds may be readily prepared by standard
techniques of organic chemistry. In the preparation of such small
molecule compounds, one skilled in the art will understand that one
may need to protect or block various reactive functionalities on
the starting compounds or intermediates while a desired reaction is
carried out on other portions of the molecule. After the desired
reactions are complete, or at any desired time, normally such
protecting groups will be removed by, for example, hydrolytic or
hydrogenolytic means. Such protection and deprotection steps are
conventional in organic chemistry. One skilled in the art is
referred to "Protective Groups in Organic Chemistry," McOmie, ed.,
Plenum Press, New York, N.Y.; and "Protective Groups in Organic
Synthesis," Greene, ed., John Wiley & Sons, New York, N.Y.
(1981) for the teaching of protective groups which may be useful in
the preparation of compounds of the inventive subject matter.
[0329] The product and intermediates of chemical synthesis may be
isolated or purified using one or more standard purification
techniques, including, for example, one or more of simple solvent
evaporation, recrystallization, distillation, sublimation,
filtration, chromatography, including thin-layer chromatography,
HPLC (e.g. reverse phase HPLC), column chromatography, flash
chromatography, radial chromatography, trituration, and the
like.
Route(s) of Administration
[0330] The route(s) of administration of the compounds and
compositions of the inventive subject matter are well known to
those skilled in the art (see, for example, "Remington's
Pharmaceutical Sciences", supra). The compounds and compositions
may be administered orally, parenterally, by inhalation spray,
topically, rectally, nasally, buccally, vaginally, or via an
implanted reservoir in dosage formulations containing conventional
non-toxic pharmaceutically-acceptable carriers, adjuvants, and
vehicles. The term parenteral as used herein includes subcutaneous,
intravenous, intraarterial, intramuscular, intraperitoneally,
intrathecally, intralesional, intraportal, intraventricularly,
intrasternal, intra-ocular, intracerebroventricular, intracerebral
(intra-parenchymal), and intracranial injection or infusion
techniques; by sustained release systems or by implantation
devices. In certain embodiments, the compositions may be
administered by bolus injection or continuously by infusion, or by
implantation device.
[0331] To be effective therapeutically as central nervous system
targets, the compounds and compositions should readily penetrate
the blood-brain barrier when peripherally administered. Compounds
which cannot penetrate the blood-brain barrier can be effectively
administered by an intraventricular route.
[0332] The compounds and compositions may be administered in the
form of sterile injectable preparations, for example, as sterile
injectable aqueous or oleaginous suspensions. These suspensions,
may be formulated according to techniques known in the art using
suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparations may also be sterile injectable
solutions or suspensions in non-toxic parenterally-acceptable
diluents or solvents, for example, as solutions in 1,3-butanediol.
Among the acceptable vehicles and solvents that may be employed are
water, Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as
solvents or suspending mediums. For this purpose, any bland fixed
oil such as a synthetic mono- or di-glyceride may be employed.
Fatty acids such as oleic acid and its glyceride derivatives,
including olive oil and castor oil, especially in their
polyoxyethylated versions, are useful in the preparation of
injectables. These oil solutions or suspensions may also contain
long-chain alcohol diluents or dispersants.
[0333] Additionally, in on aspect of the inventive subject matter,
the compounds and compositions may be administered orally in the
form of capsules, tablets, aqueous suspensions, or solutions.
Agents that are administered in this fashion can be formulated with
or without carriers customarily used in the compounding of solid
dosage forms such as tablets and capsules. Tablets may contain
carriers such as lactose and corn starch, and/or lubricating agents
such as magnesium stearate. Capsules may contain diluents including
lactose and dried corn starch. Aqueous suspensions may contain
emulsifying and suspending agents combined with the active
ingredient. The oral dosage forms may further contain sweetening,
flavoring, coloring agents, or combinations thereof. In certain
embodiments, a capsule may be designed to release the active
portion of the formulation at the point in the gastrointestinal
tract when bioavailability is maximized and pre-systemic
degradation is minimized. Additional substituents can be included
to facilitate absorption of the agent of this invention.
[0334] A pharmaceutical composition of the invention is preferably
provided to comprise an effective quantity of one or a plurality of
the agents of this invention in a mixture with non-toxic excipients
that are suitable for the manufacture of tablets. By dissolving the
tablets in sterile water, or another appropriate vehicle, solutions
may be prepared in unit-dose form. Suitable excipients include, but
are not limited to, inert diluents, such as calcium carbonate,
sodium carbonate or bicarbonate, lactose, or calcium phosphate; or
binding agents, such as starch, gelatin, or acacia; or lubricating
agents such as magnesium stearate, stearic acid, or talc.
[0335] The pharmaceutical compositions of the invention can be
selected for parenteral delivery. The compositions may be selected
for inhalation or for delivery through the digestive tract, such as
orally. Preparation of such pharmaceutically acceptable
compositions is within the skill of the art.
[0336] The formulation components are present preferably in
concentrations that are acceptable to the site of administration.
In certain embodiments, buffers are used to maintain the
composition at physiological pH or at a slightly lower pH,
typically within a pH range of from about 5 to about 8.
[0337] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention may be provided
in the form of a pyrogen-free, parenterally acceptable aqueous
solution comprising the desired agent in a pharmaceutically
acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which the anti-IL-1R1
antibody is formulated as a sterile, isotonic solution, properly
preserved. In certain embodiments, the preparation can involve the
formulation of the desired molecule with an agent, such as
injectable microspheres, bio-erodible particles, polymeric
compounds (such as polylactic acid or polyglycolic acid), beads or
liposomes, that may provide controlled or sustained release of the
product which can be delivered via depot injection. In certain
embodiments, hyaluronic acid may also be used, having the effect of
promoting sustained duration in the circulation. In certain
embodiments, implantable drug delivery devices may be used to
introduce the desired antibody molecule.
[0338] Pharmaceutical compositions of the invention can be
formulated for inhalation. In these embodiments, agents are
formulated as a dry powder for inhalation. In preferred
embodiments, inhalation solutions may also be formulated with a
propellant for aerosol delivery. In certain embodiments, solutions
may be nebulized. Pulmonary administration and formulation methods
therefore are further described in International Patent Publication
No. WO94/20069, incorporated by reference, which describes
pulmonary delivery of chemically modified proteins.
[0339] The compounds may also be administered rectally in the form
of suppositories. These compositions can be prepared by mixing the
drug with a suitable non-irritating excipient which is solid at
room temperature, but liquid at rectal temperature and, therefore,
will melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax, and polyethylene glycols.
[0340] Furthermore, the compounds may be administered topically,
especially when the conditions addressed for treatment involve
areas or organs readily accessible by topical application,
including the lower intestinal tract. Suitable topical formulations
can be readily prepared for such areas or organs. For example,
topical application to the lower intestinal tract can be effected
in a rectal suppository formulations (see above) or in suitable
enema formulations.
[0341] It is envisioned that the continuous administration or
sustained delivery of the compounds and compositions of the
inventive subject matter may be advantageous for a given condition.
While continuous administration may be accomplished via a
mechanical means, such as with an infusion pump, it is contemplated
that other modes of continuous or near continuous administration
may be practiced. For example, such administration may be by
subcutaneous or muscular injections as well as oral pills.
[0342] Techniques for formulating a variety of other sustained- or
controlled-delivery means, such as liposome carriers, bio-erodible
particles or beads and depot injections, are also known to those
skilled in the art.
[0343] Additional pharmaceutical compositions will be evident to
those skilled in the art, including formulations involving agents
of this invention in sustained- or controlled-delivery
formulations. Techniques for formulating a variety of other
sustained- or controlled-delivery means, such as liposome carriers,
bio-erodible microparticles or porous beads and depot injections,
are also known to those skilled in the art. See for example,
International Patent Publication No. WO93/15722, incorporated by
reference, which describes controlled release of porous polymeric
microparticles for delivery of pharmaceutical compositions.
Sustained-release preparations may include semipermeable polymer
matrices in the form of shaped articles, e.g. films, or
microcapsules. Sustained release matrices may include polyesters,
hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919
and European Patent Application Publication No. EP 058481),
copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman
et al., 1983, Biopolymers 22:547-556), poly
(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.
Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),
ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (European Patent Application
Publication No. EP 133,988). Sustained release compositions may
also include liposomes that can be prepared by any of several
methods known in the art. See e.g., Eppstein et al., 1985, Proc.
Natl. Acad. Sci. USA 82:3688-3692; European Patent Application
Publication Nos. EP 036,676; EP 088,046 and EP 143,949.
[0344] Pharmaceutical compositions used for in vivo administration
are typically provided as sterile preparations. Sterilization can
be accomplished by filtration through sterile filtration membranes.
When the composition is lyophilized, sterilization using this
method may be conducted either prior to or following lyophilization
and reconstitution. Compositions for parenteral administration can
be stored in lyophilized form or in a solution. Parenteral
compositions generally are placed into a container having a sterile
access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
[0345] Once the pharmaceutical composition has been formulated, it
may be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or as a dehydrated or lyophilized powder. Such
formulations may be stored either in a ready-to-use form or in a
form (e.g., lyophilized) that is reconstituted prior to
administration.
[0346] The invention also provides kits for producing a single-dose
administration unit. The kits of the invention may each contain
both a first container having a dried protein and a second
container having an aqueous formulation. In certain embodiments of
this invention, kits containing single and multi-chambered
pre-filled syringes (e.g., liquid syringes and lyosyringes) are
provided.
[0347] The composition also may be administered locally via
implantation of a membrane, sponge or another appropriate material
onto which the desired molecule has been absorbed or encapsulated.
In certain embodiments, where an implantation device is used, the
device may be implanted into any suitable tissue or organ, and
delivery of the desired molecule may be via diffusion,
timed-release bolus, or continuous administration.
[0348] It also may be desirable to use pharmaceutical compositions
according to the invention ex vivo. In such instances, cells,
tissues or organs that have been removed from the patient are
exposed to pharmaceutical compositions after which the cells,
tissues and/or organs are subsequently implanted back into the
patient.
[0349] In particular, agents of this invention can be delivered by
implanting certain cells that have been genetically engineered,
using methods such as those described herein, to express and
secrete the polypeptide. In certain embodiments, such cells may be
animal or human cells, and may be autologous, heterologous, or
xenogeneic. In certain embodiments, the cells may be immortalized.
In other embodiments, in order to decrease the chance of an
immunological response, the cells may be encapsulated to avoid
infiltration of surrounding tissues. In further embodiments, the
encapsulation materials are typically biocompatible, semi-permeable
polymeric enclosures or membranes that allow the release of the
protein product(s) but prevent the destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissues.
[0350] The effective amount of a pharmaceutical composition to be
employed therapeutically will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will
appreciate that the appropriate dosage levels for treatment will
vary depending, in part, upon the molecule delivered, the
indication for which the agent of this invention is being used, the
route of administration, and the size (body weight, body surface or
organ size) and/or condition (the age and general health) of the
patient. In certain embodiments, the clinician may titer the dosage
and modify the route of administration to obtain the optimal
therapeutic effect. A typical dosage may range from about 0.1 g/kg
to up to about 100 mg/kg or more, depending on the factors
mentioned above. In preferred embodiments, the dosage may range
from 0.1 g/kg up to about 100 mg/kg; more preferably from 1 g/kg up
to about 100 mg/kg; or even more preferably from 5 g/kg up to about
100 mg/kg.
[0351] Dosing frequency will depend upon the pharmacokinetic
parameters of the particular agent in the formulation used.
Typically, a clinician administers the composition until a dosage
is reached that achieves the desired effect. The composition may
therefore be administered as a single dose, or as two or more doses
(which may or may not contain the same amount of the desired
molecule) over time, or as a continuous infusion via an
implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in
the art and is within the ambit of tasks routinely performed by
them. Appropriate dosages may be ascertained through use of
appropriate dose-response data.
Dosage
[0352] Dosage levels on the order of about 0.001 mg to about 100 mg
per kilogram body weight of the active ingredient compounds or
compositions are useful in the treatment of the above conditions,
with preferred levels ranging from 200 mg per day to 1600 mg per
day. The compounds and compositions of the inventive subject matter
may usually be given in two or three doses daily. Starting with a
low dose (200-300 mg) twice daily and slowly working up to higher
doses if needed is a preferred strategy. The amount of active
ingredient that may be combined with the carrier materials to
produce a single dosage form will vary depending upon the host
treated and the particular mode of administration.
[0353] It is understood, however, that a specific dose level for
any particular patient will depend upon a variety of factors,
including the activity of the specific compound employed; the age,
body weight, general health, sex and diet of the patient; the time
of administration; the rate of excretion; drug combination; the
severity of the particular disorder being treated; and the form of
administration. One of ordinary skill in the art would appreciate
the variability of such factors and would be able to establish
specific dose levels using no more than routine
experimentation.
EXAMPLES
[0354] The following examples are illustrative of the inventive
subject matter and are not intended to be limitations thereon.
Unless otherwise indicated, all percentages are based upon 100% by
weight of the final composition.
General Experimental Procedures
[0355] Experimental Animals. Four- to six-week-old wild type,
C57B/6 mice, used as controls, were purchased from a commercial
supplier. WSX-1.sup.-/- mice were bred and maintained as
homozygotes in a specific-pathogen free environment. Four- to
six-week-old IL-12p40.sup.-/- and RAG-2.sup.-/- mice were purchased
from a commercial supplier and IL-10.sup.-/- mice were bred
in-house. Mice deficient in IFN-.gamma. were purchased from a
commercial supplier. All animals were maintained under specific
pathogen free conditions, in accordance to institutional
guidelines. In all experiments, mice were infected at five-eight
weeks of age, and experimental groups contained three-five
animals.
[0356] Toxoplasma gondii Infections. The ME49 strain of T. gondii
was maintained in Swiss Webster and CBA/CaJ mice. ME49 bradyzoite
cysts were prepared from donor mice as described previously (Cai et
al., 2000). Mice were challenged with 20 T. gondii cysts either by
i.p. or oral administration. Throughout the manuscript, all
infections were performed i.p. unless otherwise noted. To assess
parasite burden, mice were infected i.p. with T. gondii and, after
7 days, peritoneal lavage was performed. Cells were collected for
cytospin preparation and the number of infected cells estimated by
microscopy (n=3 per group and at least 500 cells counted per
mouse). For histological examinations, lungs, heart, spleen, and
liver were collected from animals that were infected with T. gondii
for 0 (uninfected) or 12 days. Organs were fixed in 10% formalin,
embedded in paraffin, sectioned, and stained with hematoxylin and
eosin. For in vivo depletion of T cells, WSX-1.sup.-/- mice were
treated with the indicated antibody at days 7, 8, and 9
postinfection. Endotoxin free .alpha.CD4 (GK1.5) and .alpha.CD8
(H35-17.2) mAbs were grown from hybridomas.
[0357] T. muris Infection and Antigen. T. muris was maintained in
genetically susceptible or immuno-compromised animals. Between days
35-42 post-infection, adult worms were isolated and cultured in
RPMI containing 500 U/ml penicillin and 500 g/ml streptomycin for
24 hours. T. muris excretory-secretory Ag was isolated at 4 hours
and 24 hours, dialyzed, sterile filtered, and protein
concentrations determined by Bradford Assay. Antigen preparations
were used in lymphocyte restimulations, 50 g/ml. Deposited eggs
were collected after 24 hours of culture, washed three times in
sterile water, incubated at room temperature for six weeks, and
stored at 4 C. Mice were infected on day zero with 150-200
embryonated eggs and worm burdens assessed on various days
post-infection.
[0358] Detection of IL-27 and WSX-1 mRNA Levels. IL-27 and WSX-1
levels were determined by RT-PCR. For ex vivo analysis of mRNA
expression following T. gondii infection, whole splenocytes were
isolated from wild type mice that had been infected for 0
(uninfected) and 7 days. For ex vivo analysis of mRNA expression
following T. muris infection, mRNA was isolated from whole
mesenteric lymph node (hereinafter "MLN") cell suspensions using
Trizol. After using standard procedures known in the art for
isolating mRNA, PCR was utilized for 34 cycles: 95.quadrature.C, 30
seconds/60.quadrature.C 30 seconds/72.quadrature.C 1 minute, to
quantify message levels. .beta.-actin expression was used as an
internal control to assure equal loading of every reaction. Primers
specific for IL-27p28 (two 20-mers), EBI3 (one 20-mer and one
23-mer), and WSX-1 (two 20-mers) were used. Specific sequences are
found in Applicants' publication, Villarino, et al., The IL-27R
(WSX-1) Is Required to Suppress T Cell Hyperactivity during
Infection, Immunity, 19:645-655 (2003), which is incorporated by
reference in its entirety.
[0359] Isolation and Culture of Splenocytes for ex vivo Recall
Assays. Spleens from infected/uninfected wild type and
WSX-1.sup.-/- mice were harvested, dissociated into a single cell
suspension, and depleted of erythrocytes using 0.86% (wt/vol)
ammonium chloride (Sigma). Cells were washed three times and
resuspended in complete RPMI 1640 (10% heat-inactivated fetal
bovine serum, 100 U/ml penicillin, 1 mg/ml streptomycin,
nonessential amino acids, and .beta.-mercapthoethanol) before being
plated at a cell density of 2.times.10.sup.5 cells per well in a
final volume of 200 .mu.l in 96-well plates (Costar). Where
indicated, cells were stimulated with plate bound .alpha.CD3
antibody (1 .mu.g/ml) or cultured with soluble Toxoplasma antigen
(stag, 25 .mu.g/ml). For flow cytometry experiments, cells were
cultured at a final density of 2.times.10.sup.6 cells/well in a
final volume of 1 ml in 24-well plates (Costar).
[0360] Cytokine Production Analyses. Levels of IL-12, IFN-.gamma.,
IL-10, TNF-.alpha., IL-23, and IL-2 were measured by ELISA. For
both splenocyte recall assays and in vitro differentiation assays,
supernatants were collected after 72 hr of culture. For detection
of intracellular IFN-.gamma. by flow cytometry, all cells were
treated with brefeldin A (BFA, 10 .mu.g/ml) for 2 hr prior to
fixation and permeabilization with saponin. Cytokine was detected
using APC-conjugated_IFN-.gamma. mAb (BD Pharmingen) in combination
with surface staining for CD4 or CD8 (PE-conjugated, BD
Pharmingen).
[0361] Ex vivo Activation and Proliferation Analyses. Splenocytes
were stained directly ex vivo for surface expression of activation
markers CD25 (PE) and CD62L (APC) in combination with either CD4 or
CD8 (FITC)(BD Pharmingen). For BrdU incorporation studies, mice
were treated with BrdU (0.8 mg/mouse i.p.) for 3 days prior to
analysis. At the indicated time points after infection, mesenteric
lymph nodes were isolated and cells were stained for surface
expression of CD4 or CD8 prior to fixation. To detect incorporated
BrdU, cells were permeabilized with Tween-20 (.05%), treated with
DNAse I solution, and stained with a FITC-conjugated_BrdU mAb (BD
Pharmingen).
[0362] In Vitro Differentiation of Naive Splenocytes. For in vitro
assays, splenocytes were isolated form naive animals, red blood
cells lysed using ammonium chloride and depleted of CD8.sup.+ and
NK1.1_cells by magnetic bead separation (Mullen et al., 2001).
Cells were labeled with CFSE (5 .mu.g/ml, Sigma) according to
standard protocols (Mullen et al, 2001) and then stimulated with
soluble .alpha.CD3 antibody (0.1 .mu.g/ml), soluble .alpha.CD28
antibody (0.5 .mu.g/ml), and recombinant human IL-2 (10 U/ml,
Chiron). For nonpolarizing conditions, cells were cultured with
neutralizing antibodies to IL-12 (10 .mu.g/ml) and IL-4 (10
.mu.g/ml). For Th1 polarizing conditions, cultures were
supplemented with recombinant mouse IL-12 (5 ng/ml, Genetics
Institute) and neutralizing .alpha.IL-4 (10 .mu.g/ml) antibody.
[0363] First, after 3 days of culture, supernatants were collected
to measure IFN-.gamma. concentration by ELISA. Next, the remaining
cells were then stimulated with Phorbol 12-myristate 13-acetate
(PMA 50 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) for 4 hr,
treated with BFA (10 .mu.g/ml, Sigma) for 2 hr, and then stained
for intracellular IFN-.gamma. in combination with surface CD4 and
CFSE incorporation.
[0364] In Vitro Signaling Assays. Naive CD4.sup.+CD45RB.sup.hi T
cells were purified from wild type spleens by FACS sorting (Pflanz
et al., 2002). Cells were rested in media overnight and then
stimulated with recombinant cytokines for 15 min. Cytokines used
were IL-2, 50 ng/ml (R&D Systems); IL-12, 200 ng/ml (R&D
Systems); IFN-.alpha., 50 ng/ml (R&D Systems); IFN-.gamma., 50
ng/ml (R&D Systems); IL-27, 50 ng/ml (DNAX, in-house
hyperkines). After stimulation, cells were lysed and probed for
total and tyrosine isolated phosphorylated STAT proteins by Western
blot (Hibbert et al., 2003). All phospho-STAT antibodies from NEB
(Cell Signaling Technology), STAT-1 from Transduction Labs, STAT-3,
STAT-4, and STAT-5 from Santa Cruz.
[0365] In vivo depletions. Neutralizing .alpha.IL-12 mAb, C17.8,
.alpha.IFN-.gamma. mAb, XMG.6, and .alpha.IL-4 mAb, 11B11, 2 mg per
dose, were administered intraperitoneally on days 0, 4, 8, and 12
post-infection. Control mice received equivalent amounts of
purified rat IgG (Sigma Chemical Co., St. Louis, Mo.).
[0366] Lymphocyte proliferation and cytokine assays. Lymphocytes
were harvested from MLN, depleted of CD8.sup.+ and NK1.1.sup.+
cells using magnetic beads in combination with .alpha.CD8 and NK1.1
FITC conjugated mAb. Cells were resuspended in RPMI 1640
supplemented with 10% heat-killed fetal bovine serum, 100 U/ml
penicillin, 100 g/ml streptomycin, non-essential amino acids and
P-mercapthoethanol, and plated at 4.times.10.sup.5 cells/well in
96-well plates. For antigen specific recall responses, cells were
cultured alone or in the presence of T. muris Ag, 50 g/ml, for 72
hours. Secreted IL-4, IL-5, IL-13 and IFN-.gamma. was assayed by
sandwich ELISA. For detection of intracellular IFN-.gamma. after 14
days of infection, lymphocytes were purified as above, stimulated
with .alpha.CD3 and .alpha.CD28, both 1 g/ml, in the presence of
rIL-4, 50 ng/ml, and .alpha.IL-12 mAb, C17.8; 10 g/ml. After 72
hours, cells were pulsed with PMA, 50 ng/ml, ionomycin, 500 ng/ml,
and Brefeldin A (hereinafter "BfA"), 10 g/ml, for 3-5 hours and
stained for intracellular IFN-.gamma. in combination with surface
CD4. For detection of intracellular IFN-.gamma. after 21 days of
infection, MLN cells were stimulated with .alpha.CD3 for 18 hrs and
the incubated with BFA for 2 hours before staining for
intracellular IFN-.gamma.. Cells were acquired on a FACSCalibur
cytometer, and analyzed using CellQuest software. All dot plots
shown have a log axis of 10.sup.0 to 10.sup.4.
[0367] In vitro differentiation assays. Splenocytes were isolated
from naive animals, labeled with CFSE, 5 g/ml, and stimulated for
3-4 days with soluble .alpha.CD3 mAb, 0.1 g/ml, soluble .alpha.CD28
mAb, 0.5 g/ml, and rIL-2, 10 IU/ml, under Th2 polarizing
conditions, rIL-4, 50 ng/ml, and .alpha.IL-12 mAb, 10 g/ml, with or
without recombinant murine IL-27. For primary stimulations
CD4.sup.+ T cell proliferation and intracellular IL-4 production
were determined by flow cytometry. For secondary stimulations,
cells were harvested at day 4, washed and restimulated with
.alpha.CD3 for 24 hours under neutral conditions. Secreted levels
of IL-4, IL-5 and IL-13 were then determined by ELISA.
[0368] Estimation of parasite specific IgG2.alpha. responses.
Parasite-specific IgG2.alpha. responses were determined by capture
ELISA. Immulon IV plates were coated with T. muris ES Ag, 5 g/ml,
in carbonate/bicarbonate buffer overnight at 4 C. After blocking,
3% BSA in PBS, 0.05% Tween, eight serial 2-fold dilutions of sera,
from an initial 20-fold dilution, were added to the plates.
Parasite-specific antibody was detected using biotinylated rat
.alpha.-mouse IgG2.alpha. in combination with streptavadin-HRP.
[0369] Analysis of goblet cell responses. One-centimeter segments
of mid-cecum were removed, washed in sterile PBS, and fixed for 24
hours in 10% neutral buffered formalin. Tissues were processed
routinely and paraffin embedded using standard histological
techniques. Five m sections were cut and stained with haematoxylin
and eosin or alcian blue-periodic acid Schiffs for detection of
intestinal goblet cells. Enumeration of intestinal goblet cell
responses was carried out by counting numbers of goblet cells per
100 crypt units. The anti-mRELM.beta. antibody, its use in
immunoblotting, as well as the conditions used to isolate stool
proteins have been described previously.
[0370] Analysis of mast cell responses for histology. 1 cm lengths
of cecum were isolated, washed in PBS and fixed in Carnoy's
solution before subsequent processing and sectioning. Mast cells
were detected by staining 5 m sections overnight in 0.5% Toluidine
Blue in 0.5 mol/liter HCl, pH 0.5, and counterstaining in 1% eosin
solution. Enumeration of Toluidine Blue-positive mast cells per 40
fields was carried out for three to four mice per time point.
Immunohistochemical detection of MMCP-1+ mast cells was carried out
on paraformaldehyfe fixed tissues. Serum mast cell protease-1,
MMCP-1, was measured using a commercially available kit.
[0371] Growth of mast cells: Passive Cutaneous Anaphylaxis. Mice
were anesthetized by intraperitoneal injection of 300 1 of 2.5%
2,2,2-tribromoethanol in tert-amyl alcohol:PBS, 1:40. In vivo mast
cells were then sensitized with 25 ng of anti-DNP IgE in 25 L of
PBS by intradermal injection into the base of the dorsal aspect of
the right ear and 25 l of PBS was injection into base of the dorsal
aspect the left ear. 24 hours later mice were again anesthetized
and challenged with 100 g DNP-HSA in 200 l of 1% Evans blue by
intravenous retro-orbital injection. 30 minutes after challenge,
mice were euthanized and ears were collected and incubated at
55.degree. C. for 48 hrs in 1 mL of formamide. OD of 300 L of
formamide from each sample was then measured at 610 nm in a 96 well
plate reader.
Example 1
WSX-1 is Required for Resistance to Toxoplasma gondii
[0372] To assess the role of IL-27/WSX-1 in the development and
regulation of resistance to T. gondii, studies were carried out to
determine whether infection resulted in increased expression of
this cytokine or its receptor. Wild type C57BL/6 mice were
inoculated intraperitoneally (i.p.) with 20 cysts of the ME49
strain of T. gondii. After 7 days, mRNA was isolated from whole
splenocytes of infected and uninfected mice. Reverse transcription
PCR (RT-PCR) was used to assess levels of mRNA for IL-27p28, EBI3,
and WSX-1 in the spleen. After 7 days of infection, there was an
upregulation in levels of mRNA for IL-27p28 and EBI3, while the
constitutive level of WSX-1mRNAin unchallenged mice was not
appreciably altered by infection (FIG. 1A). To determine the
significance of this infection-induced increase of IL-27 mRNA, wild
type, WSX-1.sup.-/-, and IL-12p40.sup.-/- mice, n=4 mice per group,
representative of three experiments, were infected with T. gondii
and their survival monitored. While wild type mice were able to
survive the acute phase of this infection, WSX-1.sup.-/- animals,
like IL-12p40.sup.-/- mice, succumbed by day 15 (FIG. 1B). Similar
results were observed whether mice were infected orally or
intraperitoneally.
[0373] Since early mortality of mice deficient in IL-12 is
associated with an inability to control parasite numbers, infected
WSX-1.sup.-/- mice were examined for signs of parasite replication.
At 7 days postinfection, peritoneal lavage was performed, cells
were collected for cytospin preparation (n=3 per group), and the
percentage of cells infected with T. gondii estimated. In contrast
to the high parasite burdens found in infected IL-12p40.sup.-/-
mice, analysis of peritoneal exudates in wild type and
WSX-1.sup.-/- mice revealed few infected cells and no obvious
parasite replication was present in the heart, lungs, spleen, or
liver of infected wild type or WSX-1.sup.-/- animals (FIG. 1C; data
not shown). Wild type and WSX-1.sup.-/- animals were infected for
12 days before livers were removed and prepared for histological
analysis; in contrast to wild type mice, WSX-1.sup.-/- mice
developed prominent immune infiltrates and necrosis in the liver
and lungs after 12 days of infection (FIGS. 1E-1H; data not shown).
Pathology in the liver was further characterized by areas of
extramedullary haematopoesis and a loss of hepatocytes leading to
the development of telangiectasia (FIG. 1H). Moreover, the spleens
of infected WSX-1.sup.-/- mice contained disorganized follicular
structures and increased numbers of apoptotic cells.
[0374] Because previous work has associated CD4.sup.+ T cells with
the development of lethal immune pathology in experimental models
of toxoplasmosis, studies were performed to determine if T cells
mediated the acute mortality of infected WSX-1.sup.-/- mice.
WSX-1-deficient mice were challenged with T. gondii, treated with
antibodies to deplete CD4.sup.+ or CD8.sup.+ T cells, and the
course of infection was monitored. On days 7, 8, and 9
postinfection, mice were treated with PBS (Ctl.), 500 .mu.pg of
.alpha.CD4 mAB, or 500 .mu.g .alpha.CD8 mAb (n=3 per group,
representative of three experiments). Although administration of
.alpha.CD8 on days 5, 6, and 7 postinfection did not alter the time
to death of infected WSX-1.sup.-/- mice, the same regime using
.alpha.CD4 prevented early mortality (FIG. 1D). Together, these
studies demonstrate that, unlike IL-12p40.sup.-/- mice, the
enhanced susceptibility of WSX-1.sup.-/- mice to T. gondii is not
due to an inability to control parasite replication, but rather, is
a consequence of a CD4.sup.+ T cell-dependent immune-mediated
pathology.
Example 2
Increased Cytokine Production in WSX-1.sup.-/- Mice Infected with
T. gondii
[0375] To determine how the absence of WSX-1 affected the immune
response to T. gondii, a kinetic analysis was performed to monitor
the production of cytokines associated with resistance to this
infection. At 0, 7, and 11 days postinfection (X axes), serum was
collected from wild type and WSX-1.sup.-/- mice and ELISA used to
measure circulating levels of IL-12p40 (A) or IFN-.gamma. (D). At
the indicated time points (X-axes), whole splenocytes from wild
type and WSX-1.sup.-/- mice were cultured with soluble Toxoplasma
antigen (sTAg, 25 .mu.g/ml) or plate bound .alpha.CD3 antibody (1
.mu.g/ml) for 72 hr and assayed for IL-12p40 (B and C), IFN-.gamma.
(E and F), IL-10 (G and H) and IL-2 (I) production (n=3 mice per
group, representative of three separate experiments). Wild type,
WSX-1.sup.-/-, and IL-10.sup.-/- mice were infected with T. gondii
and survival was monitored. Infection of wild type and
WSX-1.sup.-/- mice led to highly elevated serum IL-12
concentrations that were downregulated by day 11 post-infection
(FIG. 2A). A similar profile for IL-12 production was obtained by
stimulating whole splenocytes from infected mice with soluble
Toxoplasma antigen (sTAg) or .alpha.CD3 (FIGS. 2B and 2C).
Likewise, analysis of TNF-.alpha. and IL-23 levels in the serum
revealed no significant differences between wild type and
WSX-1.sup.-/- mice. This acute inflammatory response led to a
marked increase in systemic IFN-.gamma. levels that, after 7 days,
was comparable between wild type and WSX-1.sup.-/- mice.
[0376] However, by day 11 postinfection, wild type mice had
down-regulated serum levels of IFN-.gamma., whereas WSX-1.sup.-/-
animals still had high concentrations of circulating IFN-.gamma.
(FIG. 2D). A similar trend was observed in splenic recall responses
from infected mice. Again, at day 0 and day 7 postinfection,
splenocytes from both groups produced similar amounts of
IFN-.gamma. when stimulated with sTAg or .alpha.CD3 (FIGS. 2E and
2F). At day 11 postinfection, stimulation with sTAg or .alpha.CD3
induced WSX-1.sup.-/- splenocytes to produce remarkable levels of
IFN-.gamma. when compared to wild type cohorts (FIGS. 2E and 2F).
In addition, WSX-1.sup.-/- splenocytes also produced 4 times as
much IL-2 as wild type T cells in these cultures (FIG. 2I).
[0377] The phenotype of infected WSX-1.sup.-/- mice, particularly
the overproduction of IFN-.gamma. associated with lethal immune
pathology, is similar to that of IL-10.sup.-/- mice challenged with
T. gondii (FIG. 2J). However, as splenocytes from infected wild
type and WSX-1.sup.-/- mice produced similar amounts of IL-10 when
stimulated with sTAg or .alpha.CD3 (FIGS. 2G and 2H), a defect in
this regulatory system is unlikely to contribute to the acute
mortality of WSX-1.sup.-/- mice. Moreover, while WSX-1.sup.-/- mice
are able to downregulate acute, infection-induced production of
IL-12, IL-10.sup.-/- mice maintained high levels of this cytokine
(FIG. 2K). This distinction between the WSX-1.sup.-/- and
IL-10.sup.-/- mice shows that the enhanced IFN-.gamma. response
noted in infected WSX-1.sup.-/- mice was not due to the failure of
IL-10 to suppress infection induced IL-12 production (FIG. 2L).
Example 3
Enhanced T Cell Responses in T. gondii-Infected WSX-1.sup.-/-
Mice
[0378] Since CD4.sup.+ T cells are involved in the susceptibility
of WSX-1.sup.-/- mice to acute toxoplasmosis and splenocytes from
infected WSX-1-deficient mice secreted elevated levels of
IFN-.gamma., single cell analysis was utilized to assess
IFN-.gamma. production by T cells. Splenocytes from wild type and
WSX-1.sup.-/- mice infected for 0, 7, and 10 days were isolated and
stimulated with plate bound .alpha.CD3 antibody for 18 hr before
staining for CD4 and intracellular IFN-.gamma.. CD4.sup.+ T cells
from uninfected wild type and WSX-1.sup.-/- mice produced little
IFN-.gamma. after 18 hr of stimulation with .alpha.CD3 and at 7
days postinfection, a similar percentage of cells produced
IFN-.gamma. in both wild type and WSX-1.sup.-/- animals (FIG. 3A).
However, by 11 days postinfection 2-fold more WSX-1.sup.-/-
CD4.sup.+ T cells were producing IFN-.gamma. when compared to wild
type cohorts (85% versus 42%) (FIG. 3A) . Moreover, CD4.sup.+ T
cells from 10 day infected WSX-1.sup.-/- mice produced more
cytokine per cell than wild type cells (FIG. 3A). While wild type
splenocytes required ex vivo stimulation to produce IFN-.gamma.,
12.0% of CD4.sup.+ T cells from WSX-1.sup.-/- mice stained positive
for IFN-.gamma. after 12 hr of culture in media alone (FIG. 3B).
Splenocytes were isolated from wild type and WSX-1.sup.-/- mice
that were infected with T. gondii for 11 days. Cells were either
rested in media for 12 hr or stimulated with .alpha.CD3 antibody
for 72 hr, before staining for CD4 and intracellular IFN-.gamma..
For flow cytometry, only CD4.sup.+ events are displayed and
rectangular gates indicate specific IFN-.gamma. staining compared
to control mAb; the percentage of IFN-.gamma..sup.+ cells are
oriented horizontally while mean fluorescence intensity (MFI)
values are oriented vertically. WSX-1.sup.-/- Th1 cells were also
able to maintain IFN-.gamma. production for longer than their wild
type counterparts. When stimulated with .alpha.CD3 for 72 hr, 25%
of CD4.sup.+ T cells from 14 day infected WSX-1.sup.-/- mice were
still secreting IFN-.gamma., while few wild type IFN-.gamma.
producers remained (FIG. 3B).
[0379] To further assess how the development of the T cell response
was affected by the absence of WSX-1, receptor-deficient mice were
infected with T. gondii and the expression of various activation
markers by T cells was determined. Wild type and WSX-1.sup.-/- mice
were infected for 0, 7, and 10 days before splenocytes were
isolated and stained for expression of CD4, CD25, and CD62L
directly ex vivo. Numbers in the figures represent the percentage
of CD4.sup.+ cells in each indicated quadrant with the percentage
of CD25high/CD62Llow in bold type. In accord with the production of
IFN-.gamma. shown in FIGS. 2 and 3, by day 7 postinfection, a
comparable rise in the number of activated T cells
(CD25high/CD62Llow) was observed in wild type and WSX-1.sup.-/-
mice (FIG. 4A). While the number of activated T cells in wild type
mice was decreased after 10 days of infection, consistent with
decreased production of IFN-.gamma. and a general downregulation of
the anti-Toxoplasma response, the frequency of CD25high/CD62Llow
CD4.sup.+ T cells in WSX-1.sup.-/- mice increased further (FIG.
4A). A similar profile for the production of IFN-.gamma. and
expression of activation markers was observed for CD8.sup.+ T cells
from infected WSX-1.sup.-/- mice.
[0380] Although there were elevated numbers of activated T cells in
WSX-1.sup.-/- mice infected with T. gondii, the basis of this
accumulation remained unclear. Since previous reports have shown
that WSX-1.sup.-/- CD4.sup.+ T cells have enhanced proliferative
responses in vitro, studies were performed to evaluate in vivo
proliferation of WSX-1.sup.-/- T cells. Wild type and WSX-1.sup.-/-
mice were infected orally with T. gondii and treated with BrdU for
3 days prior to sacrifice at days 0, 10, and 14 postinfection, and,
at different times postinfection, the incorporation of this
synthetic nucleotide was determined in CD4.sup.+ T cells. Wild type
and WSX-1.sup.-/- mice were infected for 0, 7, and 10 days before
splenocytes were isolated and stained for expression of CD4, CD25,
and CD62L directly ex vivo. Numbers represent the percentage of
CD4.sup.+ cells in each indicated quadrant with the percentage of
CD25high/CD62Llow in bold type. Again, as with IFN-.gamma.
production and activation marker expression, the amount of BrdU
incorporated was comparable between wild type and WSX-1.sup.-/-
CD4.sup.+ lymphocytes at days 0 and 10 postinfection (FIG. 4B) .
After 2 weeks, suppression of the wild type immune response was
reflected in decreased numbers of CD4.sup.+ T cells that had
incorporated BrdU. In contrast, at this later time point, the
population of WSX-1.sup.-/- CD4.sup.+ T cells that had incorporated
BrdU continued to expand (FIG. 4B). This enhanced proliferative
response, a phenomenon previously reported in vitro, is likely to
contribute to the accumulation of pathogenic CD4.sup.+ T cells
occurring during acute T. gondii infection of WSX-1.sup.-/- mice.
Together, these studies indicate that while WSX-1 is not necessary
for the generation of highly activated Th1 effector T cells
following challenge with T. gondii, this receptor is required to
regulate the intensity and duration of infection induced Th1
responses.
Example 4
WSX-1.sup.-/- T Cells Exhibit Intrinsic Hyperactivity after
Infection with T. gondii
[0381] Although infection with T. gondii led to an expanded
population of activated Th1 cells in WSX-1.sup.-/- mice, it was
unknown whether this enhanced persistence was cell autonomous or
mediated through altered accessory cell function. To address this
issue, splenocytes were isolated from uninfected mice, depleted of
adherent cells, and 75 x 10 wild type or WSX-1.sup.-/- cells were
adoptively transferred into RAG-2.sup.-/- mice. Seven days later,
mice were infected with T. gondii and at 11 days postinfection,
whole splenocytes from wild type and WSX-1.sup.-/- mice were
cultured with soluble Toxoplasma antigen or plate bound .alpha.CD3
antibody for 72 hr and assayed for IFN-.gamma. production by ELISA
(ng/ml). At day 11 postinfection, splenocytes were isolated and
stained for expression of CD4, CD25, and CD62L directly ex vivo.
Numbers represent the percentage of CD4.sup.+ cells in each
indicated quadrant with the percentage of CD25high/CD62Llow in bold
type. Analysis of T cell responses in these reconstituted mice
revealed that, as in infected WSX-1.sup.-/- mice, adoptively
transferred WSX-1.sup.-/- T cells produced elevated levels of
IFN-.gamma. during recall responses (FIG. 5A). Furthermore,
WSX-1.sup.-/- CD4.sup.+ T cells were 3 times more likely to produce
IFN-.gamma. (67% versus 20%) and had increased expression of
activation markers when compared to wild type cohorts (FIGS. 5B and
5C). These data indicate that the hyperactivity that follows
infection is intrinsic to the T cells and not due to defects in the
accessory cell compartment of WSX-1.sup.-/- mice.
Example 5
WSX-1 is Not Required for in Vitro Th1 Differentiation
[0382] In contrast to the data presented above, previous reports
suggested that IL-27/WSX-1 is required for optimal Th1
differentiation. Therefore, in vitro studies were performed to
further assess the impact of WSX-1 deficiency on CD4.sup.+ T cell
responses. Naive CD4.sup.+ T cells were purified from uninfected
wild type or WSX-1.sup.-/- spleens, stained with CFSE, and
activated with soluble .alpha.CD3 (0.1 .mu.g/ml) and .alpha.CD28
(0.5 .mu.g/ml) under either (A) nonpolarizing conditions
(.alpha.-IL-12 and .alpha.-IL-4) or (B) Th1 polarizing conditions
(rIL-12 plus .alpha.-IL-4). First, after 72 hr of culture,
supernatants were collected and secreted levels of IFN-.gamma.
determined by ELISA. Then, remaining cells from the same cultures
were stimulated with PMA and ionomycin for 4 hr before performing
intracellular staining for IFN-.gamma. in combination with CFSE and
CD4. Based on CFSE profiles, the number of cells in each individual
generation was calculated and data are presented in each table for
wild type and WSX-1.sup.-/- T cells under neutral (A) and Th1 (B)
polarizing conditions. RT-PCR analysis revealed increased
expression of IL-27p28 and EBI3 mRNA in all cultures, indicating
the likely presence of IL-27 in these studies. When naive, wild
type CD4.sup.+ T cells were activated under nonpolarizing
conditions (.alpha.IL-4, .alpha.IL-12), a small percentage of wild
type cells became competent to produce IFN-.gamma. and a low
concentration of protein was detected in the supernatants (FIG.
6A). In parallel cultures, a reduced percentage of WSX1.sup.-/-
CD4.sup.+ T cells stained positive for IFN-.gamma. while almost no
IFN-.gamma. was secreted (FIG. 6A). By using CFSE labeling to track
cell divisions, a small but reproducible increase in the ability of
WSX-1.sup.-/-CD4.sup.+ T cells to proliferate was noted (FIG. 6A).
Thus, in accord with previous studies, IL-27/WSX-1 is crucial for
the optimal production of IFN-.gamma. by naive CD4.sup.+ T cells
that have been activated under nonpolar conditions. However, a role
for IL-27/WSX-1 in the regulation of other effector functions was
demonstrateed by a small but reproducible increase in the ability
of WSX-1.sup.-/- CD4.sup.+ T cells to proliferate (FIG. 6A).
[0383] When naive wild type and WSX-1.sup.-/- CD4.sup.+ T cells
were differentiated under Th1 polarizing (.alpha.IL-12,
.alpha.IL-4), there was no significant difference in the percentage
of wild type and WSX-1.sup.-/- cells that produced IFN-.gamma.
(FIG. 6B). However, analysis of supernatants from the same cultures
revealed that WSX1.sup.-/- CD4.sup.+ T cells secreted 2 to 3 times
more IFN-.gamma. than wild type cohorts (FIG. 6B).
[0384] Moreover, a marked increase in proliferation was also noted
in CD4.sup.+ T cells from the WSX-1.sup.-/- cultures (FIG. 6B).
These data indicate that, when activated under Th1 conditions, a
similar frequency of IFN-.gamma. wild type and WSX-1.sup.-/- T
cells arise, but due to enhanced proliferation, more total
WSX-1-deficient IFN-.gamma. cells accumulate thereby leading to a
significant increase in the secreted protein concentrations. Thus,
in the presence of strongly polarizing IL-12 concentrations in
vitro or during in vivo infection with T. gondii, the ability of
IL-27/WSX-1 to augment IFN-.gamma. production becomes redundant.
Furthermore, these data demonstrate that, while IL-27/WSX-1 plays
an important role in the regulation of several effector functions,
like proliferation and cytokine production, WSX-1-deficient
CD4.sup.+ T cells do not have an intrinsic defect in Th1
differentiation.
Example 6
IL-27 Signaling Leads to Heterogeneous STAT Activation
[0385] The data presented above demonstrate that both wild type and
WSX-1.sup.-/- mice develop a vigorous, protective Th1 type response
following infection with T. gondii, but while wild type mice can
downregulate this response, WSX-1.sup.-/- mice are unable to do so.
Furthermore, the failure to downregulate CD4.sup.+ T cell responses
contributes to the infection-induced mortality in WSX-1.sup.-/-
mice. While these studies provide a cellular mechanism for the
severe immune pathology observed in WSX-1.sup.-/- mice (FIG. 1H),
they are inconsistent with previous studies that showed that
recombinant IL-27 could enhance CD4.sup.+ T cell IFN-.gamma.
responses. Therefore, to explore the basis for the stimulatory and
inhibitory effects of IL-27/WSX-1 on T cell function, naive
CD45RBHi CD4.sup.+ T cells were treated with rIL-27 in vitro and
the signaling pathways activated by this cytokine were examined.
Naive CD4.sup.+CD45RB.sup.hi T cells were sorted from wild type
spleens, rested overnight, and then stimulated with r IFN-.gamma.,
IFN-.gamma., or rIL-27 (all 50 ng/ml) for 15 min. Cells were then
lysed and total or tyrosine phosphorylated STAT-1, STAT-3, and
STAT-5 were detected by Western blot. Based on structural homology
of WSX-1 with other class I cytokine receptors, it was likely that
IL-27 would activate the Jak/STAT signaling pathway. Cells were
stimulated with various cytokines and the ability to phosphorylate
STATs 1, 3, and 5 was assessed. Stimulation of naive CD4.sup.+ T
cells with IL-2 or IL-12 failed to activate STAT1, STAT3, or STATS
, while exogenous IFN-.alpha. and IFN-.gamma. resulted in
activation of STAT1 and STAT3 but not STATS.
[0386] In contrast, stimulation with IL-27 led to increased
tyrosine phosphorylation of STAT1, STAT3, and STAT5 (FIG. 7). These
data are in accord with recent reports that WSX-1 signaling leads
to STAT-1 phosphorylation (Hibbert et al., 2003; Takeda et al.,
2003), but the finding that IL-27 can also activate STAT3 and STAT5
in naive CD4.sup.+ T cells extends our knowledge of the signaling
pathways used by IL-27/WSX-1.
Example 7
Infection with T. muris Leads to Increased IL-27 mRNA
Expression
[0387] Previous studies demonstrated that macrophage and DC
lineages increase expression of IL-27 mRNA following LPS
stimulation. In addition, upregulation of IL-27/WSX-1 mRNA has been
reported in vivo following infection with the protozoan pathogen T.
gondii. However, little was known about expression of IL-27/WSX-1
following exposure to Th2-inducing stimuli such as infection with
helminth parasites. Wild type C57B/6 mice were infected orally with
T. muris, and after 0 (i.e. uninfected), 7, and 14 days
post-infection, mRNA was isolated from MLN and RT-PCR performed to
quantify expression of IL-27p28, EBI3, WSX-1 and .beta.-actin. Wild
type and WSX-1.sup.-/- mice were infected with T. muris for 14 days
and the intestinal larval worm burden determined by microscopy.
Wild type and WSX-1.sup.-/- mice were infected with T. muris for 14
days, MLN cells isolated and stimulated with T. muris antigen, 50
g/ml, for 48 hours. Concentrations of secreted IL-4 and IL-5 were
determined by ELISA. Oral challenge of wild type C57BL/6 mice with
T. muris resulted in the generation of a protective Th2 response
which leads to worm expulsion between day 18-21 post-infection. The
use of RT-PCR to analyze levels of mRNA for WSX-1, EBI3 and
IL-27p28 mRNA in the draining MLN of C57BL/6 mice on days 0, 7, and
14 post-infection revealed that while levels of mRNA for WSX-1 and
EBI3 was constitutive, there was a marked increase in the levels of
IL-27p28 mRNA following T. muris infection. Together, these data
demonstrate that in response to infection with T. muris there is
the production of IL-27 that may regulate the immune response to
this parasite.
Example 8
Infection of WSX-1.sup.-/- Mice Leads to Accelerated Worm Expulsion
Mediated by Enhanced Th2 Responses
[0388] To assess the role of the IL-27/WSX-1 interaction in
immunity to T. muris, wild type C57BL/6 and WSX-1.sup.-/- mice were
inoculated with infective eggs and the worm burden measured at day
21 post-infection. At this time point, wild type C57BL/6 mice had
expelled worms and WSX-1.sup.-/- mice displayed a similar
phenotype. Although these data demonstrated that WSX-1.sup.-/- mice
can develop protective immunity to T. muris, it was unclear if the
absence of WSX-1 affected the kinetics of this response. Therefore,
wild type and WSX-1.sup.-/- mice were challenged with T. muris and
worm burden assessed at day 14 post-infection. At this time point,
infected wild type mice harbored high numbers of T. muris,
indicating that protective Th2 responses had not yet been
established. In contrast, at this early time point, infected
WSX-1.sup.-/- mice had expelled larval parasites.
[0389] To determine if enhanced resistance to T. muris in
WSX-1.sup.-/- mice was associated with increased Th2 responses, MLN
cells were isolated from wild type and WSX-1.sup.-/- mice at day 14
post-infection and parasite-specific cytokine responses analyzed.
MLN cells from infected WSX-1.sup.-/- mice secreted significantly
higher levels of IL-4 and IL-5 compared to cells from infected wild
type mice. To confirm that the enhanced resistance to T. muris
observed in WSX-1.sup.-/- mice was due to a type 2 effector
cytokine response, .alpha.IL-4 mAb was administered to infected
WSX-1.sup.-/- mice. After 14 days of infection, treatment with
.alpha.IL-4 led to the equivalent establishment of infection in
wild type and WSX-1.sup.-/- mice. These data indicate that in
WSX-1.sup.-/- mice, elevated Th2 responses mediate enhanced
resistance to T. muris.
Example 9
Enhanced Goblet Cell and Mast Cell Responses in the Absence of
WSX-1
[0390] We have established that the development of protective Th2
type responses required for resistance to T. muris are associated
with a goblet cell hyperplasia and mastocytosis. To better
understand the basis for the enhanced resistance of WSX-1.sup.-/-
mice to T. muris several approaches were used to compare intestinal
goblet cell and mast cell responses in wild type and WSX-1.sup.-/-
mice. Analysis of histological sections of gut tissue mice stained
to visualize goblet cells mucin revealed that in uninfected wild
type and WSX-1.sup.-/- mice, similar numbers of goblet cells were
observed. However at day 14 post-infection, while wild type mice
displayed no signs of infection-induced goblet cell hyperplasia,
WSX-1.sup.-/- mice demonstrated a dramatic increase in intestinal
goblet cell hyperplasia and mucin production.
[0391] To further characterize these goblet cell responses, western
blots and immunohistochemistry were used to examine the expression
of RELM.beta., a goblet cell. specific protein that is stimulated
during Th2 responses. These studies revealed that while uninfected
wild type mice expressed negligible levels of RELMP uninfected
WSX1.sup.-/- mice expressed higher levels of this protein.
Furthermore, following 14 days of infection wild type mice
expressed low levels of this protein and WSX-1.sup.-/- mice
displayed a marked elevation in the levels of RELM.beta.. Together,
these studies demonstrate that WSX-1.sup.-/- mice display enhanced
goblet cell responses following infection with T. muris and these
data are consistent with the elevated Th2 responses and enhanced
resistance to this parasite in these mice.
[0392] Analsysis of histological sections stained to visualize
intestinal mast cells revealed that uninfected wild type and
WSX-1.sup.-/- mice had similar numbers of mast cells. After 14 days
of infection, mid-cecum sections were stained for detection of
intestinal goblet cells and the number of goblet cells per 100
crypt units enumerated by microscopy. At 14 days post-infection in
wild type mice these numbers were not significantly increased
whereas at this time point, WSX-1.sup.-/- mice displayed a marked
increase in mast cell numbers. This infection-induced mastocytosis
was accompanied by elevated serum levels of mast cell protease that
was not observed in wild type mice at the same time point. These
data establish that WSX-1.sup.-/- mice infected with T. muris
develop a strong mast cell response that correlates with the
establishment of a parasite-specific Th2 response and enhanced
resistance to this parasite. While it seemed likely that the
mastocytosis observed in infected WSX-1.sup.-/- mice would be a
consequence of the enhanced Th2 responses, a comparison of the
levels of WSX-1 on mast cell and splenic CD4.sup.+ T cells revealed
that mast cells expressed high levels of WSX-1 and these results
indicated that WSX-1 may have a direct role in the regulation of
mast cell function.
[0393] To more directly assess the role of WSX-1 in the regulation
of mast cell response in vivo, wild type and WSX-1.sup.-/- mice
were used to assay mast cell responses in an IgE-mediated mast cell
dependent model of passive cutaneous anaphylaxis. In these studies,
wild type and WSX-1.sup.-/- mice were primed with intradermal
injections of anti-DNP-IgE and challenged 24 hours later with
DNP-BSA in a solution of Evans blue. Rechallenge leads to
anaphylaxis and the rapid release of mast cell derived mediators
that results in profound changes in vascular permeability and the
extravasation of Evans blue provides a surrogate marker of plasma
exudation. In these assays, rechallenge led to the extravasation of
Evans blue in wild type mice but these levels were significantly
increased in the absence of WSX-1: wild type, n=11; WSX-1.sup.-/-,
n=10, p=0.0036 Student T test. Taken together these studies
demonstrate that WSX-1 can act as a negative regulator of mast cell
responses.
Example 10
Enhanced Th2 Responses and Rapid Expulsion of T. muris in
WSX-1.sup.-/- Mice are Independent of a Defect in IFN-.gamma.
Production
[0394] Together, the studies discussed above demonstrate that
WSX-1.sup.-/- mice develop enhanced resistance to T. muris, but
these findings do not address the mechanism that underlies the
elevated Th2 responses. Since IL-27/WSX-1 can promote the
production of IFN-.gamma., it is possible that a primary defect in
Th1 responses in WSX-1.sup.-/- mice would lead to unopposed Th2
responses in infected mice. This hypothesis is supported by studies
demonstrating that IL-12 and IFN-.gamma. can impair the development
of protective Th2 cytokine responses following chronic infection
with T. muris.
[0395] To address whether a defect in the production of IFN-.gamma.
contributed to the enhanced Th2 responses in infected WSX-1.sup.-/-
mice, the ability of MLN cells from wild type or WSX-1.sup.-/- mice
infected for 14 days to produce IFN-.gamma. was assessed. MLN cells
from wild type and WSX-1.sup.-/- mice infected for 14 days were
isolated and stimulated with .alpha.CD3/.alpha.CD28 for 48 hours as
described above. Cells were stained for intracellular IFN-.gamma.
in combination with surface CD4. In these experiments, stimulation
with T. muris Ag resulted in the production of negligible levels of
IFN-.gamma.. However, stimulation of MLN cells from these animals
with .alpha.CD3 revealed that there was a higher frequency of
WSX-1.sup.-/- CD4.sup.+ T cells producing IFN-.gamma. when compared
to wild type controls. These findings demonstrate that there was no
early defect in the production of IFN-.gamma. in WSX-1.sup.-/- mice
infected with T. muris and are consistent with recent reports that
identified WSX-1-independent IFN-.gamma. responses in vivo.
Nevertheless, to address whether a defect in IFN-.gamma. production
would result in accelerated type 2 immunity, studies were performed
to assess how the absence of a Th1 response would affect cytokine
production and worm expulsion in wild type mice infected with T.
muris.
[0396] Wild type mice were treated with .alpha.IL-12 plus
.alpha.IFN-.gamma. prior to and during infection and response
monitored at day 14 post-infection. Wild type mice were challenged
with T. muris and treated with neutralizing .alpha.IL-12 and
.alpha.IFN-.gamma. mAb, 2 mg/dose, on days 0, 4, 8 and 12
post-infection. After 14 days of infection, MLN cells were isolated
from untreated wild type, untreated WSX-1.sup.-/-, and mAb treated
wild type mice. For all groups, mean intestinal larval worm burden
was determined by microscopy, and are representative of three
independent experiments. Cells were stimulated with T. muris Ag and
the concentrations of secreted IL-5 and IL-13 quantified by ELISA.
In these studies, the administration of .alpha.IL-12 and
.alpha.IFN-.gamma. resulted in a small increase in the production
of IL-5 and IL-13, but these levels were markedly lower than those
observed in WSX-1.sup.-/- mice. Moreover, in contrast to
WSX-1.sup.-/- mice, treatment with .alpha.IL-12 plus
.alpha.IFN-.gamma. did not result in goblet cell hyperplasia,
enhanced expression of RELMP, or rapid expulsion of T. muris.
Similar results were obtained when IFN-.gamma..sup.-/- mice were
challenged with T. muris. Together, these studies demonstrate that
the enhanced type 2 responses observed in the WSX-1.sup.-/- mice
are not reproduced following in vivo blockade of Th1 responses in
wild type mice and demonstrate that the enhanced Th2 responses
observed in WSX-1.sup.-/- mice infected with T. muris are
independent of a defect in IFN-.gamma. production.
Example 11
Inhibition of Th2 Responses by IL-27/WSX-1 In Vitro
[0397] Although the data presented above demonstrate an enhanced
Th2 response in WSX-1.sup.-/- mice infected with T. muris, it was
unclear whether this was due to a direct effect on T cell function.
To address this issue, studies were performed to determine how the
absence of WSX-1 or the addition of IL-27 affected the development
of CD4.sup.+ T cell responses under Th2 polarizing conditions, in
which endogenous Th1 responses are blocked.
[0398] Naive wild type and WSX-1.sup.-/- splenocytes were
stimulated with .alpha.CD3 plus .alpha.CD28 under Th2 polarizing
conditions, .alpha.IL-12, rIL-4, and CD4.sup.+ T cell proliferation
and cytokine production assayed. Polyclonal stimulation was
associated with increased levels of IL-27 mRNA in these cultures
and sustained expression of mRNA for WSX-1 in wild type cells,
suggesting the presence of functional IL-27/WSX-1 signaling in
vitro. For primary stimulations, wild type and WSX-1.sup.-/-
splenocytes were isolated from uninfected mice and activated with
soluble .alpha.CD3/.alpha.CD28 under Th2 polarizing conditions.
After 72 hours, cells were stained for intracellular IL-4 in
combination with surface CD4. After three days of primary
stimulation, intracellular staining for IL-4 revealed that there
were equivalent frequencies of wild type and WSX-1.sup.-/-
CD4.sup.+ T cells that were IL-4.sup.+.
[0399] For secondary stimulations, wild type or WSX-1.sup.-/-
splenocytes were activated as above for 96 h, washed, counted, and
restimulated with plate bound .alpha.CD3, 1 g/ml, under neutral
conditions. After 24 hours, secreted concentrations of IL-5 and
IL-13 were determined by ELISA. For proliferation assays, naive
wild type and WSX-1.sup.-/- splenocytes were labeled with CFSE
before undergoing primary stimulation as before. Upon secondary
stimulation of these cells, although negligible levels of IL-4
protein were detected, there was a significant increase in the
production of IL-5 and IL-13 in WSX-1.sup.-/- compared to wild type
cultures. After 3 or 4 days in culture, CD4.sup.+ T cell
proliferation was visualized by flow cytometry. The number of cells
in proliferative generations 1 through 6 are enumerated for primary
stimulations of 3 or 4 days. Results are representative of four
independent experiments. Analysis of the proliferative response
during primary stimulation revealed that there were increased
numbers of WSX-1.sup.-/- CD4.sup.+ T cells present in the 1st to
the 4th proliferative generations on day 3 after stimulation. By
day 4 post-stimulation, the enhanced proliferative responses of
WSX-1.sup.-/- CD4.sup.+ T cells over wild type controls were even
more apparent, with a marked increase in the percentage of
WSX-1.sup.-/- T cells observed in the 5th and 6th generations.
Furthermore, the CD4.sup.+ T cell responder frequency, an indicator
of the percentage of cells that have responded to antigenic
stimulation by undergoing at least one proliferative cycle, was
higher in WSX-1.sup.-/- compared to wild type cultures: wild type,
34%; WSX-1.sup.-/-, 46%. Consistent with the enhanced Th2 responses
observed in the absence of WSX-1, when recombinant IL-27 was added
to cultures of wild type CD4+ T cells, a marked reduction in the
levels of IL-4 being produced was observed. However, in these
experiments these effects were not associated with a reduction on
the levels of proliferation. Taken together, these in vitro studies
indicate that the IL-27/WSX-1 interaction is a negative regulator
of Th2 effector cell function and is the basis for the enhanced Th2
responses observed in WSX-1.sup.-/- mice following infection with
T. muris.
[0400] FIGS. 9-13 are graphs which depict a Flow Cytometry analysis
of whole blood from wild type and WSX-1 knockout mice stimulated
with conditioned medium from either mock-transfected or
IL-27-transfected 293 cells. The cells are stained for
intracellular phospho-STAT1 as a measure of IL-27 signaling and a
surface maker to identify the cell lineage of the responding cells.
The data show that, in whole blood, two major cell types respond;
CD4 T cells and a cell type that is currently characterized by its
scatter properties only, but appears to be of non-lymphoid nature:
mast cells or possibly basophils. FIG. 14 shows the OD read at 610
nm, a measure of vascular permeability. This data shows that these
two major cell types not only express IL-27 receptor, but that
IL-27R is functionally competent on both cell types.
[0401] The invention being thus described, it will be obvious that
the same may be modified or varied in many ways. Such modifications
and variations are not to be regarded as a departure from the
spirit and scope of the invention and all such modifications and
variations are intended to be included within the scope of the
following claims.
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