U.S. patent application number 16/240630 was filed with the patent office on 2019-08-29 for therapeutic peptides and methods for treating autoimmune related disease.
This patent application is currently assigned to OP-T LLC. The applicant listed for this patent is OP-T LLC. Invention is credited to Charles W. Henry, David Hal Wagner, JR., Martin Glenn Yussman.
Application Number | 20190263888 16/240630 |
Document ID | / |
Family ID | 67685588 |
Filed Date | 2019-08-29 |
![](/patent/app/20190263888/US20190263888A1-20190829-D00000.png)
![](/patent/app/20190263888/US20190263888A1-20190829-D00001.png)
![](/patent/app/20190263888/US20190263888A1-20190829-D00002.png)
![](/patent/app/20190263888/US20190263888A1-20190829-D00003.png)
![](/patent/app/20190263888/US20190263888A1-20190829-D00004.png)
![](/patent/app/20190263888/US20190263888A1-20190829-D00005.png)
![](/patent/app/20190263888/US20190263888A1-20190829-D00006.png)
![](/patent/app/20190263888/US20190263888A1-20190829-D00007.png)
![](/patent/app/20190263888/US20190263888A1-20190829-D00008.png)
![](/patent/app/20190263888/US20190263888A1-20190829-D00009.png)
![](/patent/app/20190263888/US20190263888A1-20190829-D00010.png)
View All Diagrams
United States Patent
Application |
20190263888 |
Kind Code |
A1 |
Wagner, JR.; David Hal ; et
al. |
August 29, 2019 |
THERAPEUTIC PEPTIDES AND METHODS FOR TREATING AUTOIMMUNE RELATED
DISEASE
Abstract
Methods and materials for preventing and modulating
atherosclerosis. In particular, small peptides that are capable of
interacting with CD40, thereby interfering with the ability of CD40
to interact with CD154, which impacts inflammation and
atherosclerosis. The use of such peptides in reducing
atherosclerosis, and in particular, the autoimmune inflammatory
response that may be a driving factor thereof. The use of such
short peptides to lower LDL cholesterol. Methods and materials for
detecting T-cells that express CD40 (Th40 cells). Methods and
materials for preventing, modulating, reducing and/or reversing
type 2 diabetes and auto-inflammatory disease. In particular, small
peptides that are capable of interacting with CD40, thereby
interfering with the ability of CD40 to interact with CD154, which
impacts inflammation and type 2 diabetes. The use of such peptides
in reducing type 2 diabetes, and in particular, the autoimmune
inflammatory response that may be a driving factor thereof. The use
of such short peptides to lower IL2, INF.gamma., and IL17a. The use
of such peptides to increase glucose transport protein (GLUT4).
Methods and materials for detecting T-cells that express CD40 (Th40
cells).
Inventors: |
Wagner, JR.; David Hal;
(Denver, CO) ; Yussman; Martin Glenn; (Denver,
CO) ; Henry; Charles W.; (Denver, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OP-T LLC |
Denver |
CO |
US |
|
|
Assignee: |
OP-T LLC
Denver
CO
|
Family ID: |
67685588 |
Appl. No.: |
16/240630 |
Filed: |
January 4, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16184129 |
Nov 8, 2018 |
|
|
|
16240630 |
|
|
|
|
15423822 |
Feb 3, 2017 |
|
|
|
16184129 |
|
|
|
|
13880387 |
May 23, 2013 |
9562088 |
|
|
PCT/US2011/056860 |
Oct 19, 2011 |
|
|
|
15423822 |
|
|
|
|
15423822 |
Feb 3, 2017 |
|
|
|
16184129 |
|
|
|
|
13880387 |
May 23, 2013 |
9562088 |
|
|
PCT/US11/56860 |
Oct 19, 2011 |
|
|
|
15423822 |
|
|
|
|
62614262 |
Jan 5, 2018 |
|
|
|
61394699 |
Oct 19, 2010 |
|
|
|
61394699 |
Oct 19, 2010 |
|
|
|
62584595 |
Nov 10, 2017 |
|
|
|
62669918 |
May 10, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/08 20130101;
A61K 38/10 20130101; A61K 38/1774 20130101; C07K 14/70575
20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. (canceled)
42. (canceled)
43. (canceled)
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. A method for one or more of preventing, modulating, reducing,
treating, or reversing type 2 diabetes in a subject, comprising
administering to a subject having type 2 diabetes; a
therapeutically effective amount of a peptide that affects the
interaction of CD40 with CD154/CD40-ligand.
53. The method of claim 52, the peptide binding to CD40.
54. The method of claim 52, said peptide binding to a CD40 protein
with a Kd of greater than 10.sup.-6.
55. The method of claim 52, said peptide affecting the interaction
of CD40 and CD154.
56. The method of claim 52, said peptide inhibiting the binding of
CD40 to CD154.
57. The method of claim 52, said peptide binding CD40 at the site
where CD40 interacts with CD154.
58. The method of claim 52, said peptide affecting the interaction
of CD40 with CD154 to prevent the expansion of Th40 cells.
59. The method of claim 52, said peptide affecting the interaction
of CD40 with CD154 reduce the number of Th40 cells.
60. The method of claim 52, said peptide affecting the interaction
of CD40 with CD154 to alter the cytokine expression profile of a
cell population treated with said peptide.
61. The method of claim 52, said peptide affecting an amino acid
sequence selected from the group of SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID
NO:27.
62. (canceled)
63. The method of claim 52, said peptide being an amino acid
sequence selected from the group of SEQ ID NO:4-9 and 24-30.
64. (canceled)
65. (canceled)
66. The method of claim 52, the peptide interacting with the CD40
protein to modulate glucose transport protein 4 (GLUT 4).
67. (canceled)
68. (canceled)
69. The method of claim 52, said peptide binding to multiple bone
marrow derived cell types which express CD40 of approximately 45
kDa, including splenic CD4hi cells, CD8 cells, and other antigen
presenting cells.
70. (canceled)
71. A method of modulating type 2 diabetes in a patient comprising
administering to a patient a peptide that binds to a CD40 protein
at the CD154-binding site and modulates type 2 diabetes.
72. (canceled)
73. (canceled)
74. The method of claim 71, further comprising, administering to
the subject, a peptide that interacts with a CD40 protein to
modulate IFN.gamma..
75. The method of claim 71, the peptide being selected from SEQ ID
NOs 3-9 and 27-30.
76. The method of claim 71, further comprising administering a
peptide selected from SEQ ID NOs 3-9 and 27-30, in an amount
sufficient to reduce or inhibit interleukin-2 signaling.
77. The method of claim 71, further comprising administering a
peptide selected from SEQ ID NOs 3-9 and 27-30, in an amount
sufficient to reduce or inhibit interleukin 17 (IL-17)
signaling.
78. (canceled)
79. (canceled)
80. (canceled)
81. A method to one or more of prevent, modulate, or reduce type 2
diabetes, the method comprising administering to a subject in need
thereof, a therapeutically effective amount of a peptide which
specifically binds to a CD40 presenting cells at the CD154 binding
site.
82. (canceled)
83. (canceled)
84. (canceled)
85. (canceled)
86. (canceled)
87. (canceled)
88. (canceled)
89. (canceled)
90. The method of claim 81, said peptide comprising an amino acid
sequence selected from the group of SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.
91. (canceled)
92. (canceled)
93. (canceled)
94. (canceled)
95. (canceled)
96. (canceled)
97. (canceled)
98. (canceled)
99. (canceled)
100. (canceled)
101. (canceled)
102. (canceled)
103. (canceled)
104. (canceled)
105. (canceled)
106. (canceled)
107. (canceled)
108. (canceled)
109. (canceled)
110. (canceled)
111. (canceled)
112. (canceled)
113. (canceled)
114. (canceled)
Description
REFERENCE TO SEQUENCE LISTING
[0001] This application contains a Sequence Listing submitted as an
electronic text file entitled "US_16/240,630_Sequence_Listing.txt"
having a size in bytes of 11 kb and created May 1, 2019. The
information contained in this electronic file is hereby
incorporated by reference in its entirety pursuant to 37 CFR
1.52(e)(5).
[0002] The present disclosure relates to methods or uses for
prevention, modulation, and reduction of cardiovascular disease
and/or atherosclerosis in a subject in need of a therapeutically
effective amount of a peptide that inhibits the interaction of CD40
and CD154, and the use of such compounds in modulating T-cell
activity and in treating disease. Furthermore, the present
disclosure relates to methods of preventing, modulating, reducing,
treating and/or reversing of type 2 diabetes mellitus and/or
auto-inflammatory disease, via administration of a therapeutically
effective amount of a CD40-binding peptide that inhibits,
influences, disrupts, blocks, and/or changes the interaction of
CD40 and CD154 are disclosed.
BACKGROUND
[0003] Autoimmune diseases are conditions arising from an abnormal
immune response to a normal body part. More than 80 diseases occur
because of the immune system attacking the body's own organs,
tissues, and cells. Type 1 diabetes, rheumatoid arthritis, systemic
lupus erythematosus, and inflammatory bowel disease are common
autoimmune diseases that affect a wide range of people across
entire populations. Significantly, the autoimmune disorders
mentioned above afflict substantial number of people affecting
their daily lives and routines and require significant monetary and
healthcare resources, time and care from healthcare providers.
[0004] According to the World Health Organization (WHO), an
estimated 17.7 million people died from cardiovascular diseases
(CVDs) in 2015, which represented 31% of all global deaths. CVDs,
may also be known as heart and blood vessel disease, and includes
numerous problems, many of which are related to a process called
atherosclerosis. Moreover, according to the Healthcare Cost and
Utilization Project (HCUP) which is sponsored by the Agency for
Healthcare Research and Quality (AHRQ), in 2011, coronary
atherosclerosis alone accounted for more than $10.4 billion in
hospital costs. Accordingly, in 2011, coronary atherosclerosis
alone was one of the ten most expensive conditions for inpatient
hospitalizations in the United States.
[0005] Cardiovascular Disease and Atherosclerosis
[0006] Atherosclerosis is defined by arterial plaque formation that
may lead to heart attack and stroke. Arterial plaque formation is
caused by the deposition of cells, substances, waste products, and
cellular debris including, but not limited to: cholesterol, dead
cells, dendritic cells, foam cells, macrophages, mast cells,
monocytes, smooth muscle cells, T-cells, collagen, calcium, and
fibrin. Inflammatory changes within the arterial wall and plaque
may play a crucial and causative role in atherosclerotic disease
development. Consequently, the concept of atherosclerosis as an
autoimmune and inflammatory disease has been investigated; however,
a therapeutic control has not been established. The importance of
controlling inflammation is highlighted by current clinical trials
targeting other aspects of autoimmune, inflammation, and
cardiovascular disease and death.
[0007] For example, CIRT (Cardiovascular Inflammation Reduction
Trial) is attempting to use methotrexate to target interleukin-6
(IL-6) to test whether methotrexate will reduce rates of myocardial
infarction, stroke, and cardiovascular death among patients with
coronary artery disease patients with type 2 diabetes. Another
example includes, CANTOS (Canakinumab Anti-inflammatory Thrombosis
Outcomes Study) which is studying whether canakinumab can block the
pro-inflammatory cytokine interleukin-1.beta. (IL-1.beta.) to
reduce rates of recurrent myocardial infarction, stroke, and
cardiovascular death rates in heart attack patients who remain at a
high risk. This risk is demarcated by elevated levels of the
inflammatory biomarker high sensitivity C-reactive protein (hsCRP).
These studies acknowledge that inflammation plays a critical role
in atherothrombosis and atherosclerosis; however, these studies
also recognize that is unknown whether inhibition of inflammation
per se will lower vascular event rates.
[0008] Mammalian and human atherosclerotic lesions are
characterized as a chronic inflammatory-fibroproliferative disease
of the blood vessel wall containing monocytes, macrophages,
endothelial cells, smooth muscle cells, platelets, and T-cells.
Each of these cell types can express either or both of the
CD40/CD154 costimulatory pair. This dyad is responsible for
enhancing the immune response and may contribute to many chronic
inflammatory diseases including rheumatoid arthritis, multiple
sclerosis, and type 1 diabetes (T1D). However, no viable therapy
exists for this highly atherogenic dyad.
[0009] Inflammation may occur when inflammatory cells, such as
neutrophils, eosinophils, basophils, mast cells, macrophages,
platelets, and endothelial cells, respond to inflammatory events or
harmful stimuli, such as, invading microorganisms, damages cells,
or other irritants. The body's inflammatory response is beneficial
because for example, in the case of invading microorganisms, the
inflammatory response is an important step in localizing the
infecting agent for removal by the immune system. However, in
autoimmunity there is no infection, yet severe inflammation is
present or persistent. The inflammation in this case, referred to
as aseptic chronic inflammation (ACI), is detrimental since it
destroys normal tissues. The results of this aseptic inflammation
are life-altering and in some cases life-threatening. Moreover, as
with acute inflammation, this process is mediated by immune cells,
including T-cells.
[0010] A major concern for modern medicine is how to control ACI
such as that which occurs during autoimmune diseases, as well as
how to control acute inflammation resulting from trauma.
Inflammation, both chronic and acute, leads to tissue degeneration
and eventual loss of function of major organs. ACI is not limited
to a single disease, but is instrumental in numerous autoimmune
diseases, including, but not limited to: type 1 diabetes (T1D),
multiple sclerosis (MS), systemic lupus erythematosus (SLE),
rheumatoid arthritis (RA), Crohn's disease, inflammatory bowel
disease (IBS), chronic obstructive pulmonary disease (COPD)
including types of autoimmune asthma, atherosclerosis, vasculitis,
hypertension, thyroiditis including Hashimoto's and Graves
diseases, primary biliary cirrhosis, Paget's disease, Addison's
disease, acute respiratory distress syndrome (ARDS), acute lung
injury, and aseptic chronic inflammation (ACI) associated with
organ transplantation.
[0011] Autoimmune disorders are classified into two types:
organ-specific (directed mainly at one organ) and
non-organ-specific (widely spread throughout the body). Examples of
organ-specific autoimmune disorders are insulin-dependent Type 1
diabetes (T1D) which affects the pancreas; Hashimoto's thyroiditis
and Graves' disease, which affect the thyroid gland; pernicious
anemia, which affects the blood; Addison's disease, which affects
the adrenal glands; chronic active hepatitis, which affects the
liver; myasthenia gravis which affects the muscle; and multiple
sclerosis (MS), which affects tissue of the nervous system. An
example of a non-organ-specific autoimmune disorders is rheumatoid
arthritis (RA). Autoimmune diseases are often chronic,
debilitating, and life-threatening. The National Institutes of
Health (NIH) estimates that up to 23.5 million Americans suffer
from autoimmune disease and that the prevalence is rising. It has
been estimated that autoimmune diseases are among the ten leading
causes of death among women in all age groups up to 65 years.
[0012] Acute inflammation, as observed during trauma or sepsis, is
also immune cell mediated. While a comprehensive, complete, and
exhaustive list of the molecular mediators in this process have not
yet been identified, a prominent role for T-cells, lymphocytes,
neutrophils, macrophages, monocytes, neutrophils, eosinophils,
basophils, mast cells, and other inflammatory cells is strongly
implicated. Therefore, a process to modulate these cell types may
control the inflammatory response.
[0013] Type 2 Diabetes
[0014] The CD40-CD154 dyad constitutes a major inflammatory pathway
(Schonbeck U, et al. Cell Mol. Life Sci. (2001) 58(1):4-43) that
plays a significant role in type 1 diabetes (Waid D M. et al. Clin.
Immunol. (2007) 124(2):138-148). Type 2 diabetes (T2D) has
historically and scientifically been primarily categorized as a
metabolic disorder; however, type 2 diabetes is in the process of
being redefined as an autoimmune disease rather than just a
metabolic disorder (Winer, D, et al. Nature Medicine (2011)
17:610-617).
[0015] According to the United States Centers for Disease Control
National Diabetes Statistics Report for 2017 (available at
https://www.cdc.gov/diabetes/pdfs/data/statistics/national-diabetes-stati-
stics-report.pdf), an estimated 30.3 million people of all ages--or
9.4% of the U.S. population had diabetes in 2015. An estimated 23
million people--or 7.2% of the U.S. population had been diagnosed
with diabetes mellitus, and about 95% of those diagnosed with
diabetes have type 2 diabetes. Based on fasting plasma glucose
levels, one third to one half of cases of type 2 diabetes are
undiagnosed and untreated (Harris M I, Diabetes Care. (1998) 21
[Suppl. 3: C11-C14]; Howard B V, et al., Circulation. 2002;
105:e132-e137; Grundy S M, et al., Circulation. 1999; 100:
1134-1146).
[0016] Although type 2 diabetes most often develops in people over
the age of 45, type 2 diabetes increased 21% in American youth from
2001 to 2009 and a large study called SEARCH for Diabetes in Youth
found that newly diagnosed cases of Type 2 diabetes in children and
teens increased by about 4.8 percent in each year of the study's
period between 2002 and 2012 ("Rates of new diagnosed cases of type
1 and type 2 diabetes on the rise among children, teens" National
Institutes of Health, Apr. 13, 2017, available at
https://www.nih.gov/news-events/news-releases/rates-new-diagnosed-cases-t-
ype-1-type-2-diabetes-rise-among-children-teens).
[0017] Major comorbidities complicate diabetes, the most common
being cardiovascular disease (70.4 per 1,000 persons) including
those with ischemic heart disease and stroke. Overall, for the year
2012, the American Diabetes Association estimates that the total
direct and indirect estimated cost in the United States was $245
billion, including $176 billion in direct medical costs and $69
billion in reduced productivity (Yang, W. American Diabetes
Association, 2013, Diabetes Care, 36 (4): 1033-46).
[0018] Both type 1 and type 2 diabetes are powerful and independent
risk factors for coronary artery disease (CAD), stroke, and
peripheral arterial disease (Schwartz C J, et al. Diabetes Care.
(1992) 15:1156-1167; Stamler, J. et al. Diabetes Care. (1993)
16:434-444; Beckman J A, et al., Diabetes and atherosclerosis:
epidemiology, pathophysiology, and management. JAMA (2002)
287:2570-2581). Atherothrombosis accounts for 65% to 80% of all
deaths among North American patients with diabetes, compared with
about 33% of all deaths in the general North American population
(American Diabetes Association. Diabetes Care. (1993) 16:72-78).
Therefore, a therapeutic regimen that is effective and can be
tolerated for long periods of time would be beneficial to
individuals and from a public health perspective. An ideal
anti-diabetic agent may be an agent which corrects hyperglycemia,
prevents macrovascular complications, and corrects the
pathophysiological disturbances responsible for Type 2 Diabetes
("T2D"). Insulin resistance is basic to T2D, but .beta.-cell
failure eventually occurs with imbalance between insulin resistance
and insulin secretion being a further complication. Therefore,
therapeutically beneficial treatment approaches may aim to reverse
insulin resistance and improve .beta.-cell function.
[0019] T2D often conincides with obesity however genetic and
environmental factors recently have been described as disease
contributors (Comuzzie A G, Best Pract. Res. Clin. Endocrinol.
Metab. (2002) 16(4):611-21. PubMed PMID: 12468410; van Tilberg J.,
et al. J. Med. Genet. (2001) 38(9):569-78. Pub Med PMID: 11546824;
PMCID: PMC 1734947). Moreover, additional research has emerged that
indicates that T2D, like T1D, has prominent inflammation component
that is a contributing and/or driving factor of the T2D disease
commencement, development and progression. The CD40-CD154
inflammatory dyad may act as a molecular driver to propel
auto-immune inflammation and influence excessive levels of the dyad
may be an unappreciated but contributing factor to T2D. (Hseih C J,
et al., Cir. J. (2009) 73(5) 948-54; Kutlu M, et al., Clin. Invest.
Med. (2009) 32(6): E244; Santilli F, et al., J. Am. Coll. Cardiol.
(2006) 47(2):391-7; Santini, E, et al. J. Endocrinol Invest. 2008;
31(7):660-5; Varo N, et al., Circulation. 2003;
107(21):2664-9).
[0020] Generally, inflammation may occur when inflammatory cells,
such as neutrophils, eosinophils, basophils, mast cells,
macrophages, platelets, endothelial cells, and lymphocytes,
including but not limited to T cells and B cells respond to
inflammatory events or harmful stimuli, such as, invading
microorganisms, damaged cells, or other irritants. The body's
inflammatory response is beneficial because, for example, in the
case of invading microorganisms, the inflammatory response is an
important step in localizing the infecting agent for removal by the
immune system. However, in autoimmunity there is no infection, yet
severe inflammation is present or persistent. The inflammation in
this case, referred to as aseptic chronic inflammation (ACI), is
detrimental since it destroys normal tissues. The results of this
aseptic inflammation are life-altering and in some cases
life-threatening. Moreover, as with acute inflammation, this
process is mediated by immune cells, including T-cells.
[0021] A major concern for modern medicine is how to control ACI
such as that which occurs during autoimmune diseases, as well as
how to control acute inflammation resulting from trauma.
Inflammation, both chronic and acute, leads to tissue degeneration
and eventual loss of function of major organs. ACI is not limited
to a single disease, but is instrumental in numerous autoimmune
diseases, including, but not limited to: type 1 diabetes (T1D),
multiple sclerosis, systemic lupus erythematosus, rheumatoid
arthritis, Crohn's disease, inflammatory bowel disease, chronic
obstructive pulmonary disease including types of autoimmune asthma,
atherosclerosis, vasculitis, hypertension, thyroiditis including
Hashimoto's and Graves diseases, primary biliary cirrhosis, Paget's
disease, Addison's disease, acute respiratory distress syndrome,
acute lung injury, and ACI associated with organ
transplantation.
[0022] Autoimmune disorders are classified into two types:
organ-specific (directed mainly at one organ) and
non-organ-specific (widely spread throughout the body). Examples of
organ-specific autoimmune disorders are insulin-dependent Type 1
diabetes (T1D) which affects the pancreas; Hashimoto's thyroiditis
and Graves' disease, which affect the thyroid gland; pernicious
anemia, which affects the blood; Addison's disease, which affects
the adrenal glands; chronic active hepatitis, which affects the
liver; myasthenia gravis which affects the receptors at the
junction between nerves and muscles; and multiple sclerosis, which
affects tissue of the nervous system. An example of a
non-organ-specific autoimmune disorders is rheumatoid arthritis.
Autoimmune diseases are often chronic, debilitating, and
life-threatening. The National Institutes of Health (NIH) estimates
that up to 23.5 million Americans suffer from autoimmune disease
and that the prevalence is rising. It has been estimated that
autoimmune diseases are among the ten leading causes of death among
women in all age groups up to 65 years.
[0023] Acute inflammation, as observed during trauma or sepsis, is
also immune cell mediated. While a comprehensive, complete, and
exhaustive list of the molecular mediators in this process have not
yet been identified, a prominent role for T-cells, lymphocytes,
neutrophils, macrophages, monocytes, neutrophils, eosinophils,
basophils, mast cells, and other inflammatory cells is strongly
implicated. Therefore, a process to modulate these cell types may
control the inflammatory response.
[0024] A unique T cell subset has been shown to be instrumental in
the development of autoimmune disease. These cells are
phenotypically characterized as CD4loCD40+ (Waid, D. M., et al.,
Eur. J. of Immunol., 34:1488, 2004; Vaitaitis, G. M., et al.,
Cutting Edge, J. Immunol., 170:3455, 2003; Wagner, D. H., Jr., et
al., Proc. Nat'l. Acad. Sci. USA, 99:3782, 2002; Wagner, D. H.,
Jr., et al., Int'l J. of Mol. Med. 4:231, 1999), and are referred
to as Th40 cells. (Waid, D. M., et al. (2004) Eur. J. of Immunol.
34:1488; Vaitaitis, G. M., et al., Cutting Edge, J. Immunol.
170:3455, 2003; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci.
USA 99:3782, 2002; Wagner, D. H., Jr., et al., Int'l J. of Mol.
Med. 4:231, 1999). CD40 expression typically is associated with
antigen presenting cells and the majority of prior art describes
CD40 as being expressed on B cells, macrophages, monocytes, and
other cells; however, CD40 proteins are also expressed on T cells
(Waid, D. M., et al., 2004. Eur. J. of Immunol., 34:1488, 2004;
Vaitaitis, G. M., et al., Cutting Edge, J. Immunol., 170:3455,
2003; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA,
99:3782, 2002; Wagner, D. H., et al., Int'l. J. of Mol. Med.,
4:231, 1999; Bourgeois, C., et al., Science, 297:2060, 2002;
Fanslow, W. C., et al., J. of Immun., 152:4262, 1994; Ramsdell, F.,
et al., J. of Immunol. 152:2190, 1994; Grabstein, K. H., et al., J.
of Immunol., 150:3141, 1993; Armitage, R. J., et al., Sem. in
Immun., 5:401, 1993; Cooper, C. J., et al., J of Immunol.,
173:6532, 2004). While Th40 cells comprise a proportion of the
peripheral CD4+ compartment in naive, non-autoimmune mice (Waid, D.
M., et al., Eur. J. of Immunol., 34:1488, 2004; Wagner, D. H., Jr.,
et al., Int'l J. of Mol. Med., 4:231, 1999), and in humans (Waid.
D. M., et al., Clin. Immunol. 124:138, 2007), this proportion is
drastically expanded to as much as 50% of the CD4+ compartment in
autoimmune prone mice (Waid, D. M., et al., Eur. J. of Immunol.
34:1488, 2004; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci.
USA 99:3782, 2002; Wagner, D. H., et al., Int'l J. of Mol. Med.,
4:231, 1999) and humans (Waid, D. M., et al., Eur. J. of Immunol.
34:1488, 2004; Waid. D. M., et al., Clin. Immunol. 124:138, 2007;
Waid. D. M., et al., Clin. Immunol. 124:138, 2007). These T cells
do not express early activation markers and occur in the naive
phenotype of non-challenged mice.
[0025] In NOD (non-obese diabetic) mice, Th40 cells occur at
exaggerated levels in spleen, lymph nodes and the pancreas, even
prior to diabetes onset (Waid, D. M., et al., Eur. J. of Immunol.
34:1488, 2004; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci.
USA 99:3782, 2002). An elevated number and percentage of these T
cells are seen in peripheral blood of type 1 diabetic (T1D)
patients when compared to non-autoimmune controls and type 2
diabetic patients (Waid. D. M., et al., Clin. Immunol., 124:138,
2007).
[0026] The observed increase in Th40 cells could mean that those T
cells are antigen responsive or that CD40 expression is activation
induced. Furthermore, several diabetogenic T cell clones are CD40+
(Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782,
2002). Purified primary Th40 cells from NOD mice and from
pre-diabetic NOD (12-weeks of age) mice successfully transfer type
1 diabetes to NOD/scid (Non-Obese Diabetic/Severe Combined
Immunodeficiency) recipient mice, directly demonstrating
pathogenicity of the Th40 T cell subset (Waid, D. M., et al., Eur.
J. of Immunol. 34:1488, 2004; Wagner, D. H., Jr., et al., 2002.
Proc. Nat'l Acad. Sci. USA, 99:3782, 2002). It has been shown that
Th40 cells infiltrate islet beta cells destroying insulin
production thus suggesting islet antigen specificity (Waid, D. M.,
et al., Eur. J. of Immunol. 34:1488, 2004; Wagner, D. H., Jr., et
al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002). It has also been
shown that Th40 cells are required for diabetes transfer.
Peripheral (spleen and regional lymph node) T cells that were CD40
depleted, then CD25, Treg, depleted were not capable of
transferring diabetes to Scid (Severe Combined Immunodeficiency)
recipients. Even though Treg cells were removed, if the
auto-aggressive CD40+ T cells subset is absent, disease transfer
does not occur.
[0027] While Th40 cells are important in the development of
autoimmunity, another important factor is expression of the
CD40-Ligand, CD154. CD154 is temporally induced on activated
T-cells in response to CD3/TCR stimulation (Lederman, S. et al., J.
of Exp. Med., 175:1091, 1992). CD154 expression has also been
demonstrated on platelets, monocytes, basophils, eosinophils,
dendritic cells, fibroblasts, smooth muscle, and endothelial cells
(Russo, S. et al., J. Immunol. 171:5489, 2003; Stumpf, C., et al.,
Eur. J. Heart Fail., 5:629, 2003; Schonbeck, U., et al., Cell Mol.
Life Sci. 58:4, 2001). CD154 is a member of the tumor necrosis
factor (TNF) super-family and a soluble form of CD154 (sCD154) has
been described (Russo, S., et al., J. Immunol. 171:5489 2003;
Stumpf, C., et al., Eur. J. Heart Fail 5:629, 2003; Toubi, E., et
al., Autoimmunity 37:457, 2004). Therefore, sCD154 may act like a
cytokine (Stumpf, C., et al., Eur. J. Heart Fail. 5:629, 2003).
Even though CD154 has not been genetically linked in T1D studies,
sCD154 is significantly elevated in T1D and may play a role in the
disease process (Varo, N. et al., Circulation 107:2664, 2003;
Cipollone, F., et al., Diabetologia 48:1216, 2005; Devaraj, S., et
al., Diabetes 55:774, 2006). The importance of CD40-CD154
interaction in autoimmunity has been established (Wagner, D. H.,
Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002; Kobata, T.,
et al., Rev. Immunogenet. 2:74, 2000; Homann, D., et al., Immunity
16:403, 2002; Goodnow, C. C., et al., Lancet 357:2115, 2001;
Balasa, B., et al., J. of Immunol. 159:4620, 1997). Blocking
CD40-CD154 interaction may prevent collagen induced arthritis,
(Durie, F. H., et al., Science 281:1328, 1993) experimental
autoimmune encephalitis (Howard, L. M., et al., Autoimmunity
37:411, 2004), prostatitis (Grossman, M. E., et al., J. Immunother.
24:237, 2001), and type-1 diabetes in the NOD mouse model (Durie,
F. H. et al., Science 281:1328, 1993; Balasa, B. et al., Journal of
Immunology 159:4620, 1997; Howard, L. M., et al., Autoimmunity
37:411, 2004; Grossman, M. E. et al., J. Immunother. 24:237, 2001).
In the diabetes model, it was essential to administer a CD154
blocking antibody to NOD mice at 3-weeks of age because at 9-weeks,
blocking antibodies had no effect on diabetes prevention (Balasa,
B. et al., J. of Immunol. 159:4620, 1997).
[0028] Previous work has also demonstrated that the Th40 cell
subset induces RAG1 and RAG2 (Recombination-Activating Genes)
transcription, translation and nuclear translocation (Vaitaitis, G.
M., et al., Cutting Edge, J. Immunol. 170:3455, 2003) when CD40 is
engaged (Vaitaitis, G. M. et al., Cutting Edge, J. Immunol.
170:3455, 2003). CD3 engagement does not induce RAG1 or RAG2 in
T-cells (Vaitaitis, G. M., et al., Cutting Edge, J. Immunol.
170:3455, 2003). Subsequent to RAG1/RAG2 induction, CD40-mediated
T-cell receptor (TCR) revision occurs in peripheral T cells
(Vaitaitis, G. M. et al., Cutting Edge, J. Immunol. 170:3455,
2003). CD40 induction of TCR revision is RAG dependent. T cells
isolated from a TCR-Tg mouse undergo TCR revision when CD40
engaged, but T-cells from the TCR-Tg.RAG-/- mouse do not TCR revise
when CD40 engaged (Wagner, D. H., Jr. et al., Int'l J. of Mol. Med.
4:231, 1999).
[0029] CD40 is a 50-kDa integral membrane protein of the tumor
necrosis factor receptor (TNF-R) family. It is constitutively
expressed as a homotrimer (Foy T M, et al., Ann. Rev. of Immunol.,
14:591, 1996). In general, stimulation of all CD40-expressing cell
types induces operations which contribute to inflammation, such as
enhancement of costimulatory and adhesion molecules, and
up-regulation of proteolytic enzymes (Mach, F. et al.,
Atherosclerosis. 137 Suppl:S89-95, 1998).
[0030] CD40's ligand--CD154--is a 39-kDa protein that belongs to
the tumor necrosis factor (TNF) family. CD40 forms a trimer that
binds CD154 at the interface of the three monomers. CD154 is
expressed commonly on cells beyond the surface-expressed CD154, as
CD154 may also exist in a soluble biologically active form (sCD154)
that is shed from the cell surface after activation. The main
source of sCD154 is platelets. (Foy T M, et al., Ann. Rev. of
Immunol., 14:591, 1996).
[0031] Genetically manipulated mouse models are utilized for
research and development concerning atherosclerosis and
cardiovascular disease because wild type mice are generally highly
resistant to development and progression of atherosclerosis. Prior
studies have attempted to block the CD40/CD154 interaction by using
monoclonal antibodies and this approach has proven efficacious in
several mouse model studies utilizing the Apoe-/- or LDLr deficient
atherosclerotic models. Additionally, these same mouse models built
with a deletion of CD154 saw significant reductions in overall
plaque formation and may have also contributed to production of a
more stable plaque phenotype. Clinically, stable plaques are
identifiable and denoted by increased collagen and smooth muscle
content, a thick fibrous cap, and an observable decrease in T cell,
macrophage, and lipid accumulation.
[0032] Genetically manipulated mouse models are utilized for
research and development concerning T2D because mouse models can
portray insulin resistance and the inability of the beta cell to
sufficiently compensate, which are characteristic of T2D in humans.
Many animal models, including mouse models for T2D are obese,
reflecting the human condition where obesity is closely linked to
T2D development.
[0033] Multiple treatment options have been put forward to address
and control both chronic and acute inflammation. Many approaches
use non-steroidal anti-inflammatory drugs (NSAIDS) that attack the
production of leukotrienes and prostaglandins, cellular products
that cause localized inflammation. Other approaches use more
powerful immunosuppressant drugs such as cyclophosphamide,
methotrexate and azathioprine that suppress the immune response and
stop the progression of the disease. Still other treatments involve
the use of monoclonal antibodies (mAb) designed to alter the immune
responses to self-tissues, as occurs during autoimmune diseases.
However, all of these treatments often have severe, long-term side
effects.
[0034] Current immune-modulatory therapies may rely upon monoclonal
antibody treatments that may give rise to complications. For
example, antibodies administered to a subject may cross-react with
unintended targets and cause severe nephritic complications and
those that specifically act against CD154 may cause embolic
complications. Further, the CD40-CD154 interaction may play an
important role in antibody generation which may indicate that
administration of a monoclonal antibody could induce auto-antibody
generation and further complications, which may inhibit the
restoration of normal immune function (see generally Banchereau, J.
et al., Annu. Rev. of Immunol. 12:881, 1994).
[0035] Other studies have demonstrated that blocking the CD154
interaction by using monoclonal antibodies, or limiting the CD40
receptor by monoclonal antibodies may abrogate atherosclerosis, and
may confer a more favorable plaque phenotype characterized by lower
inflammation and higher fibrosis. These studies additionally
demonstrated that neointimal formation and restenosis may be
limited by blocking the CD154 interaction. Studies concerning lupus
nephritis may have demonstrated that blocking CD40 mediated signals
can reduce anti-double-stranded DNA (anti-dsDNA) antibodies.
Moreover, these studies may demonstrate that the reduction of
anti-dsDNA was associated with increased serum complement levels
and reduced glomerular inflammation, which may be viewed positively
from a clinical perspective. However, the use of monoclonal
antibodies to target the CD40/CD154 dyad was abandoned due to
thromboembolic events which may have been related to the
functioning of CD154 in thrombus stabilization. It is postulated
that CD154 stabilize thrombi by interaction with the integrin
.alpha..sub.IIb.beta..sub.3, and by inhibiting CD154, thrombi may
be less stable, and as a consequence shed emboli causing thrombotic
events.
[0036] Studies of small molecules have also been conducted to
attempt to inhibit the important CD40-CD154 costimulatory
interaction required for T cell activation and the development of
an effective immune response. For example, suramin, a symmetric
polysulfonated napthylamine-benzamide urea derivative was studied
for its ability to inhibit CD154 binding to its receptor and
prevented the CD154-induced proliferation of human B cells;
however, its numerous reversible toxicities (lethargy, rash,
fatigue, anemia, hyperglycemia, hypocalcemia, coagulapathies,
neutropenia, renal and hepatic complications) (Kaur, M. et al.
(2002) Invest. New Drugs 20(2):209-19), loss of activity in
protein-rich medium, and its interference with positive
costimulatory interaction (Margolles-Clark, E. et al. (2009)
Biochem. Pharmacol. 77(7):1236-45) made this and other related
small-molecule candidates unlikely sources for effective therapy
for the CD40-CD154 dyad.
[0037] Uncertainty exists regarding the primary lesion and the
relative importance of the different tissues, metabolic defects in
the liver and the peripheral tissues such as fat, muscle, and
pancreatic .beta.-cells likely all contribute to type 2 diabetes.
Furthermore, although the cause of T2D is incompletely understood,
complex and confounded by both genetic and environmental
influences, hyperglycemia itself is believed to hinder pancreatic
beta-cell function (Cernea, S., et al. Biochem. Med. (Zagreb) 2013;
23(3):266-80).
[0038] Accordingly, type 2 diabetes in humans typically develops
through a progressive series of increasingly disruptive phases or
stages. Initially, pre-diabetes is characterized by impaired
glucose tolerance, wherein the body has difficulty clearing glucose
after a meal (postprandial hyperglycemia) and/or the body may have
decreased sensitivity to insulin. In a second phase or stage,
postprandial hyperglycemia and basal hyperglycemia occur while
insulin producing beta cells of the pancreas become dysfunctional
at an increasing rate. During the next phase of the disease
progression, hyperglycemia occurs even after fasting and at a
cellular level significant beta cell atrophy takes place.
Ultimately, in the final phase or end stage of disease progression,
beta cells can no longer produce and/or release insulin and the
patient requires insulin replacement therapy.
[0039] T2D is clinically characterized by hyperglycemia and
pathologically by insulin resistance with relative insulin
secretory impairment. Individuals who are genetically prone to
developing T2D may experience insulin resistance (the earliest
detectable metabolic defect) between 15 and 25 years or more before
the clinical onset of overt diabetes. (Kahn, C R, Diabetes. (1994)
43:1066-1084). T2D classically has been associated with age and
obesity; however, the increased diagnosis of youth with T2D has
demonstrated that these two factors alone are not the sole reliable
predictors of the disease. Body mass index (BMI) is also clearly
associated with T2D, but both genetic and environmental factors are
now identified as contributory as well (Wu Y, et al. Int. J. Med.
Sci. (2014) 11(11):1185-200. Epub 2014/09/06; Cefalu, W T,
Diabetes. (2009) 58(2):307-8; Donath, M Y, et al., Nat. Rev.
Immunol. (2011) 11(2):98-107). Obesity creates inflammatory
conditions, and sustained inflammation resulting from obesity or
other conditions may be important in T2D development (Donath, M Y,
et al., Nat. Rev. Immunol. (2011) 11(2):98-107; Donath, M Y, Nat.
Rev. Drug Discov. (2014) 13(6):465-76).
[0040] Thus, there exists a need in the art for safer and more
effective methods for treatment and prevention of T2D implicated by
the autoimmune and inflammatory pathway and dyad related to
CD40-CD154. The present developments may address this need by
describing methods for treatment of T2D by administration of a
therapeutically effective amount of CD40-binding peptide.
[0041] Thus, there exists a need in the art for safer and more
effective methods for treatment and prevention of cardiovascular
diseases (CVDs) and T2D implicated by aseptic chronic inflammation
(ACI). The present developments may address this need by describing
methods for treatment of atherosclerosis by administration of a
therapeutically effective amount of a peptide that affects,
regulates, blocks, inhibits, or modulates the CD40-CD154 dyad.
Further, the present developments may provide the added benefit of
preventing auto-antibody generation, and thus allow the resumption
of normal immune function.
[0042] This statement of background is for information purposes
only and is not intended to be a complete or exhaustive explication
of all potentially relevant background.
SUMMARY
[0043] The present developments may provide novel methods for
preventing, modulating, and/or reducing atherosclerosis that arises
in a corporeal body. Atherosclerosis may arise as a result of
chronic inflammatory response of white blood cells in the walls of
arteries. It is postulated that the chronic inflammatory response
and the subsequent buildups of plaque in arteries may be caused by
elevated levels of cholesterol and triglycerides in the blood, high
blood pressure, and cigarette smoking.
[0044] The present developments are based on the knowledge that
interaction of CD40-ligand (CD154 protein) with CD40 protein
expressed on T-cells (Th40 cells), may be important in the
development of atherosclerosis and autoimmune disease. The present
developments may be based on the elucidation of the critical
residues in CD40 and CD154 that may be important for this
interaction. The present developments relate to blocking the
interaction between a CD40 protein and a CD154 protein through the
use of small peptides that interact with the CD40 protein at a site
where the CD154 protein would normally bind. The present
developments also relate to using such peptides to reduce the level
of Th40 cells, thereby reducing the severity of disease.
[0045] One embodiment of the present developments is a method for
preventing atherosclerosis comprising contacting the CD40 protein
with a peptide that interacts with the CD40 protein. Preferred
peptides may be those that are less than 25 amino acids in length,
and that bind to a CD40 protein, thereby inhibiting its interaction
with a CD154 protein; however, the length of the peptide should not
be considered a limitation on the developments herein as there are
numerous other factors that may affect the ability of the peptide
to perform its intended and desired result.
[0046] One embodiment of the present developments is a method for
preventing, modulating, and/or reducing atherosclerosis, the method
comprising inhibiting interaction between a CD40 protein and a
CD154 protein with a peptide that interacts with the CD40 protein.
Preferred peptides interact with the CD40 protein at the
CD154-binding site. Preferably such peptides are less than 25 amino
acids in length. Even more preferred peptides are those amino acid
sequences selected from SEQ ID NOs 3-9 and 25-30.
[0047] One embodiment of the present developments is a method for
preventing, modulating, and/or reducing atherosclerosis, the method
comprising administering to a subject in need thereof, a
therapeutically effective amount of a peptide that affects the
interaction of CD40 with CD154/CD40-ligand. An aspect of this
development may be that the peptide binds to CD40. In this
embodiment, the peptide may bind to a CD40 protein with a Kd of
greater than 10.sup.-6. Further, in this embodiment, the peptide
may affect the interaction between CD40 and CD154. Additionally, a
preferred embodiment may inhibit the binding of CD40 to CD154.
Moreover, in this embodiment, the peptide binds to CD40 at the site
where CD40 interacts with CD154. In this embodiment, the peptide
affects the interaction of CD40 with CD154 in such a manner as to
prevent the expansion of Th40 cells. In this embodiment, the
peptide affects the interaction of CD40 with CD154 in such a manner
as to reduce the number of Th40 cells. In this embodiment, the
peptide affects the interaction of CD40 with CD154 in such a manner
as to alter the cytokine expression profile of a cell population,
treated with said peptide.
[0048] One embodiment of the present developments is a method to
modulate and/or reduce atherosclerosis in an animal, the method
comprising administering to the animal, a peptide that interacts
with a CD40 protein in such a manner as to modulate IFN.gamma.
(interferon gamma). Preferred peptides are those that interact with
the CD40 protein at the CD154-binding site, thereby modulating
IFN.gamma.. Preferred peptides modulate inflammation by reducing
the level of Th40 cells to no more than 25% of the total T-cell
population. Such methods can be used to prevent and/or reduce
atherosclerosis and symptoms that may accompany cardiovascular
diseases, more generally.
[0049] One embodiment of the present developments is a method to
identify a patient at risk for developing cardiovascular disease
and/or atherosclerosis, the method comprising obtaining a sample
containing T-cells from a patient to be tested, contacting the
sample with a peptide that binds the CD40 protein, detecting the
CD-40 bound peptide, and determining the level of Th40 cells from
the amount of CD40 bound, wherein a level of Th40 cells greater
than 25% of the total T-cell population indicates the patient is at
risk for developing cardiovascular disease and/or developing
atherosclerosis.
[0050] Yet, another embodiment of the present developments is a
method to prevent, modulate, or reduce calcium buildup, or
calcification of vessel walls, the method comprising administering
to the subject in need thereof, a therapeutically effective amount
of a peptide which specifically binds to a CD40 presenting cells at
the CD154 binding site.
[0051] Another embodiment of the present developments, is a method
to administer a CD40-binding peptide to prevent, modulate, and/or
reduce atherosclerosis, comprising selecting a peptide that
interacts with a CD40 protein and CD154 binding site, selecting a
delivery method selected from the group comprising intramuscular
(IM) delivery, intravenous (IV) delivery, subcutaneous (SC)
delivery, oral delivery, gavage delivery, emollient/skin delivery,
or transdermal patch.
[0052] Another embodiment of the present developments, is a method
to administer a CD40-binding peptide to prevent, modulate, and/or
reduce atherosclerosis in an animal, comprising selecting a peptide
that interacts with a CD40 protein and CD154 binding site, using an
extended delivery method selected from the group comprising an
implantable device, a hydrophilic polymer formulation, a permeable
polymeric membrane, injectable gel implants, solvent extraction
system, phase inversion system, thermosensitive gels, pH dependent
in situ gels, microparticles, microspheres, nanoparticles,
nanospheres, bio-degradable implants, or photoactivated depot.
[0053] Another embodiment of the present developments, is a method
to lower LDL cholesterol in a subject, the method comprising
administering to the subject in need thereof, a therapeutically
effective amount of a peptide which specifically binds to a CD40
presenting cells at the CD154 binding site.
[0054] The present developments may provide novel methods for
preventing, modulating, reducing, treating and/or reversing T2D
that arises in a corporeal body. Moreover, the developments
disclosed herein are therapeutic methods which may additionally be
used for the prevention, control, and treatment of diseases,
disorders, and conditions, in particular immune and inflammatory
diseases.
[0055] The present developments are based on the knowledge that
interaction of CD40-ligand (CD154 protein) with CD40 protein
expressed on T-cells (Th40 cells), may be important in the
development of type 2 diabetes and autoimmune diseases. The present
developments may be based on the elucidation of the critical
residues in CD40 and CD154 that may be important for this
interaction. The present developments relate to blocking and/or
disrupting the interaction between a CD40 protein and a CD154
protein through the use of small synthesized peptides that interact
and/or associate with the CD40 protein at a site where the CD154
protein would normally bind. The present developments also relate
to using such peptides to reduce the level of Th40 cells, thereby
reducing the severity of disease. The peptides of the current
developments may bind directly and/or alternatively associate with
the CD40 molecule in such a way as to alter CD40 function.
[0056] In autoimmune diseases and conditions, CD40 engagement may
promote inflammation. Accordingly, in one embodiment of the present
developments the peptide may alter CD40 signals to no longer be
inflammatory. Thus, in one embodiment the peptides of the current
development may block, disrupt, interfere, and/or inhibit CD40
function at sites including but not limited to Th40 cells, pancreas
beta cells, endothelial cells, B cells, monocytes, and/or
macrophages. The current developments contemplate the use of the
small interfering peptides (SIPs) disclosed herein to interfere
with the CD40-signaling pathway of any cells that may present with
CD40. These peptides may be able to interfere at specific sites
depending on the route of administration.
[0057] One embodiment of the present developments is a method for
preventing, modulating, reducing, and/or reversing type 2 diabetes
comprising contacting the CD40 protein with a peptide that
interacts with the CD40 protein. Preferred peptides are those that
are less than 25 amino acids in length, and that bind to a CD40
protein, thereby inhibiting its interaction with a CD154
protein.
[0058] One embodiment of the present developments is a method for
preventing, modulating, reducing and/or reversing type 2 diabetes,
the method comprising inhibiting interaction between a CD40 protein
and a CD154 protein with a peptide that interacts with the CD40
protein. Preferred peptides interact with the CD40 protein at the
CD154-binding site. Preferably such peptides are less than 25 amino
acids in length. Even more preferred peptides are those amino acid
sequences selected from SEQ ID NOs 3-9 and 25-30.
[0059] In one aspect the present development provides a method for
modulating and/or increasing glucose transport protein 4 (GLUT4)
the method comprising administering to a patient a therapeutically
sufficient dose of a peptide selected from SEQ ID NOs 3-9 and
25-30.
[0060] One embodiment of the present developments is a method to
prevent, modulate, reduce and/or reverse type 2 diabetes in an
animal, the method comprising administering to the animal, a
peptide that interacts with a CD40 protein in such a manner as to
modulate glucose transport protein 4 ("GLUT4"). Preferred peptides
are those that interact with the CD40 protein at the CD154-binding
site, thereby modulating GLUT4. Preferred peptides may modulate,
upregulate, or increase GLUT4 in both adipose and muscle tissue
compared to untreated populations. Such methods can be used to
prevent and/or reduce T2D and symptoms that may accompany T2D and
autoimmune related inflammation, more generally.
[0061] Another embodiment of the present developments, is a method
to administer a CD40-binding peptide to prevent, modulate, reduce
and/or reverse type 2 diabetes, comprising selecting a peptide that
interacts with a CD40 protein and CD154 binding site, selecting a
delivery method selected from the group comprising intramuscular
(IM) delivery, intravenous (IV) delivery, subcutaneous (SC)
delivery, oral delivery, gavage delivery, emollient/skin delivery,
transdermal patch, or nasal administration.
[0062] Another embodiment of the present developments, is a method
to administer a CD40-binding peptide to prevent, modulate, reduce
and/or reverse type 2 diabetes in an animal, comprising selecting a
peptide that interacts with a CD40 protein and CD154 binding site,
using an extended delivery method selected from the group
comprising an implantable device, a hydrophilic polymer
formulation, a permeable polymeric membrane, injectable gel
implants, solvent extraction system, phase inversion system,
thermosensitive gels, pH dependent in situ gels, microparticles,
microspheres, nanoparticles, nanospheres, bio-degradable implants,
or photoactivated depot.
[0063] Another embodiment of the present developments, is a method
of modulating, controlling, and/or increasing GLUT4 in a subject,
the method comprising administering to the subject in need thereof,
a therapeutically effective amount of a peptide selected from SEQ
ID NOs 3-9 and 25-30.
[0064] Another embodiment of the present developments, is a method
to modulate, affect and/or reduce interleukin-2 signaling in a cell
or a subject comprising administering a peptide selected from SEQ
ID NOs 3-9 and 25-30, in an amount sufficient to reduce or inhibit
interleukin-2 signaling, wherein, the interleukin-2 signaling is
associated with a condition selected from the group comprising type
I diabetes, multiple sclerosis, systemic lupus erythematosa,
rheumatoid arthritis, Crohn's disease, inflammatory bowel disease,
chronic obstructive pulmonary disease, asthma, atherosclerosis,
vasculitis, hypertension, thyroiditis, primary biliary cirrhosis,
Paget's disease, Addison's disease, acute respiratory distress
syndrome, acute lung injury, and/or aseptic chronic inflammation,
more generally.
[0065] Another embodiment of the present developments, is a method
to modulate and/or reduce interleukin-2 signaling in a cell or a
subject comprising administering a peptide selected from SEQ ID NOs
3-9 and 25-30 in an amount sufficient to reduce or inhibit
interleukin-2 signaling, wherein, the interleukin-2 signaling is
associated with type 2 diabetes.
[0066] Another embodiment of the present developments is a method
to modulate and/or reduce IFN-.gamma. in a cell or a subject
comprising administering a peptide selected from SEQ ID NOs 3-9 and
25-30, in an amount sufficient to reduce or inhibit IFN-.gamma.
signaling, wherein, the IFN-.gamma. signaling is associated with
type 2 diabetes.
[0067] Another embodiment of the present developments, is a method
to modulate, affect, and/or induce changes of interleukin-21
(IL-21), interleukin-22 (IL-22), IFN.gamma., TNF.alpha.,
interleukin-6 (IL-6), granulocyte-macrophage colongy-stimulating
factor (GM-CSF), interleukin-4 (IL-4), interleukin-10 (IL-10) and
transforming growth factor beta (TGF.beta.) in a cell or subject
comprising administering a peptide selected from SEQ ID NOs 3-9 and
25-30, in an amount sufficient to change said interleukin-21
(IL-21), interleukin-22 (IL-22), IFN.gamma., TNF.alpha.,
interleukin-6 (IL-6), granulocyte-macrophage colongy-stimulating
factor (GM-CSF), interleukin-4 (IL-4), interleukin-10 (IL-10) and
transforming growth factor beta (TGF.beta.), wherein the said
interleukin-21 (IL-21), interleukin-22 (IL-22), IFN.gamma.,
TNF.alpha., interleukin-6 (IL-6), granulocyte-macrophage
colongy-stimulating factor (GM-CSF), interleukin-4 (IL-4),
interleukin-10 (IL-10) and transforming growth factor beta
(TGF.beta.) signaling is associated with a condition selected from
group comprising type I diabetes, multiple sclerosis, systemic
lupus erythematosa, rheumatoid arthritis, Crohn's disease,
inflammatory bowel disease, chronic obstructive pulmonary disease,
asthma, atherosclerosis, vasculitis, hypertension, thyroiditis,
primary biliary cirrhosis, Paget's disease, Addison's disease,
acute respiratory distress syndrome, acute lung injury, type 2
diabetes, and/or aseptic chronic inflammation, more generally.
[0068] Another embodiment of the present developments includes a
method to modulate and/or reduce interleukin 17 (IL-17) in a cell
or a subject comprising administering a peptide selected from SEQ
ID NOs 3-9 and 25-30 in an amount sufficient to reduce or inhibit
interleukin 17 (IL-17) signaling, wherein, the IL-17 signaling is
associated with a condition selected from the group comprising type
I diabetes, multiple sclerosis, systemic lupus erythematosa,
rheumatoid arthritis, Crohn's disease, inflammatory bowel disease,
chronic obstructive pulmonary disease, asthma, atherosclerosis,
vasculitis, hypertension, thyroiditis, primary biliary cirrhosis,
Paget's disease, Addison's disease, acute respiratory distress
syndrome, acute lung injury, and/or aseptic chronic inflammation,
more generally.
[0069] Another embodiment of the present developments includes a
method to modulate and/or reduce interleukin 17 (IL-17) in a cell
or a subject comprising administering a peptide selected from SEQ
ID NOs 3-9 and 25-30 in an amount sufficient to reduce or inhibit
interleukin 17 (IL-17) signaling, wherein, the IL-17 is associated
with type 2 diabetes.
[0070] One embodiment of the present developments is a method to
identify a patient at risk for developing type 2 diabetes, the
method comprising obtaining a sample containing T-cells from a
patient to be tested, contacting the sample with a peptide that
binds the CD40 protein, detecting the CD-40 bound peptide, and
determining the level of Th40 cells from the amount of CD40 bound,
wherein a level of Th40 cells greater than 25% of the total T-cell
population indicates the patient is at risk for developing type 2
diabetes.
[0071] One embodiment of the present developments is the
composition of matter of small interfering peptides of those in SEQ
ID NOs: 4, 27, 28, 29, and 30. These embodiments of the current
developments may be used for the for the treatment of disease
selected from the group comprising type I diabetes, multiple
sclerosis, systemic lupus erythematosa, rheumatoid arthritis,
Crohn's disease, inflammatory bowel disease, chronic obstructive
pulmonary disease, asthma, atherosclerosis, vasculitis,
hypertension, thyroiditis, primary biliary cirrhosis, Paget's
disease, Addison's disease, acute respiratory distress syndrome,
acute lung injury, type 2 diabetes, and/or aseptic chronic
inflammation, more generally.
BRIEF DESCRIPTION OF THE FIGURES
[0072] FIG. 1 is a chart of the effect of various peptides of CD154
on the development of diabetes in NOD mice. The 8-mer (SEQ ID NO:
5), 10-mer (SEQ NO: 24), 13-mer (SEQ ID NO:25), 15-mer (SEQ ID NO:
7), and 24-mer (SEQ ID NO:26) were tested.
[0073] FIG. 2A is a chart of the effect of a 15-mer peptide from
CD154 on the CD4/CD8 ratio in NOD mice.
[0074] FIG. 2B is a chart of the effect of 15-mer peptide on
beta-islet infiltration in treated versus control pancreata
excised, examined, and scored.
[0075] FIG. 3 is a graph of reversal of diabetes in NOD mice using
a 15-mer peptide from CD154.
[0076] FIG. 4 is a dot-plot of the detection of Th40 cells using a
SIP-15-mer peptide from CD154.
[0077] FIG. 5 is a dot-plot of a screening of B cells using a
SIP-15-mer peptide from CD154.
[0078] FIG. 6 is a chart demonstrating a comparison of Th40 cell
levels in diabetic and non-diabetic mice.
[0079] FIG. 7 is a chart demonstrating the effect of treatment with
the 15-mer peptide on insulin granulation of the pancreas.
[0080] FIG. 8 is a graph that shows the effect of mutations in the
15-mer peptide on the ability of the 15-mer peptide to inhibit
development of diabetes in NOD mice.
[0081] FIG. 9 is a chart showing the number of cells
(.times.10.sup.6) of CD3+CD4+CD40+ in different mice models.
[0082] FIG. 10 is a chart showing the percentage of Th40 cells in
the peripheral blood in human subjects in control and diabetic
subjects.
[0083] FIG. 11 is a dot-plot comparing CD4+ and CD40 cell data
obtained through flow cytometry. Th40 cells (CD4+CD40+) are in the
upper right quadrant.
[0084] FIG. 12 is a chart showing the percentage of Th40 cells of
CD3+CD4+ population of mice models.
[0085] FIG. 13 is a chart that shows Th40 Cell percentage of
CD3+CD4+ population within the murine aorta of C57B-6, young
non-diabetic NOD mice, and diabetic NOD mice.
[0086] FIG. 14 is an image at 200.times. magnification showing Th40
cells in the shoulder region of plaque in the ApoE-/- mouse
model.
[0087] FIG. 15 is a graph of interferon gamma control of Th40
proliferation.
[0088] FIG. 16 is a plot of CD40 stimulated proliferation of Th40
cells in the absence/presence of anti-interferon gamma antibody
(.alpha.IFN.gamma.) demonstrating that interferon gamma mediates
CD40 induced proliferation.
[0089] FIG. 17 is a sample of stains of aortic arch of the lesser
curvature of the aortic arch.
[0090] FIG. 18 is a stain of lesser curvature of aortic arch in
control and treated subjects.
[0091] FIG. 19 is a chart of area measurements of the lesser
curvature of aortic arches and plaques.
[0092] FIG. 20-23 are charts of treated and control ApoE mice
subjects.
[0093] FIG. 24 is a table of blood clot data from human blood in
the presence of 15-mer peptide compared to normal clotting.
[0094] FIG. 25A is a table providing the relative peptide stability
assessed by ExPASy analysis.
[0095] FIG. 25B is a table providing the relative peptides
stability assessed by ExPASy analysis.
[0096] FIG. 26 is a western blot comparing control and treated
samples from subject mice.
[0097] FIG. 27 is a graph of LDL cholesterol measured in treated
and untreated subjects.
[0098] FIG. 28A is an image of KGYY6 treated aortic en-face Sudan
IV staining.
[0099] FIG. 28B is an image of control aortic en-face Sudan IV
staining.
[0100] FIG. 29 is a graph demonstrating the reduction of lesion
areas of Sudan IV staining.
[0101] FIG. 30 is a graph of plaque volume reduction for area under
the curve.
[0102] FIG. 31 is a graph of plaque composition for KGYY6 (SEQ ID
NO:29) treated and control subject mice.
[0103] FIG. 32A is an image of trichrome stained sections of KGYY6
(SEQ ID NO:29) treated subject.
[0104] FIG. 32B is an image of trichrome stained sections of
control subjects.
[0105] FIG. 33(a)-(b) are graphs that show a statistically
significant improvement in glucose tolerance (GTT) and insulin
sensitivity in response to SEQ ID NO: 29.
[0106] FIG. 34 is a western blot analysis of GLUT4 (insulin
regulated glucose transport protein) comparing adipose and muscle
tissue of treated (with SEQ ID NO: 29) and untreated mice.
[0107] FIG. 35 is a graph of the percentage change of in-vitro
lymphocyte cytokines measured in spleen cells from ApoE-/- and
C57BL/6 mice as measured by flow cytometry.
DETAILED DESCRIPTION
[0108] The present subject matter is based on the discovery that a
unique subset of T-cells, which express CD40 protein, and thus are
referred to as Th40 cells, may be instrumental in autoimmune
inflammation. Moreover, involvement of Th40 cells in the autoimmune
process may be dependent on the interaction between CD40 protein
expressed on the surface of the T-cell, and CD154 protein.
Interaction of CD40 and CD154 results in activation signals being
delivered between the cells, and subsequent activation of the Th40
cell. Such activation results in propagation of the Th40 cell and
an increase in inflammation (e.g., an increase in the number of
immune cells and immunoregulatory molecules, present in the
system). Accordingly, inhibition of the CD40/CD154 interaction can
modulate Th40 cell activity, and thereby affect inflammation. Thus
the present subject matter relates to the peptides, and
administration thereof, that may affect the interaction between a
CD40 protein and a CD154 protein, thereby modulating inflammation.
Moreover, the present subject matter relates to peptides that
affect the interaction between CD40 protein expressed on the
surface of a T-cell, and a CD154 protein, thereby affecting T-cell
activity, controlling inflammation, and consequently preventing,
modulating, and reducing atherosclerosis. The present subject
matter also encompasses the use of such peptides to detect Th40
cells.
[0109] Before the present development is further described, it is
to be understood that this invention is not strictly limited to
particular embodiments described, as such may of course vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present invention
will be limited only by the claims.
[0110] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. It should
further be understood that as used herein, the term "a" entity or
"an" entity refers to one or more of that entity. For example, a
nucleic acid molecule refers to one or more nucleic acid molecules.
As such, the terms "a", "an", "one or more" and "at least one" can
be used interchangeably. Similarly, the terms "comprising",
"including" and "having" can be used interchangeably.
[0111] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited. The publications
discussed herein are provided solely for their disclosure prior to
the filing date of the present application. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such publication by virtue of prior invention.
Further, the dates of publication provided may be different from
the actual publication dates, which may need to be independently
confirmed.
[0112] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable sub-combination.
All combinations of the embodiments are specifically embraced by
the present invention and are disclosed herein just as if each and
every combination was individually and explicitly disclosed. In
addition, all sub-combinations are also specifically embraced by
the present invention and are disclosed herein just as if each and
every such sub-combination was individually and explicitly
disclosed herein.
[0113] It is further noted that the claims may be drafted to
exclude any optional element. As such, this statement is intended
to serve as antecedent basis for use of such exclusive terminology
as "solely," "only" and the like in connection with the recitation
of claim elements, or use of a "negative" limitation.
[0114] Furthermore, as used herein the term animal refers to a
vertebrate, preferably a mammal, more preferably a human. Suitable
mammals on which to use the methods of the present invention
include but are not limited farm animals, sports animals, pets,
primates, mice, rats, horses, dogs, cats, and humans. The term
animal can be used interchangeably with the terms subject or
patient.
[0115] One embodiment of the present subject matter is a peptide
that interacts with a CD40 protein in such a manner as to prevent
atherosclerosis. As used herein, the terms interact, interaction,
and the like, mean that two molecules come into sufficient physical
proximity such that they cause a modulation of inflammation. One
such type of interaction is a binding interaction. In such an
interaction the peptide associates with CD40 to form a complex. An
example of complex formation is the association of an antigen with
an antibody. According to the present subject matter, binding of a
peptide hereof to a CD40 protein can be reversible (e.g.,
non-covalent binding interactions) or non-reversible (e.g.,
covalent binding interactions). Moreover, a reversible interaction
can be strong or weak, the strength of the interaction being
determined by the forces (e.g., ionic charges, hydrogen binding,
van der Walls interactions, etc.) exerted by each protein on the
other protein in the complex. Factors affecting the strength of an
interaction between two molecules are known to those skilled in the
art. One useful measure of the strength of binding between two
molecules, such as a peptide and a protein, is the dissociation
constant (Kd). Preferred peptides of the present invention are
those that bind to a CD40 protein with a Kd of no more than about
1.times.10.sup.-6 M, about 1.times.10.sup.-7 M, or about
1.times.10.sup.-8 M. Particularly preferred peptides are those
having a Kd of less than about 1.times.10.sup.-9 M. In one
embodiment, a peptide hereof binds to a CD40 protein with a Kd of
less than 100 nM, less than 50 nM, less than 25 nM, less than 10
nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1
nM. Methods of measuring and analyzing binding interactions between
a peptide and a CD40 protein are known by those of skill in the
art.
[0116] As used herein, the change the level of Th40 cells present
in an animal, or in a culture of T cells may be indicative of
modulation of inflammation. As used herein, the terms level,
number, count and concentration can be used interchangeably.
Modulation of inflammation may mean an increase or decrease in the
number of Th40 cells present in the inflammatory environment;
however, the modulation of inflammation should not be limited to
cell numbers or counts.
[0117] Consequently, modulation can be referred to as positive or
negative. Positive modulation (also referred to as up-regulation)
of inflammation may result in an increase in the number of Th40
cells in the inflammatory environment. Negative modulation (also
referred to as down-regulation) of inflammation may result in a
reduction in the number of Th40 cells present in the inflammatory
environment. The level, count, or concentration of Th40 cells may
not be indicative of inflammation in the inflammatory environment.
A preferred peptide may be one that down-regulates inflammation,
thereby reducing the number of Th40 cells present in the
inflammatory environment. Positive and negative modulation of
inflammation may or may not result in a change in the type and
amount of immunoregulatory molecules present in the inflammatory
environment. In some instances, it is possible that the Th40 levels
will not change but the activity of those cells is altered such
that they are no longer exerting or increasing inflammatory
cytokines and other biomarkers of inflammation. Therefore, as used
herein modulating inflammation may refer to changes in the Th40
levels, numbers, or concentration in an corporeal body or sample,
and may also refer to changes in inflammation more generally that
may be associated with disease, inflammatory cytokines, and/or cell
derived inflammatory mediator molecules.
[0118] It will be appreciated by those skilled in the art that both
a cell culture system and the immune system of an animal comprise
basal levels of immune cells and immunoregulatory molecules. The
phrases basal level and normal level can be used interchangeably.
With regard to the immune system of an animal, as used herein, the
basal level of a type of immune cell (e.g., Th40 cell), or a
immunoregulatory molecule, refers to the average number of that
cell type, or immunoregulatory molecule, present in a population of
individuals considered healthy (i.e., free of metabolic,
autoimmune, or infectious disease). With regard to a cell culture
system, as used herein, the basal level of a type of immune cell,
or an immunoregulatory molecule, refers to the average level of
that cell type, or immunoregulatory molecule, present in a
population of cells that is non-activated. Those skilled in the art
are capable of determining if a T-cell, or a population of such
cells, is activated. For example, the expression of CD69, CD25
and/or CD154 proteins by a cell indicates that the cell has been
activated.
[0119] The basal level of a cell or molecule can be a specific
amount (e.g., a specific concentration) or it can encompass a range
of amounts. Basal levels, or ranges, of immune cells and
immunoregulatory molecules are known to those in the art. For
example, in a healthy individual, the normal level of CD4+ T-cells
present in human blood is 500-1500 cells/ml. Variability in this
measurement can result from differences in the method used to
determine the cell count. Furthermore, normal levels of cells can
also be reported as a percentage of a total cell population. For
example, in a healthy individual, Th40 cells make up less than 25%
of the total T cell population. Thus, as used herein, the term
inflammation refers to an inflammatory environment in which Th40
cells make up greater than about 25%, greater than about 30%,
greater than about 35%, greater than about 40%, greater than about
45%, greater than about 50%, greater than about 55%, greater than
about 60%, greater than about 65%, greater than about 70%, greater
than about 75%, or greater than about 80% of the total T-cell
population. Moreover, a preferred peptide herein is one that
reduces the level of Th40 cells to less than about 50%, less than
about 45%, less than about 40%, less than about 35%, less than
about 30%, less than about 27%, or equal to about 25% of the total
T-cell population. Methods of measuring different types of T-cells
in the T-cell population are known to those skilled in the art.
Furthermore, a novel method for detecting Th40 cells using peptides
hereof is disclosed herein.
[0120] As used herein, the phrase inflammatory environment refers
to the overall population of immune cells, and related
immunoregulatory molecules, that are present in a culture of cells,
or in the body of an animal. As such, the phrase inflammatory
environment encompasses the types, and/or the relative amounts of
immune cells and immunoregulatory molecules (e.g., cytokines)
present in a culture of cells, or in an animal, which are involved
in affecting an inflammatory reaction. Examples of cells
encompassed by the term inflammatory environment include, but are
not limited to, T cells, neutrophils, macrophages, granulocytes,
and the like. The inflammatory environment relates to cells and
molecules that mediate both acute and chronic inflammation. It will
be appreciated by those skilled in the art that the inflammatory
environment refers to the system to which peptides hereof are
administered. In one embodiment, the system is a cell culture
system. In one embodiment, the system is a whole animal.
[0121] A preferred peptide hereof is one that selectively interacts
with a CD40 protein in solution, as determined using an assay such
as an immunosorbent assay, or on the surface of a T-cell. As used
herein, the terms selectively, selective, specific, and the like,
indicate the peptide has a greater affinity for a CD40 protein than
it does for proteins unrelated to the CD40 protein. More
specifically, the terms selectively, selective, specific, and the
like indicate that the affinity of the peptide for CD40 is
statistically significantly higher than its affinity for a negative
control (e.g., an unrelated protein such as albumin) as measured
using a standard assay (e.g., ELISA). Suitable techniques for
assaying the ability of a peptide to selectively interact with a
CD40 protein are known to those skilled in the art. Such assays can
be in vitro or in vivo assays. Examples of useful assays include,
but are not limited to, an enzyme-linked immunoassay, a competitive
enzyme-linked immunoassay, a radioimmunoassay, a fluorescence
immunoassay, a chemiluminescent assay, a lateral flow assay, a
flow-through assay, an agglutination assay, a particulate-based
assay (e.g., using particulates such as, but not limited to,
magnetic particles or plastic polymers, such as latex or
polystyrene beads), an immunoprecipitation assay, an immunoblot
assay (e.g., a western blot), a phosphorescence assay, a
flow-through assay, a chromatography assay, a polyacrylamide gel
electrophoresis (PAGE)-based assay, a surface plasmon resonance
assay, a spectrophotometric assay, a particulate-based assay, an
electronic sensory assay and a flow cytometric assay. Methods of
performing such assays are well known to those skilled in the art.
In one embodiment, an assay can be performed using cells in
culture, or it can be performed in a whole animal. Assays can be
designed to give qualitative, quantitative or semi-quantitative
results, depending on how they are used and the type of result that
is desired.
[0122] Cardiovascular Disease (CVD), Atherosclerosis, and
Cholesterol Related Developments
[0123] One embodiment hereof is a peptide that interacts with a
CD40 protein in such a manner as to affect the interaction of the
CD40 protein with a CD154 protein, thereby modulating inflammation.
The effect of the peptide on the CD40/CD154 interaction can be
positive or it can be negative. For example, the peptide can
interact with the CD40 protein in such a manner that the strength
of the interaction between the CD40 protein and a CD154 protein is
increased. Alternatively, the peptide can interact with the CD40
protein such that the strength of the interaction between the CD40
protein and a CD154 protein is decreased. Methods of measuring the
strength of binding between the peptide and a CD40 protein are
known to those skilled in the art. A preferred peptide hereof is
one that reduces the strength of the interaction between a CD40
protein and a CD154 protein. Preferred peptides hereof reduce the
strength of binding between a CD40 protein and a CD154 protein by
at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, or at
least 95%. A particularly preferred peptide is one that completely
inhibits binding of CD40 to CD154. Complete inhibition of binding
between CD40 and CD154 means that when a peptide hereof is brought
into proximity with a CD40 protein and a CD154 protein under
conditions that would normally allow the interaction of CD40 and
CD154, no such interaction occurs and activation signals are not
stimulated in the CD40-expressing cell. Consequently CD40/CD154
mediated modulation of inflammation does not occur. In one
embodiment, the peptide interacts with the CD40 protein in such a
manner as to reduce the level of inflammation in the system. In one
embodiment, the peptide interacts with the CD40 protein in such a
manner as to inhibit the development of inflammation in the
system.
[0124] While peptides hereof can interact with any site on the CD40
protein, preferred peptides interact with the CD40 protein at a
location that overlaps with the CD154 binding site. In one
embodiment, a peptide hereof interacts with the CD40 protein at the
CD154 binding site. An example of such a peptide is a CD40 ligand
competitive antagonist. As used herein, peptides that interfere
with, or inhibit, the binding of a CD154 protein to a CD40 protein
are referred to as small interfering peptides (SIPs). As used
herein a small interfering peptide is a peptide that, through
physio-chemical properties, interferes with the interaction of a
CD40 protein with a CD154 protein, thereby preventing activation
signals from being delivered to the CD40-bearing cell, thus
limiting the activation of the CD40-bearing cell, and consequently,
inflammation. As demonstrated herein, the consequences of such
interference are prevention of T-cell activation and propagation,
and a prevention or reduction of inflammation. As demonstrated
herein, in some instances the results of such inhibition or
prevention of interaction between CD40 and CD154 may include
observable data that demonstrates that atherosclerosis, and
characteristics of diseases associated therewith, are prevented,
modulated, and/or reduced.
[0125] Additionally, a small interfering peptide, may, through its
physio-chemical properties, interfere with the interaction of a
CD40 protein with a CD154 protein, thereby preventing activation
signals from being delivered to the CD40-bearing cell, thus
limiting the activation of the CD40-bearing cell, and consequently,
modulating, inhibiting, and preventing atherosclerosis. As
demonstrated herein, the consequences of such interference are
prevention of T cell activation and propagation, and a prevention,
reduction, or modulation of atherosclerotic developments.
[0126] A peptide useful for practicing methods of the present
developments should be of a size sufficient to interact with CD40
protein in such a manner as to modulate atherosclerosis. It is
understood by those skilled in the art that preferred peptides are
relatively short since they are easier and less expensive to
produce. Preferred peptides may be those that are less than 25
amino acids in length; however, the length of the peptide may be
longer than 25 amino acids in some instances. A preferred peptide
may be one that is 4, 6, 8, 10, 13, 15, or 24 amino acids in
length. In one embodiment, the peptide is an amino acid selected
from the group of SEQ ID NO:3 (Core-sequence see Table 1), SEQ ID
NO:4 (6-mer see Table 1), SEQ ID NO:5 (8-mer mouse see Table 1),
SEQ ID NO:6 (8-mer human see Table 1), SEQ IN NO:7 (15-mer see
Table 1), SEQ ID NO:8 (15-mer human see Table 1), SEQ ID NO:9
(24-mer see Table 1), SEQ ID NO: 24 (10-mer see Table 1), SEQ ID
NO: 25 (13-mer see Table 1), SEQ ID NO: 26 (24-mer see Table 1),
SEQ ID NO: 27 (6-mer (Form 2) see Table 1), SEQ ID NO: 28 (6-mer
(Form 3) see Table 1), SEQ ID NO: 29 (6-mer (Form 4) see Table 1),
SEQ ID NO: 30 (6-mer (Form 4) see Table 1) and SEQ ID NO:32
(24-mer-mouse (Form 2)). The sequences of such peptides are shown
below in Table 1.
TABLE-US-00001 TABLE 1 SEQ ID NO SEQUENCE Description 1 MIETYSQPSP
RSVATGLPAS MKIFMYLLTV SwissPro FLITQMIGSV LFAVYLHRRL DKVEEEVNLH
27548.2 Mouse EDFVFIKKLK RCNKGEGSLS LLNCEEMRRQ CD40 Ligand
FEDLVKDITL NKEEKKENSF EMQRGDEDPQ (CD154 IAAHVVSEAN SNAASVLQWA
KKGYYTMKSN Protein) LVMLENGKQL TVKREGLYYV YTQVTFCSNR EPSSQRPFIV
GLWLKPSSGS ERILLKAANT HSSSQLCEQQ SVHLGGVFEL QAGASVFVNV TEASQVIHRV
GFSSFGLLKL 2 MIETYNQTSP RSAATGLPIS MKIFMYLLTV SwissPro FLITQMIGSA
LFAVYLHRRL DKIEDERNLH 29965 Human EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ
CD40 Ligand FEGFVKDIML NKEETKKENS FEMQKGDQNP (CD154 QIAAHVISEA
SSKTTSVLQW AEKGYYTMSN Protein) NLVTLENGKQ LTVKRQGLYY IYAQVTFCSN
REASSQAPFI ASLCLKSPGR FERILLRAAN THSSAKPCGQ QSIHLGGVFE LQPGASVFVN
VTDPSQVSHG TGFTSFGLLK L 3 KGYY Core-sequence 4 KKGYYT 6-mer 5
AKKGYYTM 8-mer-mouse 6 AEKGYYTM 8-mer human 7 VLQWAKKGYYTMKSN
15-mer-mouse 8 VLQWAEKGYYTMSNN 15-mer human 9
NAASVLQWAKKGYYTMKSNLVMLE 24-mer 10 ISQAVHAAHAEINEAGR 15-mer from
ovalbumin; control peptide 11 G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-1
12 V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-2 13
V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-3 14
V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-N Gly-4 15
V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N Gly-5 16
V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N Gly-6 17
V-L-Q-W-A-K-G-G-Y-Y-T-M-K-S-N Gly-7 18
V-L-Q-W-A-K-K-G-G-Y-T-M-K-S-N Gly-8 19
V-L-Q-W-A-K-K-G-Y-G-T-M-K-S-N Gly-9 20
V-L-Q-W-A-K-K-G-Y-Y-G-M-K-S-N Gly-10 21
V-L-Q-W-A-K-K-G-Y-Y-T-G-K-S-N Gly-11 22 ISQAVHAAHAEINEAGR 15-mer
from ovalbumin; control peptide 23 YVQGKANLKSKLMYT Scrambled
peptide 24 WAKKGYYTMK 10-mer mouse 25 VLQWAKKGYYTMK 13-mer mouse 26
AASVLQW AKKGYYTMKSNLVVLEN 24-mer mouse 27 KGYYTM 6-mer (Form 2) 28
AEKGYY 6-mer (Form 3) 29 AKKGYY 6-mer (Form 4) 30 AKGYYT 6-mer
(Form 5) 31 YKNVKQMAYWLTGKS Scrambled peptide 32
AASVLQWAKKGYYTMKSNLVMLEN 24-mer-mouse (Form 2)
[0127] Interaction of a CD40 protein and a CD154 protein has been
shown to occur at particular regions within each protein. The
inventors have now shown that, surprisingly, a peptide comprising
only a short portion of the CD154 region that interacts with CD40
is capable of binding to a CD40 protein, thereby modulating
atherosclerosis. Thus one embodiment hereof is a peptide that
comprises at least a portion of the amino acid sequence of a CD154
protein such that the peptide interacts with CD40 protein in such a
manner as to modulate atherosclerosis. In one embodiment,
interaction of the peptide with CD40 protein results in negative
modulation of atherosclerosis. In one aspect, the peptide comprises
at least a portion of SEQ ID NO:1 or SEQ ID NO:2.
[0128] In one aspect, the peptide is as short as possible yet
comprises enough of the CD154 protein to allow interaction with a
CD40 protein in such a manner as to modulate atherosclerosis. In
one embodiment, a peptide hereof comprises 6, 13 or 15 contiguous
amino acids from SEQ ID NO:1 or SEQ ID NO:2, and interacts with
CD40 in such a manner as to modulate atherosclerosis. A preferred
peptide comprises a core sequence of
lysine-glycine-tyrosine-tyrosine (KGYY; SEQ ID NO:3), which
corresponds to amino acids 142-145 of SEQ ID NO:1 and amino acids
143-146 of SEQ ID NO:2. Useful peptides can comprise additional
regions of sequence from SEQ ID NO:1 or SEQ ID NO:2 that are
adjacent to the core sequence, so long as the peptide is capable of
modulating atherosclerosis. In one embodiment hereof, a peptide
comprises at least one sequence selected from SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ
ID NO:30, and SEQ ID NO:32, so long as the peptide interacts with
CD40 protein in such a manner as to modulate atherosclerosis. In
one embodiment of the present subject matter, a peptide hereof is a
sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ
ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ
ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,
SEQ ID NO:30, and SEQ ID NO:32. In one embodiment of the present
subject matter, a peptide hereof is a sequence selected from SEQ ID
NOs 3-9, 25-30, and 32.
[0129] While peptides of the present subject matter may be selected
entirely of or from sequences that are responsible for the
interaction of the peptide with a CD40 protein, they may
additionally contain amino acid sequences that do not interact with
a CD40 protein, but which have other useful functions. Any useful,
additional amino acid sequence can be added to the CD40-interacting
sequence, so long as the additional sequences do not have an
unwanted effect on the ability of the CD40 interacting sequence to
interact with a CD40 protein. For example, in addition to the amino
acid sequence responsible for interacting with a CD40 protein, a
peptide hereof can contain amino acid sequences that are useful for
visualizing or purifying the peptide. Such sequences act as labels
(e.g., enzymes) or tags (antibody binding sites). Examples of such
labels and tags include, but are not limited to, B-galactosidase,
luciferase, glutathione-s-transferase, thioredoxin, HIS-tags,
biotin tags, and fluorescent tags. Other useful sequences for
labeling and tagging proteins are known to those of skill in the
art.
[0130] Likewise, peptides hereof can be modified, so long as such
modification does not significantly affect the ability of the
peptide to modulate atherosclerosis. Such modifications can be
made, for example, to increase the stability, solubility or
absorbability of the protein. Examples of such modifications
include, but are not limited to pegylation, glycosylation and
chemical modification of the peptide.
[0131] Peptides hereof may be obtained from nature (e.g., obtained
from plants, animals or microorganisms) or they may be produced in
a laboratory (e.g., recombinantly or synthetically). Preferred
peptides are those that are synthesized. Also encompassed are
peptides that are combinations of natural and synthetic molecules.
General methods for producing and isolating recombinant or
synthetic peptides are known to those skilled in the art. It should
be noted that, as used herein, an isolated, or biologically pure,
molecule, is one that has been removed from its natural milieu. As
such the terms isolated, biologically pure, and the like, do not
necessarily reflect the extent to which the protein has been
purified.
[0132] As has been described herein, interaction of the CD40
protein and the CD154 protein are necessary for involvement of Th40
cells in atherosclerosis. Consequently, inhibition of the
interaction between a CD40 and CD154 protein using peptides hereof
is a useful method of affecting atherosclerosis. Thus one
embodiment is a method to reduce the interaction between a CD40
protein and a CD154 protein comprising introducing into an
environment containing a CD40 protein and a CD154 protein, a
peptide, that interacts with the CD40 protein in such a manner as
to reduce the interaction between the CD40 protein and the CD154
protein. In one aspect hereof, the peptide reduces the interaction
between the CD40 protein and the CD154 protein by at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, or at least
95%. In one embodiment, the peptide reduces the interaction between
the CD40 protein and the CD154 protein by a factor of at least 10,
at least 100, at least 1,000, at least 10,000. Methods of measuring
the strength of the interaction between the CD40 protein and the
CD154 protein have been discussed previously, and are also know to
those of skill in the art.
[0133] One embodiment hereof is a method to modulate
atherosclerosis comprising contacting a CD40 protein with a peptide
that interacts to the CD40 protein in such a manner as to modulate
inflammation. In one aspect, interaction of the peptide with the
CD40 protein decreases the number of Th40 cells by at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, or at least
95%. In one embodiment, interaction of the peptide with the CD40
protein decreases the number of Th40 cells by a factor of at least
10, at least 100, at least 1,000, at least 10,000.
[0134] One aspect of the current developments hereof is a method to
modulate atherosclerosis, cardiovascular disease, and/or
cholesterol levels comprising contacting a CD40 protein with a
peptide that interacts with the CD40 protein in such a manner as to
ameliorate inflammation. One aspect of this alternative embodiment
is that the Th40 levels do not change in response to contacting
said CD40 protein with a peptide that interacts with the CD40
protein. In one aspect, Th40 cells treated with peptide may stop,
slow, reduce, or retard production of inflammatory cytokines. In
this aspect the number of Th40 levels may remain relatively
unchanged.
[0135] One aspect is a method to reduce atherosclerosis in a
patient, the method comprising administering a peptide hereof to
the patient. In one embodiment, the peptide comprises an amino acid
sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7, SEQ
ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ
ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID
NO:32. In one embodiment, the peptide is an amino acid sequence
selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24,
SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30 and SEQ ID NO:32. In a preferred embodiment,
interaction of the peptide with the CD40 protein decreases the
number of Th40 cells by at least 5%, at least 10%, at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, or at least 95%. In another embodiment,
interaction of the peptide with the CD40 protein decreases the
number of Th40 cells by a factor of at least 10, at least 100, at
least 1,000, at least 10,000. In a preferred embodiment, the level
of Th40 cells is reduced so that Th40 cells comprise no more than
about 20%, about 25%, about 30%, about 35%, or about 40% of the
total T-cell population.
[0136] Peptides and methods hereof are suitable for use in cell
culture as well as for treating a patient. As used herein the term
patient refers to any animal in need of such treatment. The animal
can be a human or a non-human animal. A preferred animal to treat
is a mammal. A peptide can be administered or applied per se, or as
pharmaceutical compositions. A peptide hereof, or a pharmaceutical
composition thereof, can be administered to a patient by a variety
of routes, including, but limited to, by injection (e.g.,
intravenous, intramuscular, subcutaneous, intrathecal,
intraperitoneal), by inhalation, by oral (e.g., in a pill, tablet,
capsule, powder, syrup, solution, suspension, thin film, dispersion
or emulsion.), transdermal, transmucosal, pulmonary, buccal,
intranasal, sublingual, intracerebral, intravaginal rectal or
topical administration or by any other convenient method known to
those of skill in the art.
[0137] The amount of a peptide hereof and/or a pharmaceutical
composition thereof that will be effective can be determined by
standard clinical techniques known in the art. Such an amount is
dependent on, among other factors, the patient being treated,
including, but not limited to the weight, age, and condition of the
patient, the intended effect of the compound, the manner of
administration and the judgment of the prescribing physician. Also,
in this context, it should be noted that in treating a patient
exhibiting a disorder of interest, a therapeutically effective
amount of an agent or agents such as these is administered. A
therapeutically effective dose refers to that amount of the
compound that results in amelioration of one or more symptoms or a
prolongation of survival in a patient.
[0138] A peptide hereof, or a pharmaceutical composition thereof,
can be administered alone or in combination with one or more other
pharmaceutical agents, including other compounds of the present
disclosure. The specific pharmaceutical composition depends on the
desired mode of administration, as is well known to the skilled
artisan.
[0139] Because the developments have demonstrated that Th40 cells
are intimately involved in the development of autoimmune diseases,
atherosclerosis, and cardiovascular disease, the peptides and
methods disclosed herein can be used to affect atherosclerosis
resulting from such diseases. Thus, one embodiment hereof is a
method to treat atherosclerotic disease in a patient in need of
such treatment, the method comprising administering to a patient a
peptide that interacts with the CD40 protein, thereby reducing
atherosclerosis. In one embodiment the peptide interacts with the
CD40 protein in such a manner as to affect the interaction of CD40
and CD154, thereby reducing atherosclerosis. In a preferred
embodiment, interaction of the peptide with the CD40 protein
reduces the number of Th40 cells in a patient to a level equal to
that observed in subjects that do not have cardiovascular disease.
The present developments are suitable for treating any patient
having an autoimmune disease and/or cardiovascular disease, the
development of which is dependent on Th40 cells. More specifically,
peptides hereof are suitable for reducing the level of Th40 cells
in such patients. In a preferred embodiment, a peptide hereof
reduces the level of Th40 cells in a patient suffering from a
cardiovascular disease to no more than about 25% of the total
T-cell population. In another embodiment, a peptide hereof reduces
inflammatory cytokine levels while having no effect on the Th40
levels in a patient.
[0140] One example of a disease that is particularly amenable to
treatment using a peptide of the present developments may be
atherosclerosis. In atherosclerosis, inflammatory changes of the
arterial wall occur resulting in the formation and buildup of
arterial plaque. Consequently, control of inflammatory cells and
cell signaling via CD40-CD154 interaction may be able to be used to
control, modulate, and/or reduce atherosclerotic lesions that are
characterized as chronic inflammatory-fibroproliferative disease of
the vessel wall. Several murine models of T2D and/or
atherosclerosis have been developed. The progression of lesion
formation is observable in Apolipoprotein E (ApoE) deficient
transgenic mice and can be observed by measurement of the aortic
arch, the number and type of plaque, and characterized in
accordance with the American Heart Association's staging of
atherosclerosis, ranging from AHA type I to AHA type V. AHA type 1,
may be characterized by early or initial lesions, may be comprised
of histologically "normal" cells, macrophage infiltration, and
isolated foam cells. AHA type V, may be characterized by advanced
or complicated legions, including but not limited to increased
endothelial dysfunction characterized by surface defects, hematoma,
hemorrhage, and/or thrombosis. Thus, one embodiment of the present
developments is a method to prevent atherosclerosis in an
individual at risk for developing atherosclerosis, the method
comprising administering to the individual a peptide to selectively
bind to a CD40 expressing cell.
[0141] Moreover, atherosclerosis may be particularly amenable to
treatment using a peptide of the current development. The risk for
atherosclerosis may result from familial factors (e.g.,
inheritance) or from other factors, such as the physical condition
of the individual. The level of atherosclerotic activity and
disease may vary from individual to individual depending on
numerous factors such as level of activity, diet, smoking status,
and other variable factors that are dynamic such as levels of
inflammation. Some methods of risk assessment for atherosclerosis
are known to those skilled in the art. Accordingly, in one
embodiment of the present development, the method of treatment
comprises administering to a patient in need thereof, a peptide
that comprises an amino acid sequence selected from SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, and SEQ ID NO:9, so long as
the peptide can down-regulate inflammation. In one embodiment, the
peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32.
[0142] The developments herein also show that, surprisingly,
peptides hereof can be used to reverse the disease process in
individuals already showing signs of atherosclerosis. Thus, one
aspect of the present subject matter is a method to reverse
atherosclerosis comprising administering to a patient diagnosed as
having atherosclerosis, a peptide hereof. In one embodiment, the
peptide comprises an amino acid sequence selected from SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, and SEQ ID NO:9, so long as
the peptide can down-regulate inflammation. In one embodiment, the
peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. As used herein the
phrase to reverse atherosclerosis means to reduce the aortic-arch
infiltration, plaque, and lesions of an individual with
atherosclerosis to a level comparable to that observably lower
levels or in some instances to levels that may be more common in a
non-atherosclerotic individual.
[0143] As has been described, peptides of the present invention
selectively bind to a CD40 expressing cell. Consequently, peptides
of the present subject matter can be used to identify Th40 cells.
Thus one embodiment hereof is a method to detect Th40-dependent
atherosclerosis, said method comprising contacting a T-cell
population with a peptide hereof. In a preferred embodiment, the
peptide is labeled with a detectable marker, such as, for example,
luciferase or alkaline phosphatase. Such detection can be performed
using assay techniques known to those skilled in the art. In
general, an assay for detecting Th40 cells using a peptide hereof
comprises (a) obtaining a sample of cells; (b) contacting a peptide
hereof with said cells under condition suitable to allow binding of
the peptide to Th40 cells, if present; (c) washing said cells using
conditions that disrupt non-specific interactions, and that remove
unbound peptide; and (d) detecting peptide bound to cells.
Detection of bound peptide can be achieved directly or indirectly.
For example, direct detection can be achieved using a peptide
labeled using a detectable marker, as disclosed herein. Following
the wash step listed above, the cells are then simply screened for
the presence of detectable marker. The presence of detectable
marker in the cell sample indicates the presence of Th40 cells, and
thus Th40-dependent atherosclerosis. Alternatively, indirect
detection involves the use of a second molecule, such as an
antibody, that binds to the peptide. In an indirect detection
assay, following the wash step listed above, a detection molecule
that binds the peptide is added to the cell sample. The detection
molecule is labeled with a detectable marker. After washing away
unbound detection molecule, the cells are screened for the presence
of detectable marker. The presence of detectable marker in the cell
sample indicates the presence of Th40 cells. It should be
understood that the assays described herein are meant as examples
of useful assays, and other assay techniques can be employed.
Suitable assay techniques are known to those skilled in the art,
and are also disclosed in, for example, Molecular Cloning: A
Laboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T,
Cold Spring Harbor Laboratory Press; 2nd Edition (December 1989).
All references cited herein are incorporated herein in their
entirety.
[0144] The assay technology described above can also be used to
identify other molecules that affect the interaction of a CD40
protein with a CD154 protein. Examples of such molecules include,
but are not limited to, proteins, peptides and small molecules. For
example, assays can be designed that test the ability of molecules
to compete with a peptide of the present developments for binding
to a Th40 cell. For instance, a peptide labeled with a detectable
marker, can be mixed with a test molecule and a population of cells
known to contain Th40 cells, under conditions that allow binding of
the peptide to the Th40 cells. Following an appropriate incubation
period, the cells are washed to remove unbound peptide, and the
cells screened for the presence of detectable marker.
Alternatively, the labeled peptide could be bound to Th40 cells
first, and after a wash step to remove unbound peptide, the test
molecule could be added to the cells containing bound peptide.
Following an incubating period and a wash step to remove unbound
molecule, or released peptide, the cells are screened for the
presence of detectable marker. In either case, absence of the
detectable marker in the cell sample indicates the test molecule is
able to compete with the peptide for binding to the Th40 cells,
while presence of the detectable marker would indicate the test
molecule does not inhibit binding of the peptide to Th40 cells.
Inhibition of binding need not be 100%, as such assay would also be
useful for identifying molecules that partially inhibit binding of
the peptide to Th40 cells. It is understood by those skilled in the
art that such assays would involve the use of positive controls
(e.g., unlabeled peptide) and negative controls (e.g., a
protein/molecule that is known not to bind to Th40 cells).
[0145] Because increased levels of Th40 cells are associated with
the development of autoimmune disease, the present developments can
be used to identify patients at risk for developing autoimmune
disease and autoimmune related atherosclerosis and/or
cardiovascular disease more generally. Thus, one embodiment of the
present developments is a method to identify a patient at risk for
developing autoimmune related atherosclerosis. In one embodiment,
patients at risk for developing atherosclerosis are identified by
obtaining a sample from a patient to be tested, contacting the
T-cell portion of said sample with a peptide hereof, and
determining the level of Th40 cells present in the sample, wherein
a level of Th40 cells greater than about 25% of the total T-cell
population indicates the patient is at risk for developing
atherosclerotic disease. In one embodiment, the peptide comprises
an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:8, SEQ ID NO:7, SEQ ID NO:9 SEQ ID NO:24, SEQ ID NO:25, SEQ
ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,
and SEQ ID NO:32 so long as the peptide binds to the CD40 protein.
In one embodiment, the peptide is an amino acid sequence selected
from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32. In a
preferred embodiment the peptide is labeled with a suitable
detectable marker such as, for example, luciferase or alkaline
phosphatase.
[0146] The present developments also comprise kits useful for
practicing the methods disclosed herein, the kit comprising a
peptide that interacts with a CD40 protein in such a manner as to
modulate atherosclerosis. In one embodiment, the peptide comprises
an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, so long as the peptide can
down-regulate atherosclerosis. In one embodiment, the peptide is an
amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:7, SEQ ID NO:8, and SEQ ID NO:9. Another embodiment is a kit for
determining the level of Th40 cells, the kit comprising a peptide
that interacts with a CD40 protein, and methods for detecting
CD40-bound peptide. Kits can also contain associated reagents and
components, such as, but not limited to, buffers, labels,
containers, inserts, tubing, vials, syringes, and the like.
[0147] Type 2 Diabetes Related Developments
[0148] The present subject matter is based on the discovery that a
unique subset of T-cells, which express CD40 protein, and thus are
referred to as Th40 cells, that may be instrumental in autoimmune
inflammation, including conditions such as type 2 diabetes.
Moreover, involvement of Th40 cells in the autoimmune process may
be dependent on the interaction between CD40 protein expressed on
the surface of the T-cell, and CD154 protein. Interaction of CD40
and CD154 results in activation signals being delivered between the
cells, and subsequent activation of the Th40 cell. Such activation
results in propagation of the Th40 cell and an increase in
inflammation (e.g., an increase in the number of immune cells and
immunoregulatory molecules, present in the system). Accordingly,
inhibition of the CD40/CD154 interaction can modulate Th40 cell
activity, and thereby affect inflammation. Thus the present subject
matter relates to the peptides, and administration thereof, that
may affect the interaction between a CD40 protein and a CD154
protein, thereby modulating inflammation. Moreover, the present
subject matter relates to peptides that affect the interaction
between CD40 protein expressed on the surface of a T-cell, and a
CD154 protein, thereby affecting T-cell activity, controlling
inflammation, and consequently preventing, modulating, reducing
and/or reversing type 2 diabetes. The present subject matter also
encompasses the use of such peptides to detect Th40 cells.
[0149] One embodiment of the present subject matter is a peptide
that interacts with a CD40 protein in such a manner as to prevent
type 2 diabetes. As used herein, the terms interact, interaction,
and the like, mean that two molecules come into sufficient physical
proximity such that they cause a modulation of inflammation. One
such type of interaction is a binding interaction. In such an
interaction the peptide associates with CD40 to form a complex. An
example of complex formation is the association of an antigen with
an antibody. According to the present subject matter, binding of a
peptide hereof to a CD40 protein can be reversible (e.g.,
non-covalent binding interactions) or non-reversible (e.g.,
covalent binding interactions). Moreover, a reversible interaction
can be strong or weak, the strength of the interaction being
determined by the forces (e.g., ionic charges, hydrogen binding,
van der Walls interactions, etc.) exerted by each protein on the
other protein in the complex. Factors affecting the strength of an
interaction between two molecules are known to those skilled in the
art. One useful measure of the strength of binding between two
molecules, such as a peptide and a protein, is the dissociation
constant (Kd). Preferred peptides of the present invention are
those that bind to a CD40 protein with a Kd of no more than about
1.times.10.sup.-6 M, about 1.times.10.sup.-7 M, or about
1.times.10.sup.-8 M. Particularly preferred peptides are those
having a Kd of less than about 1.times.10.sup.-9 M. In one
embodiment, a peptide hereof binds to a CD40 protein with a Kd of
less than 100 nM, less than 50 nM, less than 25 nM, less than 10
nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1
nM. Methods of measuring and analyzing binding interactions between
a peptide and a CD40 protein are known by those of skill in the
art.
[0150] As used herein, the change the level of Th40 cells present
in an animal, or in a culture of T cells may be indicative of
modulation of inflammation. As used herein, the terms level,
number, count and concentration can be used interchangeably.
Modulation of inflammation may mean an increase or decrease in the
number of Th40 cells present in the inflammatory environment;
however, the modulation of inflammation should not be limited to
cell numbers or counts. Consequently, modulation can be referred to
as positive or negative. Positive modulation (also referred to as
up-regulation) of inflammation may result in an increase in the
number of Th40 cells in the inflammatory environment. Negative
modulation (also referred to as down-regulation) of inflammation
may result in a reduction in the number of Th40 cells present in
the inflammatory environment. The level, count, or concentration of
Th40 cells may not be indicative of inflammation in the
inflammatory environment. A preferred peptide may be one that
down-regulates inflammation, thereby reducing the number of Th40
cells present in the inflammatory environment. Positive and
negative modulation of inflammation may or may not result in a
change in the type and amount of immunoregulatory molecules present
in the inflammatory environment. In some instances, it is possible
that the Th40 levels will not change but the activity of those
cells is altered such that they are no longer exerting or
increasing inflammatory cytokines and other biomarkers of
inflammation. Therefore, as used herein modulating inflammation may
refer to changes in the Th40 levels, numbers, or concentration in a
corporeal body or sample, and may also refer to changes in
inflammation more generally that may be associated with disease,
inflammatory cytokines, and/or cell derived inflammatory mediator
molecules.
[0151] It will be appreciated by those skilled in the art that both
a cell culture system and the immune system of an animal comprise
basal levels of immune cells and immunoregulatory molecules. The
phrases basal level and normal level can be used interchangeably.
With regard to the immune system of an animal, as used herein, the
basal level of a type of immune cell (e.g., Th40 cell), or a
immunoregulatory molecule, refers to the average number of that
cell type, or immunoregulatory molecule, present in a population of
individuals considered healthy (i.e., free of metabolic,
autoimmune, or infectious disease). With regard to a cell culture
system, as used herein, the basal level of a type of immune cell,
or an immunoregulatory molecule, refers to the average level of
that cell type, or immunoregulatory molecule, present in a
population of cells that is non-activated. Those skilled in the art
are capable of determining if a T-cell, or a population of such
cells, is activated. For example, the expression of CD69, CD25
and/or CD154 proteins by a cell indicates that the cell has been
activated.
[0152] The basal level of a cell or molecule can be a specific
amount (e.g., a specific concentration) or it can encompass a range
of amounts. Basal levels, or ranges, of immune cells and
immunoregulatory molecules are known to those in the art. For
example, in a healthy individual, the normal level of CD4+ T-cells
present in human blood is 500-1500 cells/ml. Variability in this
measurement can result from differences in the method used to
determine the cell count. Furthermore, normal levels of cells can
also be reported as a percentage of a total cell population. For
example, in a healthy individual, Th40 cells make up less than 25%
of the total T cell population. Thus, as used herein, the term
inflammation refers to an inflammatory environment in which Th40
cells make up greater than about 25%, greater than about 30%,
greater than about 35%, greater than about 40%, greater than about
45%, greater than about 50%, greater than about 55%, greater than
about 60%, greater than about 65%, greater than about 70%, greater
than about 75%, or greater than about 80% of the total T-cell
population. Moreover, a preferred peptide herein is one that
reduces the level of Th40 cells to less than about 50%, less than
about 45%, less than about 40%, less than about 35%, less than
about 30%, less than about 27%, or equal to about 25% of the total
T-cell population. Methods of measuring different types of T-cells
in the T-cell population are known to those skilled in the art.
Furthermore, a novel method for detecting Th40 cells using peptides
hereof is disclosed herein.
[0153] As used herein, the phrase inflammatory environment refers
to the overall population of immune cells, and related
immunoregulatory molecules, that are present in a culture of cells,
or in the body of an animal. As such, the phrase inflammatory
environment encompasses the types, and/or the relative amounts of
immune cells and immunoregulatory molecules (e.g., cytokines)
present in a culture of cells, or in an animal, which are involved
in affecting an inflammatory reaction. Examples of cells
encompassed by the term inflammatory environment include, but are
not limited to, T cells, neutrophils, macrophages, granulocytes,
and the like. The inflammatory environment relates to cells and
molecules that mediate both acute and chronic inflammation. It will
be appreciated by those skilled in the art that the inflammatory
environment refers to the system to which peptides hereof are
administered. In one embodiment, the system is a cell culture
system. In one embodiment, the system is a whole animal.
[0154] A preferred peptide hereof is one that selectively interacts
with a CD40 protein in solution, as determined using an assay such
as an immunosorbent assay, or on the surface of a T-cell. As used
herein, the terms selectively, selective, specific, and the like,
indicate the peptide has a greater affinity for a CD40 protein than
it does for proteins unrelated to the CD40 protein. More
specifically, the terms selectively, selective, specific, and the
like indicate that the affinity of the peptide for CD40 is
statistically significantly higher than its affinity for a negative
control (e.g., an unrelated protein such as albumin) as measured
using a standard assay (e.g., ELISA). Suitable techniques for
assaying the ability of a peptide to selectively interact with a
CD40 protein are known to those skilled in the art. Such assays can
be in vitro or in vivo assays. Examples of useful assays include,
but are not limited to, an enzyme-linked immunoassay, a competitive
enzyme-linked immunoassay, a radioimmunoassay, a fluorescence
immunoassay, a chemiluminescent assay, a lateral flow assay, a
flow-through assay, an agglutination assay, a particulate-based
assay (e.g., using particulates such as, but not limited to,
magnetic particles or plastic polymers, such as latex or
polystyrene beads), an immunoprecipitation assay, an immunoblot
assay (e.g., a western blot), a phosphorescence assay, a
flow-through assay, a chromatography assay, a polyacrylamide gel
electrophoresis (PAGE)-based assay, a surface plasmon resonance
assay, a spectrophotometric assay, a particulate-based assay, an
electronic sensory assay and a flow cytometric assay. Methods of
performing such assays are well known to those skilled in the art.
In one embodiment, an assay can be performed using cells in
culture, or it can be performed in a whole animal. Assays can be
designed to give qualitative, quantitative or semi-quantitative
results, depending on how they are used and the type of result that
is desired.
[0155] One embodiment hereof is a peptide that interacts with a
CD40 protein in such a manner as to affect the interaction of the
CD40 protein with a CD154 protein, thereby modulating inflammation.
The effect of the peptide on the CD40/CD154 interaction can be
positive or it can be negative. For example, the peptide can
interact with the CD40 protein in such a manner that the strength
of the interaction between the CD40 protein and a CD154 protein is
increased. Alternatively, the peptide can interact with the CD40
protein such that the strength of the interaction between the CD40
protein and a CD154 protein is decreased. Methods of measuring the
strength of binding between the peptide and a CD40 protein are
known to those skilled in the art. A preferred peptide hereof is
one that reduces the strength of the interaction between a CD40
protein and a CD154 protein. Preferred peptides hereof reduce the
strength of binding between a CD40 protein and a CD154 protein by
at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, or at
least 95%. A particularly preferred peptide is one that completely
inhibits binding of CD40 to CD154. Complete inhibition of binding
between CD40 and CD154 means that when a peptide hereof is brought
into proximity with a CD40 protein and a CD154 protein under
conditions that would normally allow the interaction of CD40 and
CD154, no such interaction occurs and activation signals are not
stimulated in the CD40-expressing cell. Consequently CD40/CD154
mediated modulation of inflammation does not occur. In one
embodiment, the peptide interacts with the CD40 protein in such a
manner as to reduce the level of inflammation in the system. In one
embodiment, the peptide interacts with the CD40 protein in such a
manner as to inhibit the development of inflammation in the system.
In one aspect, the peptide may alter the way the cells may interact
with other molecules that normally occur during cell-to-cell
interactions. Such cell-to-cell interactions may be those that
result in inflammation. The peptides of the developments hereof,
may disrupt the inflammasome complex, which may promote the
maturation of pro-inflammatory cytokines interleukin-1.beta.
(IL-1.beta.) and interleukin-18 (IL-18). Furthermore, one aspect of
the peptides hereof is that these peptides may disrupt the
inflammasome complex of caspase 1, PYCARD, NALP, caspase 5,
nucleotide-binding oligomerization domain and leucine-rich
repeat-containing receptors (NLRs) and ALRs (AIM2-like
receptors).
[0156] One aspect of the peptides described herein is that
administration of the peptide may alter the way the cell may
interact with other molecules that normally occur during cell to
cell interactions which may be indicative of or result in
inflammation. One aspect of the peptides described herein is that
these peptide(s) may disrupt the inflammasome and thus alter
inflammatory outcomes.
[0157] While peptides hereof can interact with any site on the CD40
protein, preferred peptides interact with the CD40 protein at a
location that overlaps with the CD154 binding site. In one
embodiment, a peptide hereof interacts with the CD40 protein at the
CD154 binding site. An example of such a peptide is a CD40 ligand
competitive antagonist. As used herein, peptides that interfere
with, or inhibit, the binding of a CD154 protein to a CD40 protein
are referred to as small interfering peptides (SIPs). As used
herein a small interfering peptide is a peptide that, through
physio-chemical properties, interferes with the interaction of a
CD40 protein with a CD154 protein, thereby preventing activation
signals from being delivered to the CD40-bearing cell, thus
limiting the activation of the CD40-bearing cell, and consequently,
inflammation. As demonstrated herein, the consequences of such
interference are prevention of T-cell activation and propagation,
and a prevention or reduction of inflammation.
[0158] Additionally, a small interfering peptide, may, through its
physio-chemical properties, interfere with the interaction of a
CD40 protein with a CD154 protein, thereby preventing activation
signals from being delivered to the CD40-bearing cell, thus
limiting the activation of the CD40-bearing cell, and consequently,
modulating, inhibiting, preventing, and/or reversing type 2
diabetes. As demonstrated herein, the consequences of such
interference are prevention of T cell activation and propagation,
and a prevention, reduction, modulation, and reversal of type 2
diabetic developments.
[0159] A peptide useful for practicing methods of the present
developments should be of a size sufficient to interact with CD40
protein in such a manner as to modulate type 2 diabetes. It is
understood by those skilled in the art that preferred peptides are
relatively short since they are easier and less expensive to
produce. Preferred peptides are those that are less than 20 amino
acids in length. A preferred peptide is one that is 4, 6, 8, 10,
13, 15, or 24 amino acids in length. In one embodiment, the peptide
is an amino acid selected from the group of SEQ ID NO:3
(Core-sequence see Table 1), SEQ ID NO:4 (6-mer see Table 1), SEQ
ID NO:5 (8-mer mouse see Table 1), SEQ ID NO:6 (8-mer human see
Table 1), SEQ ID NO:7 (15-mer mouse see Table 1), SEQ ID NO:8
(15-mer human see Table 1), SEQ ID NO:9 (24-mer see Table 1), SEQ
ID NO: 24 (10-mer see Table 1), SEQ ID NO: 25 (13-mer see Table 1),
SEQ ID NO: 26 (24-mer see Table 1), SEQ ID NO: 27 (6-mer (Form 2)
see Table 1), SEQ ID NO: 28 (6-mer (Form 3) see Table 1), SEQ ID
NO: 29 (6-mer (Form 4) see Table 1), SEQ ID NO: 30 (6-mer (Form 4)
see Table 1) and SEQ ID NO:32 (24-mer-mouse (Form 2)). The
sequences of such peptides are shown below in Table 1 (which is an
exact duplicate of the previously listed Table 1, but repeated here
for the convenience of the reader).
TABLE-US-00002 TABLE 1 SEQ ID NO SEQUENCE Description 1 MIETYSQPSP
RSVATGLPAS MKIFMYLLTV SwissPro FLITQMIGSV LFAVYLHRRL DKVEEEVNLH
27548.2 Mouse EDFVFIKKLK RCNKGEGSLS LLNCEEMRRQ CD40 Ligand
FEDLVKDITL NKEEKKENSF EMQRGDEDPQ (CD154 IAAHVVSEAN SNAASVLQWA
KKGYYTMKSN Protein) LVMLENGKQL TVKREGLYYV YTQVTFCSNR EPSSQRPFIV
GLWLKPSSGS ERILLKAANT HSSSQLCEQQ SVHLGGVFEL QAGASVFVNV TEASQVIHRV
GFSSFGLLKL 2 MIETYNQTSP RSAATGLPIS MKIFMYLLTV SwissPro FLITQMIGSA
LFAVYLHRRL DKIEDERNLH 29965 Human EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ
CD40 Ligand FEGFVKDIML NKEETKKENS FEMQKGDQNP (CD154 QIAAHVISEA
SSKTTSVLQW AEKGYYTMSN Protein) NLVTLENGKQ LTVKRQGLYY IYAQVTFCSN
REASSQAPFI ASLCLKSPGR FERILLRAAN THSSAKPCGQ QSIHLGGVFE LQPGASVFVN
VTDPSQVSHG TGFTSFGLLK L 3 KGYY Core-sequence 4 KKGYYT 6-mer 5
AKKGYYTM 8-mer-mouse 6 AEKGYYTM 8-mer human 7 VLQWAKKGYYTMKSN
15-mer-mouse 8 VLQWAEKGYYTMSNN 15-mer human 9
NAASVLQWAKKGYYTMKSNLVMLE 24-mer 10 ISQAVHAAHAEINEAGR 15-mer from
ovalbumin; control peptide 11 G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-1
12 V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-2 13
V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-3 14
V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-N Gly-4 15
V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N Gly-5 16
V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N Gly-6 17
V-L-Q-W-A-K-G-G-Y-Y-T-M-K-S-N Gly-7 18
V-L-Q-W-A-K-K-G-G-Y-T-M-K-S-N Gly-8 19
V-L-Q-W-A-K-K-G-Y-G-T-M-K-S-N Gly-9 20
V-L-Q-W-A-K-K-G-Y-Y-G-M-K-S-N Gly-10 21
V-L-Q-W-A-K-K-G-Y-Y-T-G-K-S-N Gly-11 22 ISQAVHAAHAEINEAGR 15-mer
from ovalbumin; control peptide 23 YVQGKANLKSKLMYT Scrambled
peptide 24 WAKKGYYTMK 10-mer mouse 25 VLQWAKKGYYTMK 13-mer mouse 26
AASVLQW AKKGYYTMKSNLVVLEN 24-mer mouse 27 KGYYTM 6-mer (Form 2) 28
AEKGYY 6-mer (Form 3) 29 AKKGYY 6-mer (Form 4) 30 AKGYYT 6-mer
(Form 5) 31 YKNVKQMAYWLTGKS Scrambled peptide 32
AASVLQWAKKGYYTMKSNLVMLEN 24-mer-mouse (Form 2)
[0160] Interaction of a CD40 protein and a CD154 protein has been
shown to occur at particular regions within each protein. The
inventors have now shown that, surprisingly, a peptide comprising
only a short portion of the CD154 region that interacts with CD40
is capable of binding to a CD40 protein, thereby modulating type 2
diabetes. Thus one embodiment hereof is a peptide that comprises at
least a portion of the amino acid sequence of a CD154 protein such
that the peptide interacts with CD40 protein in such a manner as to
modulate type 2 diabetes. In one embodiment, interaction of the
peptide with CD40 protein results in negative modulation,
reduction, or inhibition of type 2 diabetes. In one aspect, the
peptide comprises at least a portion of SEQ ID NO:1 or SEQ ID
NO:2.
[0161] In one aspect, the peptide is as short as possible yet
comprises enough of the CD154 protein to allow interaction with a
CD 40 protein in such a manner as to modulate type 2 diabetes. In
one embodiment, a peptide hereof comprises 6, 13 or 15 contiguous
amino acids from SEQ ID NO:1 or SEQ ID NO:2, and interacts with
CD40 in such a manner as to modulate type 2 diabetes. A preferred
peptide comprises a core sequence of
lysine-glycine-tyrosine-tyrosine (KGYY; SEQ ID NO:3), which
corresponds to amino acids 142-145 of SEQ ID NO:1 and amino acids
143-146 of SEQ ID NO:2. Moreover, another preferred peptide
comprises a core sequence of
lysine-glycine-tyrosine-tyrosine-threonine-methionine (KGYYTM; SEQ
ID NO:27), which corresponds to amino acids 142-147 of SEQ ID NO:1
and amino acids 143-148 of SEQ ID NO:2. Useful peptides can
comprise additional regions of sequence from SEQ ID NO:1 or SEQ ID
NO:2 that are adjacent to the core sequence, so long as the peptide
is capable of modulating type 2 diabetes. In one embodiment hereof,
a peptide comprises at least one sequence selected from SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32 so
long as the peptide interacts with CD40 protein in such a manner as
to modulate type 2 diabetes. In one embodiment of the present
subject matter, a peptide hereof is a sequence selected from SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID
NO:32.
[0162] While peptides of the present subject matter can be selected
entirely of or from sequences that are responsible for the
interaction of the peptide with a CD40 protein, they may
additionally contain amino acid sequences that do not interact with
a CD40 protein, but which have other useful functions. Any useful,
additional amino acid sequence can be added to the CD40-interacting
sequence, so long as the additional sequences do not have an
unwanted effect on the ability of the CD40 interacting sequence to
interact with a CD40 protein. For example, in addition to the amino
acid sequence responsible for interacting with a CD40 protein, a
peptide hereof can contain amino acid sequences that are useful for
visualizing or purifying the peptide. Such sequences act as labels
(e.g., enzymes) or tags (antibody binding sites). Additionally, the
developments presented herein demonstrate that substitutions of
amino acids at any position other than the position-7 (K) of the
15-mer and the position-9 (Y) of the 15-mer may be made and the
integrity and function of the peptide may be maintained (see FIG.
8). Accordingly, these developments may contemplate that numerous
substitutions may be possible while still maintaining the
beneficial aspects of the peptide.
[0163] Any useful, additional amino acid sequence can be added to
the CD40-interacting sequence, so long as the additional sequences
do not have an unwanted effect on the ability of the CD40
interacting sequence to interact with a CD40 protein. For example,
in addition to the amino acid sequence responsible for interacting
with a CD40 protein, a peptide hereof can contain amino acid
sequences that are useful for visualizing or purifying the peptide.
Examples of such labels and tags include, but are not limited to,
B-galactosidase, luciferase, glutathione-s-transferase,
thioredoxin, HIS-tags, biotin tags, and fluorescent tags. Moreover,
acetyl groups and amides may be appended on the N-terminus or
C-terminus, and the developments hereof contemplate these and other
variations that may enhance stability or other traits desired of
such a peptide. Other useful sequences for labeling and tagging
proteins are known to those of skill in the art.
[0164] Likewise, peptides hereof can be modified, so long as such
modification does not significantly affect the ability of the
peptide to modulate type 2 diabetes. Such modifications can be
made, for example, to increase the stability, solubility or
absorbability of the protein. Examples of such modifications
include, but are not limited to pegylation, glycosylation and
chemical modification of the peptide.
[0165] Peptides hereof may possibly be derived from nature (e.g.,
obtained from plants, animals or microorganisms) or they can be
produced in a laboratory (e.g., recombinantly or synthetically).
Preferred peptides are those that are synthesized. Also encompassed
are peptides that are combinations of natural and synthetic
molecules. General methods for producing and isolating recombinant
or synthetic peptides are known to those skilled in the art. It
should be noted that, as used herein, an isolated, or biologically
pure, molecule, is one that has been removed from its natural
milieu. As such the terms isolated, biologically pure, and the
like, do not necessarily reflect the extent to which the protein
has been purified.
[0166] The peptides hereof do not arise naturally in a corporeal
body, but rather must be constructed and synthesized to obtain the
small interfering peptides. Certain aspects of the design and
synthesis may affect the peptides stability and ability to perform
its intended use. The peptides hereof may vary in length from four
amino acids in length as in SEQ ID NO:3, five amino acids in
length, six amino acids in length as in SEQ ID NOs: 4, 27, 28, 29,
and 30, seven amino acids in length, eight amino acids in length as
in SEQ ID NOs: 5 and 6, nine amino acids in length, ten amino acids
in length as in SEQ ID NOs: 24, eleven amino acids in length,
twelve amino acids in length, thirteen amino acids in length as in
SEQ ID NO: 25, fourteen amino acids in length, fifteen amino acids
in length as in SEQ ID NOs: 7, 8, 11, 12, 13, 14, 15, 16, 17, 18,
and 20, sixteen amino acids in length, seventeen amino acids in
length, eighteen amino acids in length, nineteen amino acids in
length, twenty amino acids in length, twenty-one amino acids in
length, twenty-two amino acids in length, twenty-three amino acids
in length, twenty-four amino acids in length as in SEQ ID NO: 26
and 32, and twenty-five amino acids in length. In some instances,
the embodiments of this development may be up to fifty or more
amino acids in length and in such instances, repeats of the core
sequence of SEQ ID NO: 3, may occur one, two, three, four, five,
six, seven, eight, nine, ten, eleven or more times in such a
peptide. In other instances, the embodiments of this development
may be up to fifty or more amino acids in length, and in such
instances repeats of the core sequence of SEQ ID NO:3, may be
varied with other sequences known to provide the desired effect
such as those of SEQ ID NOS: 4-9, 25-30, and 32. The repeats and
sequences of said variations are countless; however, the
developments herein contemplate a peptide that maintains its
ability to perform its intended use of interacting with CD40 in
such a manner as change, control, or affect inflammation in the
subject.
[0167] As has been described herein, interaction of the CD40
protein and the CD154 protein are necessary for involvement of Th40
cells in type 2 diabetes. Consequently, inhibition of the
interaction between a CD40 and CD154 protein using peptides hereof
is a useful method of affecting type 2 diabetes. Thus, one
embodiment is a method to reduce the interaction between a CD40
protein and a CD154 protein comprising introducing into an
environment containing a CD40 protein and a CD154 protein, a
peptide, that interacts with the CD40 protein in such a manner as
to reduce the interaction between the CD40 protein and the CD154
protein. In one aspect hereof, the peptide reduces the interaction
between the CD40 protein and the CD154 protein by at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, or at least
95%. In one embodiment, the peptide reduces the interaction between
the CD40 protein and the CD154 protein by a factor of at least 10,
at least 100, at least 1,000, at least 10,000. Methods of measuring
the strength of the interaction between the CD40 protein and the
CD154 protein have been discussed previously, and are also know to
those of skill in the art.
[0168] One embodiment hereof is a method to modulate type 2
diabetes comprising contacting a CD40 protein with a peptide that
interacts to the CD40 protein in such a manner as to modulate
inflammation. In one aspect, interaction of the peptide with the
CD40 protein increases the number of Th40 cells by at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, or at least
95%. In one embodiment, interaction of the peptide with the CD40
protein increases the number of Th40 cells by a factor of at least
10, at least 100, at least 1,000, at least 10,000.
[0169] One aspect is a method to reduce type 2 diabetes in a
patient, the method comprising administering a peptide hereof to
the patient. In one embodiment, the peptide comprises an amino acid
sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ
ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:25, SEQ
ID NO:26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:30
and SEQ ID NO:32. In one embodiment, the peptide is an amino acid
sequence selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ
ID NO:27, SEQ ID NO: 28, and SEQ ID NO: 29. In a preferred
embodiment, interaction of the peptide with the CD40 protein
decreases the number of Th40 cells by at least 5%, at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, or at least 95%. In
another embodiment, interaction of the peptide with the CD40
protein decreases the number of Th40 cells by a factor of at least
10, at least 100, at least 1,000, at least 10,000. In a preferred
embodiment, the level of Th40 cells is reduced so that Th40 cells
comprise no more than about 20%, about 25%, about 30%, about 35%,
or about 40% of the total T-cell population.
[0170] Peptides and methods hereof are suitable for use in cell
culture as well as for treating a patient. As used herein the term
patient refers to any animal in need of such treatment. The animal
can be a human or a non-human animal. A preferred animal to treat
is a mammal. A peptide can be administered or applied per se, or as
pharmaceutical compositions. A peptide hereof, or a pharmaceutical
composition thereof, can be administered to a patient by a variety
of routes, including, but limited to, by injection (e.g.,
intravenous, intramuscular, subcutaneous, intrathecal,
intraperitoneal), by inhalation, by oral (e.g., in a pill, tablet,
capsule, powder, syrup, solution, suspension, thin film, dispersion
or emulsion.), transdermal, transmucosal, pulmonary, buccal,
intranasal, sublingual, intracerebral, intravaginal rectal or
topical administration or by any other convenient method known to
those of skill in the art.
[0171] The amount of a peptide hereof and/or a pharmaceutical
composition thereof that will be effective can be determined by
standard clinical techniques known in the art. Such an amount is
dependent on, among other factors, the patient being treated,
including, but not limited to the weight, age, and condition of the
patient, the intended effect of the compound, the manner of
administration and the judgment of the prescribing physician. Also,
in this context, it should be noted that in treating a patient
exhibiting a disorder of interest, a therapeutically effective
amount of an agent or agents such as these is administered. A
therapeutically effective dose refers to that amount of the
compound that results in amelioration of one or more symptoms or a
prolongation of survival in a patient.
[0172] A peptide hereof, or a pharmaceutical composition thereof,
can be administered alone or in combination with one or more other
pharmaceutical agents, including other compounds of the present
disclosure. The specific pharmaceutical composition depends on the
desired mode of administration, as is well known to the skilled
artisan.
[0173] Because the inventors have discovered that Th40 cells are
intimately involved in the development of autoimmune diseases and
type 2 diabetes, the peptides and methods disclosed herein can be
used to affect conditioning resulting from such diseases. Thus, one
embodiment hereof is a method to treat type 2 diabetes in a patient
in need of such treatment, the method comprising administering to a
patient a peptide that interacts with the CD40 protein, thereby
reducing type 2 diabetes. In one embodiment the peptide interacts
with the CD40 protein in such a manner as to affect the interaction
of CD40 and CD154, thereby reducing type 2 diabetes. In a preferred
embodiment, interaction of the peptide with the CD40 protein
reduces the number of Th40 cells in a patient to a level equal to
that observed in subjects that do not have type 2 diabetes. In
another embodiment, interaction of the peptide with the CD40
protein reduces the inflammatory cytokine levels in a patient. In
another embodiment, interaction of the peptide with the CD40
protein reduces the inflammatory cytokine levels in a patient to a
level equal or similar to those observed in subjects that do not
have type 2 diabetes. The present developments are suitable for
treating any patient having an autoimmune disease and/or
cardiovascular disease, the development of which may be related to,
correlated with, or dependent on Th40 cells. The present
developments may also be suitable for treating any patient having
an autoimmune disease and/or cardiovascular disease, the
development of which is not related to, correlated with, or
dependent on Th40 cell counts, levels, and or concentrations.
[0174] In one embodiment, more specifically, peptides hereof may be
suitable for reducing the level of Th40 cells in such patients. In
this embodiment, a peptide hereof may reduce the level of Th40
cells in a patient suffering from an autoimmune disease to no more
than about 25% of the total T-cell population.
[0175] One example of a disease that is particularly amenable to
treatment using a peptide of the present developments may be type 2
diabetes. In type 2 diabetes, glucose tolerance is reduced, insulin
sensitivity is decreased, and plasma insulin levels are increased.
Consequently, control of inflammatory cells and cell signaling via
CD40-CD154 interaction may be able to be used to control, modulate,
reduce and/or reverse type 2 diabetes symptoms that are
characterized as by glucose intolerance, insulin resistance, and
increased plasma insulin levels. Several murine models of T2D
and/or atherosclerosis have been developed. For initial studies,
ApoE-/- mice were selected due to their ability to develop type 2
diabetes from a high fat diet. Glucose tolerance and insulin
testing were performed on all of the mice. The mice were
administered the 6-mer peptide (SEQ ID NO:29) at a rate of 1 mg/kg
weekly via I.V. injection and monitored. Peptide treated ApoE
deficient mice demonstrated significantly increased glucose
tolerance as well as significantly improved insulin sensitivity,
improve insulin resistance, and lowered plasma insulin levels
compared to controls. Thus, one embodiment of the present
developments is a method to prevent type 2 diabetes in an
individual at risk for developing type 2 diabetes, the method
comprising administering to the individual a peptide to selectively
bind to a CD40 expressing cell. In such an embodiment, the peptide
may be selected from SEQ ID NOS: 3-9 and 24-30.
[0176] The developments hereof have also shown that, surprisingly,
peptides hereof can be used to reverse the disease process in
individuals already showing signs of type 2 diabetes. Thus, one
aspect of the present subject matter is a method to reverse type 2
diabetes comprising administering to a patient diagnosed as having
type 2 diabetes, a peptide hereof. In one embodiment, the peptide
comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, and SEQ ID NO:30, so long as the peptide can
down-regulate or reduce inflammation. In one embodiment, the
peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, and SEQ ID NO:30. As used herein the phrase to
reverse type 2 diabetes means to increase glucose tolerance,
decrease insulin resistance, and decrease plasma insulin levels to
levels closer to or more comparable to those observed in
individuals who do not have type 2 diabetes.
[0177] In yet another development hereof has shown that,
surprisingly, peptides hereof can be used to reverse the disease
process in individuals already showing signs of type 2 diabetes.
Thus, one aspect of the present subject matter is a method to
reverse type 2 diabetes comprising administering to a patient
diagnosed as having type 2 diabetes, a peptide hereof. In one
embodiment, the peptide comprises an amino acid sequence selected
from SEQ ID NOS: 3-9 and 24-30, so long as the peptide can control,
modulate, reduce and/or reverse inflammation. In one embodiment,
the peptide is an amino acid sequence selected from SEQ ID NOS: 3-9
and 24-30, so long as the peptide can control, modulate, reduce
and/or reverse type 2 diabetes. Furthermore, in one aspect of this
embodiment a peptide hereof may reduce the level of Th40 cells in a
patient suffering from an autoimmune disease to no more than about
25% of the total T-cell population. In an alternative embodiment,
interaction of the peptide with the CD40 protein reduces the number
of Th40 cells in a patient to a level equal to that observed in
subjects that do not have type 2 diabetes. In another embodiment,
interaction of the peptide with the CD40 protein reduces the
inflammatory cytokine levels in a patient. In another embodiment,
interaction of the peptide with the CD40 protein reduces the
inflammatory cytokine levels in a patient to a level equal or
similar to those observed in subjects that do not have type 2
diabetes. The present developments may be suitable for treating any
patient having an autoimmune disease and/or cardiovascular disease,
the development of which may be related to, correlated with, or
dependent on Th40 cells. The present developments may also be
suitable for treating any patient having an autoimmune disease
and/or cardiovascular disease, the development of which is not
related to, correlated with, or dependent on Th40 cell counts,
levels, and or concentrations.
[0178] As has been described, peptides of the present invention
selectively bind to a CD40 expressing cell. Consequently, peptides
of the present subject matter can be used to identify Th40 cells.
Thus one embodiment hereof is a method to detect Th40-dependent
type 2 diabetes, said method comprising contacting a T-cell
population with a peptide hereof. In a preferred embodiment, the
peptide is labeled with a detectable marker, such as, for example,
fluorescein, luciferase or alkaline phosphatase. Such detection can
be performed using assay techniques known to those skilled in the
art. In general, an assay for detecting Th40 cells using a peptide
hereof comprises (a) obtaining a sample of cells; (b) contacting a
peptide hereof with said cells under condition suitable to allow
binding of the peptide to Th40 cells, if present; (c) washing said
cells using conditions that disrupt non-specific interactions, and
that remove unbound peptide; and (d) detecting peptide bound to
cells. Detection of bound peptide can be achieved directly or
indirectly. For example, direct detection can be achieved using a
peptide labeled using a detectable marker, as disclosed herein.
Following the wash step listed above, the cells are then simply
screened for the presence of detectable marker. The presence of
detectable marker in the cell sample indicates the presence of Th40
cells, and thus Th40-dependent type 2 diabetes. Alternatively,
indirect detection involves the use of a second molecule, such as
an antibody, that binds to the peptide. In an indirect detection
assay, following the wash step listed above, a detection molecule
that binds the peptide is added to the cell sample. The detection
molecule is labeled with a detectable marker. After washing away
unbound detection molecule, the cells are screened for the presence
of detectable marker. The presence of detectable marker in the cell
sample indicates the presence of Th40 cells. It should be
understood that the assays described herein are meant as examples
of useful assays, and other assay techniques can be employed.
Suitable assay techniques are known to those skilled in the art,
and are also disclosed in, for example, Molecular Cloning: A
Laboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T,
Cold Spring Harbor Laboratory Press; 2nd Edition (December 1989).
All references cited herein are incorporated herein in their
entirety.
[0179] The assay technology described above can also be used to
identify other molecules that affect the interaction of a CD40
protein with a CD514 protein. Examples of such molecules include,
but are not limited to, proteins, peptides and small molecules. For
example, assays can be designed that test the ability of molecules
to compete with a peptide of the present developments for binding
to a Th40 cell. For instance, a peptide labeled with a detectable
marker, can be mixed with a test molecule and a population of cells
known to contain Th40 cells, under conditions that allow binding of
the peptide to the Th40 cells. Following an appropriate incubation
period, the cells are washed to remove unbound peptide, and the
cells screened for the presence of detectable marker.
Alternatively, the labeled peptide could be bound to Th40 cells
first, and after a wash step to remove unbound peptide, the test
molecule could be added to the cells containing bound peptide.
Following an incubating period and a wash step to remove unbound
molecule, or released peptide, the cells are screened for the
presence of detectable marker. In either case, absence of the
detectable marker in the cell sample indicates the test molecule is
able to compete with the peptide for binding to the Th40 cells,
while presence of the detectable marker would indicate the test
molecule does not inhibit binding of the peptide to Th40 cells.
Inhibition of binding need not be 100%, as such assay would also be
useful for identifying molecules that partially inhibit binding of
the peptide to Th40 cells. It is understood by those skilled in the
art that such assays would involve the use of positive controls
(e.g., unlabeled peptide) and negative controls (e.g., a
protein/molecule that is known not to bind to Th40 cells).
[0180] The assay technology above can also be used to identify
other molecules that affect the interaction of a CD40 protein with
a CD154 proteins. Examples of such molecules include, but are not
limited to, proteins, peptides and small molecules. For example,
assays can be designed that test the ability of molecules to
compete with a peptide of the present developments for binding to a
CD40 protein of cells other than T cells, such as neutrophils,
eosinophils, basophils, mast cells, macrophages, platelets,
endothelial cells, and lymphocytes, including natural killer cells
and B cells. For instance, a peptide labeled with a detectable
marker, can be mixed with a test molecule and a population of cells
known to contain CD40 containing cells, under conditions that allow
binding of the peptide to the CD40 bearing cells. Following an
appropriate incubation period, the cells are washed to remove
unbound peptide, and the cells screened for the presence of
detectable marker. Alternatively, the labeled peptide could be
bound to CD40 bearing cells first, and after a wash step to remove
unbound peptide, the test molecule could be added to the cells
containing bound peptide. Following an incubating period and a wash
step to remove unbound molecule, or released peptide, the cells are
screened for the presence of detectable marker. In either case,
absence of the detectable marker in the cell sample indicates the
test molecule is able to compete with the peptide for binding to
the CD40 bearing cells, while presence of the detectable marker
would indicate the test molecule does not inhibit binding of the
peptide to CD40 bearing cells. Inhibition of binding need not be
100%, as such assay would also be useful for identifying molecules
that partially inhibit binding of the peptide to CD40 bearing
cells. It is understood by those skilled in the art that such
assays would involve the use of positive controls (e.g., unlabeled
peptide) and negative controls (e.g., a protein/molecule that is
known not to bind to CD40 bearing cells).
[0181] Because increased levels of Th40 cells are associated with
the development of autoimmune disease, the present developments can
be used to identify patients at risk for developing autoimmune
disease and autoimmune related type 2 diabetes. Thus, one
embodiment of the present developments is a method to identify a
patient at risk for developing autoimmune related type 2 diabetes.
In one embodiment, patients at risk for developing type 2 diabetes
are identified by obtaining a sample from a patient to be tested,
contacting the T-cell portion of said sample with a peptide hereof,
and determining the level of Th40 cells present in the sample,
wherein a level of Th40 cells greater than about 25% of the total
T-cell population indicates the patient is at risk for developing
type 2 diabetes. In one embodiment, the peptide comprises an amino
acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,
and SEQ ID NO:30, so long as the peptide binds to the CD40 protein.
In one embodiment, the peptide is an amino acid sequence selected
from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.
In a preferred embodiment the peptide is labeled with a suitable
detectable marker such as, for example, fluorescein, luciferase or
alkaline phosphatase. In yet another embodiment, the peptide
comprises an amino acid sequence selected from SEQ ID NOs: 4-9,
24-30, and 32, so long as the peptide binds to the CD40
protein.
[0182] The present developments also comprise kits useful for
practicing the methods disclosed herein, the kit comprising a
peptide that interacts with a CD40 protein in such a manner as to
modulate, reduce, prevent, treat, or otherwise improve symptoms of
type 2 diabetes. In one embodiment, the peptide comprises an amino
acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,
and SEQ ID NO:30, so long as the peptide can modulate type 2
diabetes. In one embodiment, the peptide is an amino acid sequence
selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID
NO:30. Another embodiment is a kit for determining the level of
Th40 cells, the kit comprising a peptide that interacts with a CD40
protein, and methods for detecting CD40-bound peptide. Kits can
also contain associated reagents and components, such as, but not
limited to, buffers, labels, containers, inserts, tubing, vials,
syringes, and the like.
[0183] The present developments also comprise kits useful for
practicing the methods disclosed herein, the kit comprising a
peptide that interacts with a CD40 protein in such a manner as to
modulate, reduce, prevent, treat, or otherwise improve symptoms of
type 2 diabetes. In one embodiment, the peptide comprises an amino
acid sequence selected from SEQ ID NOs: 3-9 and 24-30, so long as
the peptide can modulate type 2 diabetes. In one embodiment, the
peptide is an amino acid sequence selected from SEQ ID NOs: 3-9 and
24-30. Another embodiment is a kit for determining the level of
Th40 cells, the kit comprising a peptide that interacts with a CD40
protein, and methods for detecting CD40-bound peptide. Kits can
also contain associated reagents and components, such as, but not
limited to, buffers, labels, containers, inserts, tubing, vials,
syringes, and the like.
[0184] The following examples are provided for the purposes of
illustration and are not intended to limit the scope of the present
invention.
EXAMPLES
Example 1
[0185] This Example demonstrates the effect of various peptide
fragments of CD154 on CD4/CD8 ratios and the development of
diabetes in NOD mice.
[0186] Peptides were designed based on the amino acid sequence of
mouse CD154 protein (SEQ ID NO:1) in the SwissPro database. The
peptides (8-mer (SEQ ID NO: 5; SEQ ID NO: 6), 10-mer (SEQ ID
NO:24), 13-mer (SEQ ID NO:25), 15-mer (SEQ ID NO: 7), 24-mer (SEQ
ID NO:26), scrambled (SEQ ID NO: 23), and RGD
(arginylglycylaspartic acid) were then ordered from New England
Peptide. The RGD peptide is a 15-amino acid sequence from the CD154
sequence that does not include the CD40 binding motif. The
lyophilized peptides were suspended in sterile saline at 1 mg/ml.
25 ug in 100 ul (1 mg/kg) of a particular peptide was then injected
into the tail vein of 6-week old NOD mice. Control mice received
100 ul of sterile saline. This is well before the onset of diabetes
(and atherosclerosis), but after damage to pancreatic islets has
begun. Weekly after the initial injection, another 25 ug of peptide
(or 100 ul of saline in the case of the Control mice) was injected
into the tail vein. At 10 weeks of age, mice were monitored for
diabetes, as indicated by a blood glucose level greater than 250
mg/dL for three consecutive days. The results of this study are
shown in FIG. 1. During this time, blood was also taken from the
tail vein, or by sub-mandibular venal puncture, and the level of
CD4+ and CD8+ cells determined by flow cytometry using antibodies
for CD4 protein and CD8 protein. The results of this analysis are
shown in FIG. 2A.
[0187] Pancreata were excised and examined by histology for
cellular infiltrates and assigned scores based on observable,
measurable, and quantifiable data: 0=no infiltrate; 1=one pole
infiltrate; 2=peri-insulitis, bi-polar-infiltrates; 3=75%
infiltrate and 4=full infiltration. The results of this analysis
are shown in FIG. 2B.
[0188] The results demonstrate that treatment with a peptide
unrelated to the CD154 protein did not reduce the development of
diabetes in NOD mice. In contrast, treatment of mice with a 15-mer
peptide derived from the CD154 protein prevented the onset of
diabetes. Further, the 13-mer peptides derived from the CD154
protein had significant effects on the development of diabetes. In
addition, the data demonstrates that the 15-mer peptide did not
result in compromise of the immune system, as determined by the
CD4/CD8 ratio.
Example 2
[0189] This Example demonstrates the effect of the 15-mer peptide
on hyperglycemia in newly diabetic NOD mice.
[0190] Six mice from Example 1 that were not treated were allowed
to subsequently develop diabetes. These mice were injected
intravenously with 100 ug of the 15-mer peptide. These mice were
then given weekly injections of the 15-mer peptide into their tail
veins, and their blood glucose levels monitored twice-weekly. The
15-mer peptide was administered for a total of ten weeks, after
which the treatment was stopped. The results of this study are
shown in FIG. 3.
[0191] This study demonstrates that injection of the 15-mer peptide
into already diabetic mice can reverse hyperglycemia. It also
demonstrates that cessation of the treatment results in return of
hyperglycemia within 5 weeks.
Example 3
[0192] This study demonstrates the ability of the 15-mer peptide to
bind to Th40 cell and B cells.
[0193] Total lymphocytes were isolated from 9 week old NOD mice.
The lymphocytes were incubated with anti-CD4, anti-CD8, anti-CD40
and an FITC-labeled 15-mer peptide, and then analyzed by flow
cytometry. Cells were gated for CD4 (both CD4hi and CD4lo
populations were included) and CD40 versus the 15-mer peptide. The
results of this analysis are shown in FIG. 4.
[0194] B cells were isolated from the spleens of NOD mice. Sorted
MHC-II+ cells were purified from total lymphocytes. Cells were
stained with FITC-labeled 15 mer peptide, anti-CD40, and B cell
markers CD19 and CD21. MHC-II+ cells were gated for CD19+ and CD21+
and then 15-mer peptide versus CD40 antibody was measured. The
results of this study are shown in FIG. 5.
[0195] This study shows that a substantial majority, 90% of CD40+
T-cells, also bind the 15-mer peptide, thereby demonstrating that
the 15-mer peptide is highly specific for CD40+ cells. It also
shows that while 90% of B cells were CD40 positive, only 8% of B
cells bound the 15-mer peptide.
Example 4
[0196] This example demonstrates the level of CD40 positive cells
in the blood of type-I diabetic subjects and non-diabetic (control)
subjects.
[0197] 1 ml of whole blood was obtained from each individual and
incubated with biotin-conjugated, 15-mer peptide. The cells were
then exposed to horseradish peroxidase (HRP)-avidin, washed and the
absorbance at 405 nm determined using a spectrophotometer. The
results of this study are shown in FIG. 6.
[0198] This study demonstrates that blood cells from patients
having type-I diabetes had higher 15-mer peptide binding activity
than cells from non-diabetic controls.
Example 5
[0199] This example demonstrates the level of insulin granulation
observed in the pancreas of NOD mice treated with either the 15-mer
peptide or a peptide from ovalbumin.
[0200] At the onset of diabetes, six NOD mice were injected with
100 ug/ml of the 15-mer peptide, resulting in the reversal of
hyperglycemia in 80% of the recipients. Six weeks after reversal of
hyperglycemia, mice were sacrificed, and the pancreas removed for
analysis. The pancreas was fixed, sectioned and then stained using
an aldehyde/fuschsin stain that allows detection of insulin
granules. Granulation of the tissue was scored as follows:
4=completely granulated; 3=75% of islet granulated; 2=50% of islet
granulated, and peri-insulitis; 1=25% of islet granulated; 0=no
insulin granules detected. The results of this analysis are shown
in FIG. 7.
[0201] This analysis demonstrates that the 15-mer peptide preserved
insulin granules in the majority of the mice, and was significantly
improved in peptide-reversed diabetic mice compared to diabetic
mice that received an irrelevant peptide.
Example 6
[0202] This example demonstrates the effect of mutations in the
15-mer peptide on its ability to prevent the onset of diabetes.
FIG. 8 provides results related to this Example 6.
[0203] Peptide were designed and produced as described in Example
1. Variant peptides were produced so that in each variant, a
glycine was substituted for an amino acid corresponding to an amino
acid in positions 1-7 or 9-12 of SEQ ID NO:7, as follows:
TABLE-US-00003 Gly-1 (SEQ ID NO: 11) G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-N
Gly-2 (SEQ ID NO: 12) V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-3 (SEQ ID
NO: 13) V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-4 (SEQ ID NO: 14)
V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-N Gly-5 (SEQ ID NO: 15)
V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N Gly-6 (SEQ ID NO: 16)
V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N Gly-7 (SEQ ID NO: 17)
V-L-Q-W-A-K-G-G-Y-Y-T-M-K-S-N Gly-9 (SEQ ID NO: 18)
V-L-Q-W-A-K-K-G-G-Y-T-M-K-S-N Gly-10 (SEQ ID NO: 19)
V-L-Q-W-A-K-K-G-Y-G-T-M-K-S-N Gly-11 (SEQ ID NO: 20)
V-L-Q-W-A-K-K-G-Y-Y-G-M-K-S-N Gly-12 (SEQ ID NO: 21)
V-L-Q-W-A-K-K-G-Y-Y-T-G-K-S-N
[0204] NOD mice were placed in groups of 10, and the mice in each
group injected IV weekly with 25 ug of either wild-type (WT;
Legend) peptide or a variant peptide (in PBS, ph 7.2) listed above.
The development of diabetes was monitored by measuring blood
glucose levels on a weekly basis. Mice were considered "diabetic"
when blood glucose was 250 mg/dl or greater for 3 consecutive
readings. Injections began at 6 weeks of age=pre-diabetes.
[0205] This example demonstrates that substitution of a glycine at
any of positions 1-7, or 9-12, reduces the ability of the 15-mer
peptide to inhibit the development of diabetes. It also shows that
such mutations do not completely abolish the ability of the mutated
15-mer peptide to inhibit the development of diabetes.
Substitutions at the Tyr position 9, Thr position 11, and Met
position 12 proved differential for hyperglycemic prevention
activity, as shown in FIG. 8. Therefore it is also postulated that
a second 6-mer derivative--SEQ ID NO:27-6-mer (Form 2), SEQ ID
NO:28-6-mer (Form 3), SEQ ID NO:29-6-mer (Form 4), and SEQ ID
NO:30-6-mer (Form 5) may also provide increased therapeutic
efficacy.
Example 7
[0206] This example demonstrates that the same elevation of Th40
cell levels in the ApoE deficient mouse model of atherosclerosis is
also notably elevated in human Type 1 Diabetes (T1D).
[0207] The peripheral blood was measured was measured for total
count of CD3+CD4+CD40+ cell numbers in NOD, NOR (non-obese diabetic
resistant), and BALB/c (control) mice as in FIG. 9. This was
compared to the percentage of Th40 cells in peripheral blood in
human subjects for control, diabetic/new onset, and long term
diabetic populations as in FIG. 10. Further, lymphocytes were
isolated from 9-week old NOD mice. The lymphocytes were incubated
with anti-CD, anti-CD8, and an FITC-labeled 15-mer peptide, and
then analyzed by flow cytometry. Cells were gated for CD4 (both
CD4hi and CD4lo populations were included) and CD4 versus the
15-mer peptide. These results are displayed in FIG. 11.
[0208] ApoE deficient mice on a normal chow diet were selected to
receive a dose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by
IV tail injection, three times a week over a period of 26 weeks,
beginning at 9 weeks of age and also utilized a control. At 25
weeks, the animals were euthanized, weighed, and then had blood,
spleen, and pancreas removed for analysis. The subjects were then
perfused through cardiac puncture with 4% paraformaldehyde. Aortic
arches were dissected, dehydrated in sucrose gradient and then
flash frozen. Approximately thirty-five 8 um longitudinal sections
were obtained per mouse for various staining procedures. Flow
cytometry was performed utilizing a MACSQuant.RTM. Analyzer 10
(Miltenyi Biotec Inc.). Additional analysis was performed using
FlowJo.RTM. (FlowJo, LLC wholly owned by BectonDickinson, Inc.)
single-cell flow cytometry software.
[0209] C57BL/6 and ApoE-/- mice demonstrated increased levels of
Th40 cells relative to all CD3+CD4+ cells prior to hyperglycemia as
demonstrated in FIG. 12.
[0210] Further, NOD mice tested in this study demonstrated
significant Th40 infiltration in the aorta compared with control
and young non-diabetic NOD mice populations, as shown in FIG.
13.
[0211] An exemplar aortic plaque of one of the longitudinal
sections was observed at 200.times. magnification using oil-red-0,
trichrome stain and immune-fluorescence, and is shown in FIG. 14.
This microscopy and stain of the aorta showed that not only are the
Th40 cells increased in the aorta similarly to the peripheral blood
as demonstrated in FIGS. 10-14, but also the Th40 cells are found
within the shoulder region of plaque in the ApoE-/-model (the
growth region of plaque/atherosclerosis). In FIGS. 14, 10 and 20
identify cells that represent CD3+, CD4+, and CD40+ (Th40 cells)
that have significant intracellular CD40. 30 demonstrates Th40 cell
with extracellular expression and no demonstrative CD40
intracellularly. 40 identifies CD3+, CD4+, CD40neg cell.
Example 8
[0212] This example demonstrates that CD3+CD4+CD40+ cells appear to
produce interferon gamma (INF.gamma.) in abundance. Additionally,
interferon gamma controls Th40 proliferation.
[0213] ApoE deficient mice on a normal chow diet were selected to
receive a dose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by
IV tail injection, three times a week over a period of 26 weeks,
beginning at 9 weeks of age and also utilized a control. At 25
weeks, the animals were euthanized, weighed, and then had blood,
spleen, and pancreas removed for analysis. The subjects were then
perfused through cardiac puncture with 4% paraformaldehyde. Aortic
arches were dissected, dehydrated in sucrose gradient and then
flash frozen. Approximately thirty-five 8 m longitudinal sections
were obtained per mouse for various staining procedures. Flow
cytometry was performed utilizing a MACSQuant.RTM. Analyzer 10
(Miltenyi Biotec Inc.). Additional analysis was performed using
FlowJo.RTM. (FlowJo, LLC wholly owned by BectonDickinson, Inc.)
single-cell flow cytometry software.
[0214] As demonstrated in FIG. 14 through confocal microscopy, CD40
can be internal or external to the CD3+CD4+ cell. Flow cytometry
was further performed and demonstrated that while most CD3+ cells
appear to have ability to produce CD40, the CD3+CD4+CD40+ cells
appear to produce interferon gamma (IFN.gamma.) in abundance. This
flow cytometry study incorporated both the external and internal
staining of CD3, CD4, CD40, and IFN.gamma..
[0215] FIG. 16 demonstrates that interferon gamma controls Th40
proliferation. Isolated Th40 cells were cross-linked by antibody to
CD40. The graph in FIG. 16 denotes proliferation of CD40 stimulated
(CD40XL: activated) Th40 cells in the absence/presence of antibody
to INF.gamma. (.alpha.IFN.gamma.) and non-stimulated controls (UN).
Additionally, by blocking IFN.gamma., activated Th40 cells do not
proliferate.
Example 9
[0216] This example demonstrates that KGYY.sub.15 (15-mer--SEQ ID
NO:7) and KGYY.sub.6 (6-mer--SEQ ID NO:29) abrogates
atherosclerosis.
[0217] ApoE deficient mice on a normal chow diet were selected to
receive a dose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by
IV tail injection, three times a week over a period of 26 weeks,
beginning at 9 weeks of age and also utilized a control. At 25
weeks, the animals were euthanized, weighed, and then had blood,
spleen, and pancreas removed for analysis. The subjects were then
perfused through cardiac puncture with 4% paraformaldehyde. Aortic
arches were dissected, dehydrated in sucrose gradient and then
flash frozen.
[0218] FIG. 17 provides an example of the lesser curvature of the
aortic arch, defined proximally from the aortic outflow (AO). Of
the segments seen in the trichrome stain, an intimal distance of
2.4 mm was measured distally. The aortic-arch wall area subtended
by this 2.5 mm stretch of the intima was calculated for each
section of all mice, with maximal area of the inner-aortic-arch
wall of each mouse used to compute averages per group. The luminal
surface (L), aortic arch (AO), and innominate artery (I) are
labelled in this FIG. 17.
[0219] FIG. 18 demonstrates the lesser curvature of the aortic arch
of the control ApoE mice compared to the lesser curvature of the
aortic arch of mice treated with the 15-mer peptide, in accordance
with the steps outlined in this example.
[0220] FIG. 19 demonstrates the reduction of the total area
achieved by peptide treatment. The total area of the 2.5 mm segment
(as described in FIG. 17) was substantially reduced.
[0221] FIG. 20 demonstrates the reduction of number of plaque,
including early lesions and advanced plaque. The total number of
plaque was significantly decreased in the treatment group. Plaque
was subdivided based on morphology of early lesions (observable as
fatty streaks containing macrophage derived foam cells with varying
degrees of lipid accumulation) compared with more advanced
fibroatheromas (containing varying degrees of lipid or necrotic
core and fibrous caps). All plaque within the designated 2.5 mm
segment were included. Both the total number and type of plaque
were significantly decreased in those subjects treated with the
peptide.
[0222] This study demonstrates that administration of the peptide
abrogates atherosclerosis.
Example 10
[0223] This example demonstrates administration of the KGYY.sub.15
(15-mer--SEQ ID NO:7) augments cap formation while reducing
advancement of existing disease.
[0224] In this study, six ApoE-/- mice received a normal chow diet
from 0 to 20 weeks of age. At 20 weeks of age, three mice were
randomly assigned to receive dose of 1 mg/kg of KGYY.sub.15
(15-mer--SEQ ID NO:7) by IV tail injection, once a week for a
period of 4 weeks. Control mice received vehicle only. After 4
weeks of treatment, animals were euthanized then perfused through
cardiac puncture with 4% paraformaldehyde. Aortic arches were
dissected in surcrose gradient and flash frozen. Approximately
fifty, 8 um longitudinal sections were obtained per mouse. Slides
were treated with trichrome stain and analyzed using cellSens
software for measurements. Total plaque was measured including 2.5
mm lesser curvature and innominate artery.
[0225] FIG. 20 shows the number of individual early plaques and
advanced plaques were reduced in the treated subjects compared to
the control subjects.
[0226] FIG. 21 shows that the cap to core ratio of advanced plaques
was reduced in the treated subjects compared to the control
subjects.
[0227] FIG. 22 shows the average cap width (cap size) was greater
in the treated subjects compared to the control subjects.
[0228] FIG. 23 shows the average core width (core size/plaque size)
was decreased in the subjects treated with the peptide compared to
the control subjects.
[0229] This example demonstrates that administration of the
KGYY.sub.15 (15-mer--SEQ ID NO:7) augments cap formation while
reducing advancement of existing disease. Moreover, this study
further demonstrates that administration of the KGYY.sub.15 peptide
trends toward results of plaque stability.
[0230] From the foregoing, it is readily apparent that TID shares
with atherosclerosis the CD40-CD154 dyad which drives autoimmune
inflammation. There are increased Th40 cell levels in peripheral
blood of NOD mice, human TID patients, and ApoE-/- mice. Th40 cells
infiltrate the aortic wall and are found within the plaque of
ApoE-/- mice. Th40 cells produce the inflammatory cytokine
IFN.gamma. at a level greater than that of other cells and this
drives inflammation. The KGYY.sub.15 peptide targets Th40 cells.
The specified peptide furthermore abrogates and modulates
atherosclerosis which may be due to Th40 blockade or general
blockade of CD40. Moreover, the administration of the specified
peptide trends toward more stable plaque types.
Example 11
[0231] Whole human blood was administered the peptide in accordance
with similar dosing levels to those used for murine studies.
[0232] FIG. 24 provides the results of clot studies observed in
humans.
[0233] This study demonstrates that the KGYY.sub.15 peptide when
administered to humans does not modify or change the clotting of
whole human blood significantly outside of normally recognized
levels.
Example 12
[0234] This example demonstrates that ApoE mice that have been
genetically modified to obtain atherosclerosis and fed a high fat
diet and treated with the 6-mer peptide may have the levels of LDL
cholesterol values decreased compared to untreated subjects.
[0235] In this study, six ApoE-/- mice received a normal chow diet
from 0 to 20 weeks of age. At 20 weeks of age, three mice were
randomly assigned to receive dose of 1 mg/kg of KGYY.sub.6
(6-mer--SEQ ID NO:29) by IV tail injection, once a week for a
period of 4 weeks. Control mice received vehicle only.
[0236] Data obtained from untreated mice and compared to those in
treated mice showed statistically significant reduction (>50%)
in LDL cholesterol values. This data is provided in FIG. 27.
Example 13
[0237] This example demonstrates atherosclerotic changes that ApoE
-/- mice experienced when treated with KGYY.sub.6 (6-mer--SEQ ID
NO: 29). ApoE -/- mice were fed a high fat diet for 16 weeks. Mice
were randomly assigned to receive dose of 1 mg/kg of KGYY.sub.6
(6-mer--SEQ ID NO:29) by IV tail injection, once a week for a
period of 4 weeks. Control mice received vehicle only.
Atherosclerosis was investigated by several methods. En-face
analysis utilizing Sudan IV stain (lipid stain) demonstrated a
significant reduction in lesion areas. FIG. 28A is an image of
KGYY6 treated aortic en-face Sudan IV staining and FIG. 28B is an
image of control (untreated) aortic en-face Sudan staining. FIG. 29
is a graph demonstrating the reduction of lesion areas of Sudan IV
staining.
[0238] Further, measurement of plaque area and morphology was
performed using the Paigen method. This method obtains sequential 5
m aortic cross sections from the aortic root beginning at the valve
leaflets into the ascending aorta. At 50 .mu.m intervals, slides
are stained after which the area of atherosclerotic lesion is
measured. Individual plaque area measurements are plotted against
the micrometer intervals and a curve is established, with the area
under the curve (AUC) giving the total volume of plaque. These
results are presented in graph format in FIG. 30, which
demonstrates that mice treated with KGYY.sub.6 (SEQ ID NO: 29)
showed reduction in plaque volume under the curve.
Moreover, characterization of plaque composition or aortic samples
was performed using trichrome staining techniques and these results
are presented in graph format in FIG. 31. Indeed, this FIG. 31
data, which was generated using Image Pro Plus software analysis,
quantifies and shows that plaque compositional changes occurred,
including increased collagen and reduced smooth muscle content.
[0239] FIG. 32A is an image of trichrome stained cells of the
cross-sections of the aorta of the KGYY.sub.6 treated subject. FIG.
32B is an image of trichrome stained cells of the cross-sections of
the control subjects. In FIG. 32A, 50 identifies areas
characteristic of plaque formation. 60 identifies aortic valve
leaflets. FIG. 32B, the control (untreated) 50 areas characteristic
of plaque (area under the curve) are greater than those in the
subjects treated with the KGYY.sub.6 peptide.
Example 15
[0240] This example demonstrates that KGYY.sub.6 (6-mer--SEQ ID
NO:29) results in significant improvement in glucose tolerance and
insulin sensitivity.
[0241] ApoE -/- mice were fed a 60% high fat diet (research diet)
for 1 week. A select population of mice were then injected with
6-mer peptide at a dose of 1 mg/kg and others were untreated and
tracked as controls. Glucose tolerance testing (GTT) was performed
by fasting 6 hours, followed by intraperitoneal injection of 1 g/kg
body weight glucose in water. Both blood glucose and blood serum
insulin were measured at 0, 15 minutes, 30 minutes, 60 minutes, and
2 hours. The results of this study are shown in FIGS. 33(a) and
33(b). Peptide treated ApoE deficient mice demonstrated
significantly increased glucose tolerance as well as significantly
improved insulin sensitivity and lowered plasma insulin levels
compared to control.
[0242] Western analysis was performed on adipose and muscle tissue
of the treated and untreated mice, demonstrating an increase in
expression of the glucose transport protein (GLUT4) in white
adipose tissue, as shown in FIG. 34. Moreover, the western analysis
also demonstrated that the muscle tissue showed increased
expression of the GLUT4 protein. GLUT4 is responsible for glucose
uptake in response to insulin and known to have reduced expression
in type 2 diabetes.
[0243] This study demonstrates that administration of the peptide
affects glucose tolerance, insulin sensitivity, and GLUT4 levels,
each of which may be important to treating type 2 diabetic
subjects.
Example 16
[0244] This example demonstrates administration of the KGYY.sub.6
(6-mer--SEQ ID NO:29) affects the inflammatory cytokine production
of IL2, INF.gamma., and IL17a.
[0245] Spleens from ApoE mice and C57BL/6 mice were processed for
lymphocytes. ApoE -/- and C57BL/6 mice were fed a 60% high fat diet
(research diet) for 1 week. Splenic lymphocytes were treated with
6-mer in vitro for 24 hours. Cells were placed in media overnight
in the presence of varying concentrations of 6-mer peptides. The
following morning, Brefeldin A was administered for 4 hours. All
cells were stained for CD3, CD4, CD40 (Th40 cells) and measured by
use of flow cytometry for their production of IL2, INF.gamma., and
IL17a. The results of this study are shown in FIG. 35.
[0246] From the foregoing, it is readily apparent that new and
useful implementations of the methods have been herein described
and illustrated which fulfill numerous desiderata in remarkably
unexpected fashions. It is, of course, understood that such
modifications, alterations and adaptations as may readily occur to
the artisan confronted with this disclosure are intended within the
spirit of this disclosure, which is limited only by the scope of
the claims appended hereto.
Sequence CWU 1
1
321260PRTMus musculus 1Met Ile Glu Thr Tyr Ser Gln Pro Ser Pro Arg
Ser Val Ala Thr Gly1 5 10 15Leu Pro Ala Ser Met Lys Ile Phe Met Tyr
Leu Leu Thr Val Phe Leu 20 25 30Ile Thr Gln Met Ile Gly Ser Val Leu
Phe Ala Val Tyr Leu His Arg 35 40 45Arg Leu Asp Lys Val Glu Glu Glu
Val Asn Leu His Glu Asp Phe Val 50 55 60Phe Ile Lys Lys Leu Lys Arg
Cys Asn Lys Gly Glu Gly Ser Leu Ser65 70 75 80Leu Leu Asn Cys Glu
Glu Met Arg Arg Gln Phe Glu Asp Leu Val Lys 85 90 95Asp Ile Thr Leu
Asn Lys Glu Glu Lys Lys Glu Asn Ser Phe Glu Met 100 105 110Gln Arg
Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val Ser Glu 115 120
125Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys Gly Tyr
130 135 140Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys
Gln Leu145 150 155 160Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr
Thr Gln Val Thr Phe 165 170 175Cys Ser Asn Arg Glu Pro Ser Ser Gln
Arg Pro Phe Ile Val Gly Leu 180 185 190Trp Leu Lys Pro Ser Ser Gly
Ser Glu Arg Ile Leu Leu Lys Ala Ala 195 200 205Asn Thr His Ser Ser
Ser Gln Leu Cys Glu Gln Gln Ser Val His Leu 210 215 220Gly Gly Val
Phe Glu Leu Gln Ala Gly Ala Ser Val Phe Val Asn Val225 230 235
240Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser Phe Gly
245 250 255Leu Leu Lys Leu 2602261PRTHomo sapiens 2Met Ile Glu Thr
Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly1 5 10 15Leu Pro Ile
Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30Ile Thr
Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45Arg
Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55
60Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser65
70 75 80Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val
Lys 85 90 95Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser
Phe Glu 100 105 110Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala
His Val Ile Ser 115 120 125Glu Ala Ser Ser Lys Thr Thr Ser Val Leu
Gln Trp Ala Glu Lys Gly 130 135 140Tyr Tyr Thr Met Ser Asn Asn Leu
Val Thr Leu Glu Asn Gly Lys Gln145 150 155 160Leu Thr Val Lys Arg
Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175Phe Cys Ser
Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190Leu
Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200
205Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
210 215 220Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe
Val Asn225 230 235 240Val Thr Asp Pro Ser Gln Val Ser His Gly Thr
Gly Phe Thr Ser Phe 245 250 255Gly Leu Leu Lys Leu 26034PRTMus
musculus 3Lys Gly Tyr Tyr146PRTArtificial SequenceSynthetic Peptide
4Lys Lys Gly Tyr Tyr Thr1 558PRTArtificial SequenceSynthetic
Peptide 5Ala Lys Lys Gly Tyr Tyr Thr Met1 568PRTArtificial
SequenceSynthetic Peptide 6Ala Glu Lys Gly Tyr Tyr Thr Met1
5715PRTArtificial SequenceSynthetic Peptide 7Val Leu Gln Trp Ala
Lys Lys Gly Tyr Tyr Thr Met Lys Ser Lys1 5 10 15815PRTArtificial
SequenceSynthetic Peptide 8Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr
Thr Met Ser Asn Asn1 5 10 15924PRTArtificial SequenceSynthetic
Peptide 9Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys Gly Tyr Tyr
Thr Met1 5 10 15Lys Ser Asn Leu Val Met Leu Glu 201017PRTArtificial
SequenceSynthetic Peptide 10Ile Ser Gln Ala Val His Ala Ala His Ala
Glu Ile Asn Glu Ala Gly1 5 10 15Arg1115PRTArtificial
SequenceSynthetic Peptide 11Gly Leu Gln Trp Ala Lys Lys Gly Tyr Tyr
Thr Met Lys Ser Asn1 5 10 151215PRTArtificial SequenceSynthetic
Peptide 12Val Gly Gln Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser
Asn1 5 10 151315PRTArtificial SequenceSynthetic Peptide 13Val Leu
Gly Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn1 5 10
151415PRTArtificial SequenceSynthetic Peptide 14Val Leu Gln Gly Ala
Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn1 5 10 151515PRTArtificial
SequenceSynthetic Peptide 15Val Leu Gln Trp Gly Lys Lys Gly Tyr Tyr
Thr Met Lys Ser Asn1 5 10 151615PRTArtificial SequenceSynthetic
Peptide 16Val Leu Gln Trp Ala Gly Lys Gly Tyr Tyr Thr Met Lys Ser
Asn1 5 10 151715PRTArtificial SequenceSynthetic Peptide 17Val Leu
Gln Trp Ala Lys Gly Gly Tyr Tyr Thr Met Lys Ser Asn1 5 10
151815PRTArtificial SequenceSynthetic Peptide 18Val Leu Gln Trp Ala
Lys Lys Gly Gly Tyr Thr Met Lys Ser Asn1 5 10 151915PRTArtificial
SequenceSynthetic Peptide 19Val Leu Gln Trp Ala Lys Lys Gly Tyr Gly
Thr Met Lys Ser Asn1 5 10 152015PRTArtificial SequenceSynthetic
Peptide 20Val Leu Gln Trp Ala Lys Lys Gly Tyr Tyr Gly Met Lys Ser
Asn1 5 10 152115PRTArtificial SequenceSynthetic Peptide 21Val Leu
Gln Trp Ala Lys Lys Gly Tyr Tyr Thr Gly Lys Ser Asn1 5 10
152217PRTArtificial SequenceSynthetic Peptide 22Ile Ser Gln Ala Val
His Ala Ala His Ala Glu Ile Asn Glu Ala Gly1 5 10
15Arg2315PRTArtificial SequenceSynthetic Peptide 23Tyr Val Gln Gly
Lys Ala Asn Leu Lys Ser Lys Leu Met Tyr Thr1 5 10
152410PRTArtificial SequenceSynthetic Peptide 24Trp Ala Lys Lys Gly
Tyr Tyr Thr Met Lys1 5 102513PRTArtificial SequenceSynthetic
Peptide 25Val Leu Gln Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys1 5
102624PRTArtificial SequenceSynthetic Peptide 26Ala Ala Ser Val Leu
Gln Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys1 5 10 15Ser Asn Leu Val
Val Leu Glu Asn 20276PRTArtificial SequenceSynthetic Peptide 27Lys
Gly Tyr Tyr Thr Met1 5286PRTArtificial SequenceSynthetic Peptide
28Ala Glu Lys Gly Tyr Tyr1 5296PRTArtificial SequenceSynthetic
Peptide 29Ala Lys Lys Gly Tyr Tyr1 5306PRTArtificial
SequenceSynthetic Peptide 30Ala Lys Gly Tyr Tyr Thr1
53115PRTArtificial SequenceSynthetic Peptide 31Tyr Lys Asn Val Lys
Gln Met Ala Tyr Trp Leu Thr Gly Lys Ser1 5 10 153224PRTArtificial
SequenceSynthetic Peptide 32Ala Ala Ser Val Leu Gln Trp Ala Lys Lys
Gly Tyr Tyr Thr Met Lys1 5 10 15Ser Asn Leu Val Met Leu Glu Asn
20
* * * * *
References