U.S. patent application number 12/177798 was filed with the patent office on 2009-09-03 for methods and compositions for treatment of graft rejection and promotion of graft survival.
This patent application is currently assigned to Regents of the University of Colorado. Invention is credited to Charles A. Dinarello, Eli C. Lewis, Leland Shapiro.
Application Number | 20090220518 12/177798 |
Document ID | / |
Family ID | 37499146 |
Filed Date | 2009-09-03 |
United States Patent
Application |
20090220518 |
Kind Code |
A1 |
Dinarello; Charles A. ; et
al. |
September 3, 2009 |
METHODS AND COMPOSITIONS FOR TREATMENT OF GRAFT REJECTION AND
PROMOTION OF GRAFT SURVIVAL
Abstract
Embodiments herein illustrate methods of treating, reducing or
preventing transplantation rejection and/or side effects associated
with transplantation. Some embodiments relate to compositions and
methods for inhibition of graft rejection and promotion of graft
survival. Other embodiments relate to modulation of cellular
activities, including graft rejection, promotion of graft survival,
graft versus host rejection and conditions commonly associated with
graft rejection. Yet other embodiments relate to the inhibitory
compounds comprising naturally occurring and man-made inhibitors of
serine protease, derivatives and fragments of the carboxy-terminus
of alpha1-antitrypsin and inducers of other alpha1-antitrypsin
activities and uses thereof.
Inventors: |
Dinarello; Charles A.;
(Boulder, CO) ; Lewis; Eli C.; (Rehovot, IL)
; Shapiro; Leland; (Denver, CO) |
Correspondence
Address: |
FAEGRE & BENSON LLP;PATENT DOCKETING - INTELLECTUAL PROPERTY
2200 WELLS FARGO CENTER, 90 SOUTH SEVENTH STREET
MINNEAPOLIS
MN
55402-3901
US
|
Assignee: |
Regents of the University of
Colorado
Denver
CO
|
Family ID: |
37499146 |
Appl. No.: |
12/177798 |
Filed: |
July 22, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11916521 |
Dec 4, 2007 |
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PCT/US06/22436 |
Jun 7, 2006 |
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12177798 |
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60687850 |
Jun 7, 2005 |
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Current U.S.
Class: |
424/141.1 ;
514/1.1; 514/2.4 |
Current CPC
Class: |
A61K 38/217 20130101;
A61P 3/10 20180101; A61K 38/57 20130101; A61K 35/39 20130101; A61P
17/02 20180101; A61K 45/06 20130101; A61K 2039/505 20130101; A61P
1/18 20180101; A61K 38/191 20130101; A61P 29/00 20180101; A61P
37/02 20180101; A61P 37/06 20180101; C07K 16/40 20130101; A61K
38/191 20130101; A61K 2300/00 20130101; A61K 38/57 20130101; A61K
2300/00 20130101; A61K 35/39 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/141.1 ;
514/8 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 38/14 20060101 A61K038/14; A61P 37/06 20060101
A61P037/06; A61P 3/10 20060101 A61P003/10 |
Goverment Interests
FEDERALLY FUNDED RESEARCH
[0002] The studies disclosed herein were supported in part by grant
number AI-15614 from the National Institutes of Health. The U.S.
government may have certain rights to practice the subject
invention.
Claims
1. A method for preventing or reducing the risk of developing an
organ or cellular transplant rejection in a subject having had or
undergoing a cellular transplant, the method comprising
administering to the subject a composition comprising alpha-1
antitrypsin (AAT), alpha-1 antitrypsin-like compound or combination
thereof.
2. The method of claim 1, wherein the composition is administered
to the subject before transplantation, during transplantation,
after transplantation or combination thereof.
3. The method of claim 1, wherein the composition further comprises
one or more anti-transplant rejection agent, anti-inflammatory
agent, immunosuppressive agent, immunomodulatory agent,
anti-microbial agent, or a combination thereof.
4. The method of claim 1, wherein the composition comprises a
carboxy-terminal peptide corresponding to AAT, an analog thereof,
any derivative of AAT carboxy terminus that binds to serpin-enzyme
complex (SEC) receptor or a combination thereof.
5. The method of claim 1, wherein the cellular transplant is
selected from the group consisting of cornea, bone marrow, liver,
stem cell, pancreatic islet, pancreas, kidney, lung, intestine, and
a combination thereof.
6. The method of claim 5, wherein the cellular transplant is
pancreatic islet cell transplant.
7. The method of claim 3, wherein the immunosuppressive agent is
chosen from inhibitors of apoptosis, serine protease inhibitors,
reducers of lymphocyte numbers, reducers of cytokine production,
reducers of cytokine activities, monoclonal antibodies, reducers of
cytokine receptors, reducers of nitric oxide production and a
combination thereof.
8. The method of claim 7, wherein reducers of cytokine production,
reducers of cytokine activities, reducers of cytokine receptors is
an inhibitor of one or more of TNF.alpha. (tumor necrosis factor
alpha), IL-1 (interleukin-1), IL-12 (interleukin-12), IL-18
(interleukin-18), IL-17 (interleukin-17), IL-23 (interleukin-23),
IL-32 (interleukin-32), IFN.gamma. (interferon gamma) or a
combination thereof.
9. A method for treating organ or cellular transplant rejection in
a subject in need of such a treatment comprising: identifying a
subject having or in need of a cellular or organ transplant;
performing cellular or organ transplantation on the subject; and
administering a therapeutically effective amount of a composition
comprising AAT, AAT-like compound, AAT analog, AAT derivative,
serine protease inhibitor, one or more carboxy-terminal peptides
derived from AAT, any derivative of AAT carboxy terminus that binds
to serpin-enzyme complex (SEC) receptor or combination thereof to
the subject.
10. The method of claim 9, wherein administration comprises
administering the composition to the organ or cells to be
transplanted before transplant, administering the composition to
the subject before transplant, administering the composition to the
subject during transplantation, administering the composition to
the subject after transplantation or a combination thereof.
11. The method of claim 9, wherein treating the subject with the
composition reduces the risk of transplantation rejection by at
least 10% compared to a subject not treated with the
composition.
12. The method of claim 9, wherein the cellular transplant is
selected from the group consisting of cornea, bone marrow, liver,
stem cell, pancreatic islet, pancreas, kidney, lung, intestine,
skin and a combination thereof.
13. The method of claim 9, wherein the composition further
comprises one or more anti-transplant rejection agent,
anti-inflammatory agent, immunosuppressive agent, immunomodulatory
agent, anti-microbial agent, or a combination thereof.
14. A method for treating diabetes in a subject in need of such a
treatment comprising: identifying a subject having or at risk of
developing diabetes; performing pancreatic islet cell
transplantation on the subject; and administering a therapeutically
effective amount of a composition comprising AAT, AAT-like
compound, AAT analog, AAT derivative, a serine protease inhibitor,
one or more carboxy-terminal peptides derived from AAT, any
derivative or fragment of AAT carboxy terminus that binds to
serpin-enzyme complex (SEC) receptor or combination thereof to the
subject.
15. The method of claim 14, wherein administration comprises
administering the composition to the organ or cells to be
transplanted before transplant, administering the composition to
the subject before transplant, administering the composition to the
subject during transplantation, administering the composition to
the subject after transplantation or a combination thereof.
16. The method of claim 14, wherein the subject has or is at risk
of developing Type 1 diabetes.
17. The method of claim 14, wherein the subject is in early phase
type 1 diabetes.
18. The method of claim 14, wherein the subject has or is at risk
of developing Type 2 diabetes.
19. The method of claim 14, wherein treating the subject with the
composition decreases the risk of transplantation rejection by at
least 10% compared to a subject not treated with the
composition.
20. The method of claim 14, wherein the compound comprises one or
more carboxy-terminal peptides derived from AAT, an analog thereof,
any derivative or fragment of AAT carboxy terminus that binds to
serpin-enzyme complex (SEC) receptor or a combination thereof.
21. A method for reducing a side-effect of cellular transplant
rejection in a subject, the method comprising administering to the
subject a composition comprising alpha-1 antitrypsin (AAT), one or
more carboxy-terminal peptides derived from AAT, or alpha-1
antitrypsin-like compound.
22. The method of claim 21, wherein administration comprises
administering the composition to the organ or cells to be
transplanted before transplant, administering the composition to
the subject before transplant, administering the composition to the
subject during transplantation, administering the composition to
the subject after transplantation or a combination thereof.
23. The method of claim 21, wherein the composition further
comprises one or more anti-transplant rejection agent,
anti-inflammatory agent, immunosuppressive agent, immunomodulatory
agent, anti-microbial agent, or a combination thereof.
24. The method of claim 21, wherein the compound comprises a
carboxy-terminal peptide corresponding to AAT, an analog thereof,
any derivative or fragment of AAT carboxy terminus that binds to
serpin-enzyme complex (SEC) receptor or a combination thereof.
25. The method of claim 21, wherein the cellular transplant is
selected from the group consisting of cornea, bone marrow, liver,
stem cell, pancreatic islet, pancreas, kidney, lung, intestine, and
a combination thereof.
26. The method of claim 21, wherein the cellular transplant is
pancreatic islet cell transplant.
27. The method of claim 21, wherein the side effects of cellular or
organ transplant are selected from the group consisting essentially
of production of pro-inflammatory cytokines, infiltration of
immunocompetent cells, infiltration of inflammatory cells,
infiltration of cytotoxic T-cells, infiltration of mature dendritic
cells, infiltration of monocytes, production of nitric oxide,
production of prostaglandins, production of reactive oxygen
species, production of super oxide radicals, infiltration of
natural killer cells, infiltration of natural killer T-cells and a
combination thereof.
28. The method of claim 23, wherein the immunosuppressive agent is
chosen from inhibitors of apoptosis, reducers of cytokine
production, reducers of cytokine activities, reducers of nitric
oxide production and a combination thereof.
29. The method of claim 21, wherein the composition inhibits the
production of TNF.alpha. (tumor necrosis factor alpha), IL-1
(interleukin-1), IL-12 (interleukin-12), IL-18 (interleukin-18),
IFN.gamma. (interferon gamma), nitric oxide (NO) or a combination
thereof.
30. A method for preventing or reducing the risk of developing
pancreatic islet cell transplant rejection in a subject having had
or undergoing a pancreatic islet cell transplant, the method
comprising administering to the subject a composition comprising
alpha-1 antitrypsin (AAT), one or more carboxy-terminal peptides
derived from AAT, engager of the SEC receptor, alpha-1
antitrypsin-like compound, serine protease inhibitor or combination
thereof.
31. The method of claim 30, wherein administration comprises
administering the composition to the organ or cells to be
transplanted before transplant, administering the composition to
the subject before transplant, administering the composition to the
subject during transplantation, administering the composition to
the subject after transplantation or a combination thereof.
32. The method of claim 30, wherein the composition further
comprises one or more anti-transplant rejection agent,
anti-inflammatory agent, immunosuppressive agent, immunomodulatory
agent, anti-microbial agent, or a combination thereof.
33. The method of claim 30, wherein the composition comprises a
carboxy-terminal peptide corresponding to AAT, an analog thereof,
any derivative or fragment of AAT carboxy terminus that binds to
serpin-enzyme complex (SEC) receptor or a combination thereof.
34. The method of claim 30, wherein the subject has juvenile or
late onset type 1 diabetes.
35. The method of claim 30, wherein the subject has type 2
diabetes.
36. The method of claim 30, wherein reducing the risk of developing
pancreatic islet cell transplant rejection in a subject comprises
reducing the risk by at least 10 percent in the subject compared to
a second subject not treated with the composition.
37. The method of claim 30, wherein the subject is a human.
38. The method of claim 30, wherein the subject is a domesticated
animal or livestock.
39. A pharmaceutical composition comprising, AAT, AAT-like
compound, serine protease inhibitor, AAT analog, AAT derivative,
one or more carboxy-terminal peptides derived from AAT, any
derivative or fragment of AAT carboxy terminus that binds to
serpin-enzyme complex (SEC) receptor or combination thereof and at
least one of an anti-transplant rejection agent, an
anti-inflammatory agent, an immunosuppressive agent, an
immunomodulatory agent, and an anti-microbial agent.
40. The pharmaceutical composition of claim 39, wherein the
compositions is AAT, serine protease inhibitor, AAT-like compound,
AAT analog, AAT derivative, carboxy-terminal peptide corresponding
to AAT, any derivative or fragment of AAT carboxy terminus that
binds to serpin-enzyme complex (SEC) receptor or combination
thereof and one or more anti-transplant rejection agents.
41. A method for inducing immunological tolerance in a subject
undergoing a cellular or organ transplant comprising: the method
comprising administering to the subject a composition comprising
alpha-1 antitrypsin (AAT), alpha-1 antitrypsin-like compound or
combination thereof.
42. The method of claim 41, wherein the subject is undergoing islet
cell transplant.
43. The method of claim 41 , wherein the composition further
comprises one or more anti-transplant rejection agent,
anti-inflammatory agent, immunosuppressive agent, immunomodulatory
agent, anti-microbial agent, or a combination thereof.
44. The method of claim 41, wherein the composition comprises one
or more carboxy-terminal peptides corresponding to AAT, an analog
thereof, any derivative or fragment of AAT carboxy terminus that
binds to serpin-enzyme complex (SEC) receptor or a combination
thereof.
45. The method of claim 41, wherein the cellular transplant is
selected from the group consisting of cornea, bone marrow, stem
cell, pancreatic islet, kidney, lung, intestine, skin and a
combination thereof.
46. The method of claim 41, wherein the cellular transplant is
pancreatic islet cell transplant.
47. The method of claim 46, wherein the immunosuppressive agent is
chosen from inhibitors of apoptosis, reducers of cytokine
production, reducers of cytokine activities, reducers of nitric
oxide production and a combination thereof.
48. A method comprising, at least one of increasing numbers of or
increasing effectiveness and/or of sustaining T-regulatory cells in
a subject by administering an AAT or AAT derivative composition to
the subject.
49. A method comprising, increasing immune tolerance in a subject
in need thereof by administering an AAT or AAT derivative
composition to the subject in need thereof.
50. The method of claim 49, further comprising inhibiting dendridic
cell maturation.
51. The method of claim 49, wherein the subject is having,
undergoing or previously having had a transplant.
52. A method comprising, reducing antigen presentation by dendritic
cells by administering an AAT or AAT derivative composition to the
cells.
53. The method of claim 52, wherein the cells are cells of a human
subject and administration is to the subject in need of reducing
antigen presentation.
54. A method comprising, inhibiting maturation of dendritic cells
by administering an AAT, any derivative or fragment of AAT carboxy
terminus that binds to serpin-enzyme complex (SEC) receptor or AAT
derivative composition to a subject having, undergoing or
previously having had a transplant.
Description
RELATED APPLICATIONS
[0001] This application is a continuation in part of U.S.
application Ser. No. 11/916,521 filed on Jun. 7, 2006, which is a
U.S. National Stage Entry of PCT/US2006/022436, filed Jun. 7, 2006,
which claims the benefit of U.S. provisional patent application
Ser. No. 60/687,850, filed on Jun. 7, 2005, all of which are
incorporated herein by reference in their entirety.
FIELD
[0003] Embodiments of the present invention relate to compositions
and methods for treatment of subjects in need of or having a
transplant. In particular, embodiments of the present invention
relate to compositions and methods for treatment of conditions
associated with transplantations in a subject, for example, graft
rejection. More particularly, the present invention relates to
compositions and uses of alpha1-antitrypsin (.alpha.1-antitrypsin)
and agents with .alpha.1-antitrypsin-like activity and/or
compositions and uses of serine protease inhibitors.
BACKGROUND
Serine Proteases
[0004] Serine proteases serve an important role in human physiology
by mediating the activation of vital functions. In addition to
their normal physiological function, serine proteases have been
implicated in a number of pathological conditions in humans. Serine
proteases are characterized by a catalytic triad consisting of
aspartic acid, histidine and serine at the active site.
[0005] Naturally occurring serine protease inhibitors have been
classified into families primarily on the basis of the disulfide
bonding pattern and the sequence homology of the reactive site.
Serine protease inhibitors, including the group known as serpins,
have been found in microbes, in the tissues and fluids of plants,
animals, insects and other organisms. At least nine separate,
well-characterized proteins are now identified, which share the
ability to inhibit the activity of various proteases. Several of
the inhibitors have been grouped together, namely
.alpha.1-antitrypsin-proteinase inhibitor, secretory leukocyte
protease inhibitor or SLPI, antithrombin III, antichymotrypsin,
C1-inhibitor, and .alpha.2-antiplasmin, which are directed against
various serine proteases, i.e., leukocyte elastase, thrombin,
cathepsin G, chymotrypsin, plasminogen activators, and plasmin.
These inhibitors are members of the .alpha.1-antitrypsin-proteinase
inhibitor class. The protein .alpha.2-macroglobulin inhibits
members of all four classes of endogenous proteases: serine,
cysteine, aspartic, and metalloproteases. However, other types of
protease inhibitors are class specific. For example, the
.alpha.1-antitrypsin-proteinase inhibitor (also known as
(.alpha.1-antitrypsin or AAT) and inter-alpha-trypsin inhibitor
inhibit only serine proteases, .alpha.1-cysteine protease inhibitor
inhibits cysteine proteases, and .alpha.1-anticollagenase inhibits
collagenolytic enzymes of the metalloenzyme class.
[0006] The normal plasma concentration of ATT ranges from 1.3 to
3.5 mg/ml although it can behave as an acute phase reactant and
increase 3-4-fold during host response to inflammation and/or
tissue injury such as with pregnancy, acute infection, and tumors.
It easily diffuses into tissue spaces and forms a 1:1 complex with
target proteases, principally neutrophil elastase. Other enzymes
such as trypsin, chymotrypsin, cathepsin G, plasmin, thrombin,
tissue kallikrein, and factor Xa can also serve as substrates. The
enzyme/inhibitor complex is then removed from circulation by
binding to serpin-enzyme complex (SEC) receptor and catabolized by
the liver and spleen. ATT appears to represent an important part of
the defense mechanism against activity by serine proteases.
[0007] .alpha.1-antitrypsin is one of few naturally occurring
mammalian serine protease inhibitors currently approved for the
clinical therapy of protease imbalance. Therapeutic
.alpha.1-antitrypsin has been commercially available since the mid
1980's and is prepared by various purification methods (see for
example Bollen et al., U.S. Pat. No. 4,629,567; Thompson et al.,
U.S. Pat. Nos. 4,760,130; 5,616,693; WO 98/56821). Prolastin is a
trademark for a purified variant of .alpha.1-antitrypsin and is
currently sold by Talectris Company (U.S. Pat. No. 5,610,285 Lebing
et al., Mar. 11, 1997). Recombinant unmodified and mutant variants
of .alpha.1-antitrypsin produced by genetic engineering methods are
also known (U.S. Pat. No. 4,711,848); methods of use are also
known, e.g., (.alpha.1-antitrypsin gene therapy/delivery (U.S. Pat.
No. 5,399,346).
Graft Rejection
[0008] There are many diseases that culminate in organ dysfunction
or failure. Representative non-limiting examples include renal
failure due to diabetes melitus, hypertension, urinary output
obstruction, drug-induced toxicity, or hypoperfusion, as well as
cardiac dysfunction due to ischemic coronary artery disease,
cardiomyopathy/infection, or valvulopathy. Pulmonary diseases
include substantial damage due to chronic obstructive pulmonary
disease (COPD, including chronic bronchitis and emphysema), AAT
deficiency, cystic fibrosis, and interstitial fibrosis. Under
certain conditions, the only therapeutic option for treatment of a
subject may be organ transplantation. Pancreatic-islet
transplantation provides diabetic patients with the only option for
a tightly-controlled blood glucose level, as proven to be essential
for prevention of diabetic complications. In the case of islets,
post-transplant inflammation, which precedes immune rejection, is a
critical determinant of graft survival. This early inflammation is
mediated by cells other than the impending allospecific immune
cells.
[0009] One challenge to therapeutic transplantation is the damaging
effects of the host immune system on the transplant. MHC molecules
exist on the surfaces of cells and the particular structures of MHC
molecules are typically unique for each individual (with the
exception of identical twins, where the MHC molecule complements
are identical). The immune system is programmed to attack foreign
or "non-self" MHC-bearing tissues. For these reasons, when an organ
or tissue is transplanted into a recipient, an effort is made to
optimize the degree of tissue matching between donor and recipient.
MHC antigens are characterized for the recipient and donors.
Matching a donor to an allograft recipient by MHC structure reduces
the magnitude of the rejection response. An archetypal example is
blood group matching. Most transplants are allografts that occur
between non-identical members of the same species. Since these
matches are imperfect, there is an expected graft rejection immune
response associated with allografts. Current methods used, in order
to enhance graft survival, include medications to suppress the
immune response which can result in graft rejection. These
medications are referred to immunosuppressant or antirejection
drugs, such as prednisone, cyclosporine A, and cyclophosphamide, to
name a few. As mentioned above, local inflammation is experienced
immediately after grafting, and cells that are particularly
sensitive to non-specific inflammation, such as islets, can endure
graft dysfunction more severely than other types.
[0010] Despite advances in the field of antirejection therapy,
graft maintenance remains a challenge since the available
antirejection therapies are imperfect. For example,
immunosuppression enhances the risk for opportunistic infection or
neoplasia. Toxicities abound and include, but are not limited to,
diabetes, organ dysfunction, renal failure, hepatic dysfunction,
hematological defects, neuromuscular and psychiatric side effects,
and many others. Therefore, there is a need for a more effective
anti-rejection medical treatment that prolong graft survival and
improve the quality of life.
[0011] Bone marrow transplantation is a unique kind of transplant
where immune cells from a donor are transferred into a recipient,
thereby conferring the donor immune system into the recipient.
Here, the graft is capable of generating an immune response against
the host, and this is termed "graft versus host" disease (GVHD).
Immunosuppressive and antimicrobial treatment is required to block
adverse consequences of GVHD, and a need exists for safer and more
effective inhibitors of the adverse effects by the graft.
[0012] Because of some of the difficulties and inadequacies of
conventional therapy for treating transplantation complications and
associated side-effects, new therapeutic modalities are needed.
SUMMARY
[0013] Embodiments of the present invention provide for methods for
treating a subject having or in need of a transplant. In accordance
with these embodiments, a subject may be treated with a composition
for reducing the risk of a transplant rejection or a side-effect of
a transplant rejection in a subject. In accordance with this method
the subject can be administered a composition including a compound
that is capable of significantly reducing serine protease activity.
The composition may be administered before transplantation, during
transplantation, after transplantation or combination thereof. In
addition, the composition may further include one or more
anti-transplant rejection agent, anti-inflammatory agent,
immunosuppressive agent, immunomodulatory agent, anti-microbial
agent, or a combination thereof.
[0014] In certain embodiments of the invention, a composition
capable of significantly reducing serine protease activity can
include alpha-1-antitrypsin, an analog thereof or a combination
thereof. A transplant of the present invention may include an organ
transplant and/or a non-organ transplant. For example lung, kidney,
heart, liver, cornea, skin, stem cells, soft tissue (e.g. facial
component transplant), intestinal transplants, bone marrow,
pancreatic islet, pancreas transplant or combination thereof are
contemplated.
[0015] Embodiments of the present invention provide for methods for
ameliorating symptoms or signs experienced by a subject having or
in need of a transplant. In accordance with these embodiments,
symptoms or signs may include conditions associated with graft
versus host disease (GVHD), or graft rejection. In one example,
methods disclosed herein may be used to treat a subject undergoing
bone marrow transplantation. In another embodiment, symptoms or
signs may include but is not limited to one or more of the
following, kidney failure, lung failure, heart failure, malaise,
fever, dry cough, anorexia, weight loss, myalgias, and chest pains,
ventilatory compromise, sweating, nausea, vomiting, fever,
abdominal pain, bloody diarrhea, mucosal ulcerations, reduced renal
function (increased creatinine, decreased urine output), reduced
pulmonary function (increased shortness of breadth, fever, cough,
sputum, hypoxemia), reduced cardiac function (shortness of breach,
chest pain, fatigue, pulmonary or peripheral edema, valvulopathy),
reduced islet function (increased glucose, diabetes melitus), graft
versus host disease (gastrointestinal (GI) ulceration, pulmonary
failure, skin ulceration, coagulopothy, CNS dysfunction (mental
status changes, coma) CMV (cytomeglovirus infection, viral, fungal
parasitic infection)).
[0016] Embodiments of the present invention provide methods for
promoting prolonged graft survival and function in a subject
including administering to a subject in need thereof a
therapeutically effective amount of a composition including a
substance exhibiting .alpha.1-antitrypsin or .alpha.1-antitrypsin
analog or inhibitor of serine protease activity or a functional
derivative thereof.
[0017] Embodiments of the present invention provide for methods for
treating a subject in need of an immunotolerance therapy. In
accordance with these embodiments, a subject may be treated with a
composition for reducing the risk of a dysfunctional immune
responses or a side-effect of a dysfunctional immune response in a
subject. In another embodiment, methods herein provide for inducing
immune tolerance specific for a graft and/or reduce the need for
immunosuppressive therapy. In accordance with this embodiment, the
immune system of the transplant recipient may have reduced or lost
the specific ability to attack the graft while maintaining its
ability to mount any other type of immune attack. In accordance
with this method the subject can be administered a composition
including a compound that is capable of significantly reducing
serine protease activity or other activity associated with
.alpha.1-antitrypsin or .alpha.1-antitrypsin analog. In certain
embodiments, a composition capable of significantly reducing serine
protease activity can include alpha-1-antitrypsin, an analog
thereof or a combination thereof. In accordance with these
embodiments, one example for immunotolerance therapy can include
inhibiting cytokine production.
[0018] Embodiments of the present invention provide for methods for
reducing TNF.alpha. (tumor necrosis factor alpha) levels in a
subject including administering a composition including
alpha-1-antitrypsin, an analog thereof or a combination thereof to
a subject in need of such a treatment.
[0019] Embodiments of the present invention provide for methods for
treating a subject in need of an immunotolerance therapy. In
accordance with these embodiments methods are provided for reducing
NO production and/or reducing apoptosis and/or inhibiting
cytomegleovirus (infection and reactivation) including
administering a composition including a compound that is capable of
significantly reducing serine protease activity and/or other
alpha-1-antitrypsin activity. In certain embodiments of the
invention, a composition capable of significantly reducing serine
protease activity and/or mimicking other alpha-1-antitrypsin
activity can include alpha-1-antitrypsin, an analog thereof, or a
combination thereof.
[0020] In certain embodiments of the present invention, the
anti-inflammatory compound or immunomodulatory drug can include but
is not limited to one or more of interferon, interferon derivatives
including betaseron, beta-interferon, prostane derivatives
including iloprost, cicaprost; glucocorticoids including cortisol,
prednisolone, methyl-prednisolone, dexamethasone; immunsuppressives
including cyclosporine A, FK-506, methoxsalene, thalidomide,
sulfasalazine, azathioprine, methotrexate; lipoxygenase inhibitors
comprising zileutone, MK-886, WY-50295, SC-45662, SC-41661A,
BI-L-357; leukotriene antagonists; peptide derivatives including
ACTH and analogs thereof, soluble TNF-receptors; TNF-antibodies;
soluble receptors of interleukins, other cytokines,
T-cell-proteins; antibodies against receptors of interleukins,
other cytokines, T-cell-proteins; and calcipotriols; Celcept.RTM.,
mycophenolate mofetil, and analogues thereof taken either alone or
in combination.
[0021] Embodiments of the present invention provide for methods for
reducing graft rejection in a subject. In accordance with these
embodiments, a subject may be treated with a composition for
reducing the risk of graft rejection responses or a side-effect of
a graft rejection response in a subject. In accordance with this
method, the subject can be administered a composition including a
compound that is capable of significantly reducing serine protease
activity. In certain embodiments, a composition capable of
significantly reducing serine protease activity can include
.alpha.1-antitrypsin, an analog thereof or a combination thereof.
In one example, reducing graft rejection may include reducing the
symptoms associated with graft rejection in a subject having an
organ transplant, such as a kidney transplant or a bowel transplant
or a non-organ transplant, such as a bone marrow transplant soft
tissue transplant.
[0022] In yet another embodiment, the present invention may include
combination therapies including compositions exhibiting
.alpha.1-antitrypsin, an analog thereof, derivative or fragment of
the carboxy-terminus of AAT that binds the serpin-enzyme complex
(SEC) receptor or substance with serine protease inhibitor
activity. For example, a composition may include
.alpha.1-antitrypsin and another serine protease inhibitor
administered simultaneously or in separate compositions.
[0023] In accordance with embodiments disclosed herein, any of the
disclosed compositions may be used to ameliorate symptoms
associated with a transplant. These symptoms may include but are
not limited to, infiltration of graft with cells and/or serum
factors (for example, complement, anti-graft antibodies), increased
cytokine and/or chemokine production, increased nitric oxide
production, increased apoptosis and cell death, and increased
immune response against the transplant tissue and/or cells.
[0024] In another aspect, the present invention provides for a
method of ameliorating a symptom or sign associated with
transplantation in a subject in need of said amelioration. In
accordance with this embodiment, a composition may be administered
to a subject such as a pharmaceutically effective amount of a
substance of .alpha.1-antitrypsin, an analog thereof or serine
protease inhibitor activity, wherein the composition is capable of
reducing, preventing or inhibiting serine protease or protease
activity and/or binds to the sec receptor or other activity.
[0025] In certain embodiments, synthetic and/or naturally occurring
peptides may be used in compositions and methods of the present
invention for example, providing serine protease inhibitor
activity. Homologues, natural peptides, with sequence homologies to
AAT including peptides directly derived from cleavage of AAT may be
used or other peptides such as, peptides that inhibit serine
proteases or have AAT-like activity. Other peptidyl derivatives,
e.g., aldehyde or ketone derivatives of such peptides are also
contemplated herein. Without limiting to AAT and peptide
derivatives of AAT, compounds like oxadiazole, thiadiazole and
triazole peptoids and substances comprising certain
phenylenedialkanoate esters, CE-2072, UT-77, and triazole peptoids
may be used. Examples of analogues are TLCK (tosyl-L-lysine
chloromethyl ketone) or TPCK (tosyl-L-phenylalanine chloromethyl
ketone).
[0026] In other embodiments, an agent that reduces the occurrence
of graft rejection, promotes prolonged graft function or promotes
prolonged allograft survival can also be an inhibitor of serine
protease activity, an inhibitor of elastase, or an inhibitor of
proteinase-3. An inhibitor of serine protease activity can include,
but is not limited to, small organic molecules including
naturally-occurring, synthetic, and biosynthetic molecules, small
inorganic molecules including naturally-occurring and synthetic
molecules, natural products including those produced by plants and
fungi, peptides, variants of .alpha.1-antitrypsin, chemically
modified peptides, and proteins.
[0027] In some embodiments, AAT peptides contemplated for use in
the compositions and methods of the present invention are also
intended to include any and all of those specific AAT peptides
other than the 10 amino acid AAT peptides of SEQ ID NO. 1 depicted
supra. Any combination of consecutive amino acids depicting a
portion of AAT or AAT-like activity may be used, such as amino
acids 2-12, amino acids 3-13, 4-14, etc. of SEQ ID NO. 1, as well
as any and all AAT peptide fragments corresponding to select amino
acids of SEQ ID NO. 1. Applicants are herein entitled to
compositions based upon any and all AAT peptide variants based upon
the amino acid sequence depicted in SEQ ID NO. 1.
[0028] In one aspect of the invention, the pharmaceutical
compositions of the present invention are administered orally,
systemically, via an implant, intravenously, topically,
intrathecally, intratracheally, intracranially, subcutaneously,
intravaginally, intraventricularly, intranasally such as
inhalation, mixed with grafts by flushing of organ or suspension of
cells, or any combination thereof.
[0029] Other embodiments concern methods for preventing or reducing
the risk of developing an organ or cellular transplant rejection in
a subject having had or undergoing a cellular transplant, the
method comprising administering to the subject a composition
comprising alpha-1 antitrypsin (AAT), alpha-1 antitrypsin-like
compound or combination thereof. Any composition herein can be
administered to the subject before transplantation, during
transplantation, after transplantation or combination thereof A
composition contemplated herein can further include one or more
anti-transplant rejection agent, anti-inflammatory agent,
immunosuppressive agent, immunomodulatory agent, anti-microbial
agent, or a combination thereof A composition herein can include,
but is not limited to a carboxy-terminal peptide corresponding to
AAT, an analog thereof, any derivative of AAT carboxy terminus that
binds to serpin-enzyme complex (SEC) receptor or a combination
thereof. Cellular transplant can be a cornea, bone marrow, liver,
stem cell, pancreatic islet, pancreas, kidney, lung, intestinal
transplant, or a combination thereof. In certain embodiments, a
cellular transplant can be a pancreatic islet cell transplant.
[0030] Immunosuppressive agent can be chosen from inhibitors of
apoptosis, serine protease inhibitors, reducers of lymphocyte
numbers, reducers of cytokine production, reducers of cytokine
activities, monoclonal antibodies, reducers of cytokine receptors,
reducers of nitric oxide production and a combination thereof.
[0031] Other agents contemplated herein can include reducers of
cytokine production, reducers of cytokine activities, reducers of
cytokine receptors is an inhibitor of one or more of TNF.alpha.
(tumor necrosis factor alpha), IL-1 (interleukin-1), IL-12
(interleukin-12), IL-18 (interleukin-18), IL-17 (interleukin-17),
IL-23 (interleukin-23), IL-32 (interleukin-32), IFN.gamma.
(interferon gamma) or a combination thereof.
[0032] In other methods contemplated herein, treating organ or
cellular transplant rejection in a subject is contemplated by
identifying a subject having or in need of a cellular or organ
transplant; performing cellular or organ transplantation on the
subject; and administering a therapeutically effective amount of a
composition comprising AAT, AAT-like compound, AAT analog, AAT
derivative, serine protease inhibitor, one or more carboxy-terminal
peptides derived from AAT, any derivative of AAT carboxy terminus
that binds to serpin-enzyme complex (SEC) receptor or combination
thereof to the subject. In accordance with these methods, treating
the subject with the composition reduces the risk of
transplantation rejection by at least 10% compared to a subject not
treated with the composition.
[0033] Other embodiments herein include treating diabetes in a
subject by identifying a subject having or at risk of developing
diabetes; performing pancreatic islet cell transplantation on the
subject; and administering a therapeutically effective amount of a
composition comprising AAT, AAT-like compound, AAT analog, AAT
derivative, a serine protease inhibitor, one or more
carboxy-terminal peptides derived from AAT, any derivative or
fragment of AAT carboxy terminus that binds to serpin-enzyme
complex (SEC) receptor or combination thereof to the subject.
Administering the composition may include administering the
composition to the organ or cells to be transplanted before
transplant, administering the composition to the subject before
transplant, administering the composition to the subject during
transplantation, administering the composition to the subject after
transplantation or a combination thereof. Certain subjects
contemplated herein have or are at risk of developing Type 1
diabetes. These subjects may have been diagnosed with early phase
type 1 diabetes. Other subjects may have or are at risk of
developing Type 2 diabetes. It is contemplated that using
compositions disclosed herein may reduce the symptoms associated
with diabetes by 10%, or 20%, or 30% or more.
[0034] Yet other embodiments herein include methods reducing a
side-effect of cellular transplant rejection in a subject, the
method comprising administering to the subject a composition
comprising alpha-1 antitrypsin (AAT), one or more carboxy-terminal
peptides derived from AAT, or alpha-1 antitrypsin-like compound. In
accordance with these embodiments, a side effects of cellular or
organ transplant can be production of pro-inflammatory cytokines,
infiltration of immunocompetent cells, infiltration of inflammatory
cells, infiltration of cytotoxic T-cells, infiltration of mature
dendritic cells, infiltration of monocytes, production of nitric
oxide, production of prostaglandins, production of reactive oxygen
species, production of super oxide radicals, infiltration of
natural killer cells, infiltration of natural killer T-cells and a
combination thereof.
[0035] Other exemplary methods include preventing or reducing the
risk of developing pancreatic islet cell transplant rejection in a
subject having had or undergoing a pancreatic islet cell
transplant, the method comprising administering to the subject a
composition comprising alpha-1 antitrypsin (AAT), one or more
carboxy-terminal peptides derived from AAT, engager of the SEC
receptor, alpha-1 antitrypsin-like compound, serine protease
inhibitor or combination thereof A subject may have juvenile or
late onset type 1 diabetes or type 2 diabetes. Reducing the risk of
developing pancreatic islet cell transplant rejection in a subject
can include reducing the risk by at least 10 percent in the subject
compared to a second subject not treated with the composition.
[0036] In certain embodiments, the subject is a human. In some
embodiments, the subject is a domesticated animal or livestock.
[0037] A pharmaceutical composition contemplated herein may
include, AAT, AAT-like compound, serine protease inhibitor, AAT
analog, AAT derivative, one or more carboxy-terminal peptides
derived from AAT, any derivative or fragment of AAT carboxy
terminus that binds to serpin-enzyme complex (SEC) receptor or
combination thereof and at least one of an anti-transplant
rejection agent, an anti-inflammatory agent, an immunosuppressive
agent, an immunomodulatory agent, and an anti-microbial agent. In
accordance with these embodiments, the pharmaceutical composition
can be AAT, serine protease inhibitor, AAT-like compound, AAT
analog, AAT derivative, carboxy-terminal peptide corresponding to
AAT, any derivative or fragment of AAT carboxy terminus that binds
to serpin-enzyme complex (SEC) receptor or combination thereof and
one or more anti-transplant rejection agents.
[0038] Other exemplary methods can include methods for inducing
immunological tolerance in a subject undergoing a cellular or organ
transplant including, but not limited to, administering to the
subject a composition comprising alpha-1 antitrypsin (AAT), alpha-1
antitrypsin-like compound or combination thereof wherein the
subject is undergoing islet cell transplant. One cellular
transplant can be pancreatic islet cell transplant. One transplant
can be a temporary cadaver transplant of skin in a burn patient
where compositions herein inhibit rejection of the temporary
cadaver treatments.
[0039] Other exemplary methods can include, at least one of
increasing numbers of or increasing effectiveness and/or of
sustaining T-regulatory cells in a subject by administering an AAT
or AAT derivative composition to the subject.
[0040] Yet other exemplary methods can include increasing immune
tolerance in a subject in need thereof by administering an AAT or
AAT derivative composition to the subject in need thereof. In
addition, these methods may further include inhibiting dendridic
cell maturation. For example, compositions contemplated herein may
be administered to a subject having, undergoing or previously
having had a transplant.
[0041] Other exemplary methods herein include reducing antigen
presentation by dendritic cells by administering an AAT or AAT
derivative composition to the cells. In accordance with these
embodiments, the cells are cells of a human subject and
administration is to the subject in need of reducing antigen
presentation. Other examples herein include inhibiting maturation
of dendritic cells by administering an AAT, any derivative or
fragment of AAT carboxy terminus that binds to serpin-enzyme
complex (SEC) receptor or AAT derivative composition to a subject
having, undergoing or previously having had a transplant.
[0042] As such, those skilled in the art will appreciate that the
conception, upon which this disclosure is based, can readily be
used as a basis for designing other methods for carrying out the
several features and advantages of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] The following drawings form part of the present
specification and are included to further demonstrate certain
embodiments of the present invention. The embodiments may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0044] FIGS. 1A-1D illustrates an exemplary method of treating
islet allografts with AAT. Islets from DBA/2 mice (H-2d) were
transplanted under the renal capsule of streptozotocin-induced
hyperglycemic C57BL/6 mice (H-2b). (A) Glucose levels from days
6-18. (B) Treatment protocols. Control and full AAT treatment are
described in panel A. Early AAT treatment consists of treatment on
days -1, 1 and 3 (2 mg, n=3). Late AAT treatment consists of
treatment from day 2 and on every 2 days (2 mg, n=3). (C) Effect of
mouse anti-human-AAT antibodies. Dashed line indicates post
transplantation glucose levels of a mouse under full AAT treatment
protocol (see A, B) that was immunized by multiple administrations
of human AAT prior to transplantation (1 representative, n=3).
Solid line indicates glucose levels of a non-immunized mouse
treated under full AAT treatment protocol (1 representative, n=10).
Arrow indicates detection of treatment-induced, anti-human-AAT
antibodies in the non-immunized representative mouse. (D)
Comparison of day 15 post-transplantation glucose levels in mice
that were under full treatment protocol with ALB (n=3) or AAT
(non-immunized n=10, immunized n=3). Of the AAT-treated group,
antibodies were detected on day 15 in 3/3 immunized mice and in
6/10 non-immunized mice.
[0045] FIGS. 2A-2D illustrates an exemplary method of the effect of
AAT on thioglycolate-elicited peritoneal cellular infiltrates. (A)
Total cell population of lavaged cells of (O) saline or (.DELTA.)
AAT-treated (5 mg) thioglycolate-injected mice. (B) Percent cell
population from saline-treated mice at 48 hours. (C) Oxidation of
AAT. (D) Identification of elicited macrophages and
neutrophils.
[0046] FIGS. 3A-3C illustrates an exemplary method of the effect of
AAT on MHC-incompatible, NIH-3T3-fibroblast-elicited peritoneal
cellular infiltrates. (A) Cell numbers. The number of cells in each
subpopulation was calculated from the percentages obtained by FACS
analysis, and total number of cells in the infiltrate. (B)
Representative FACS analysis. (C) Effect of AAT on intensity and
function of infiltrate elicited by islet allograft. Left,
Hematoxilyn and Eosin (H&E) staining of day 7 islet allografts.
Right, Immunohistochemistry (IHC) with anti-insulin antibodies of
day 15 islet grafts. R, renal parenchyma, G, graft, C, renal
capsule.
[0047] FIGS. 4A-4H illustrates an exemplary method of the effect of
AAT on islet responses. (A-D) Mean.+-.SEM of A. nitric levels, B.
Cell viability and C. MIP-1.alpha. levels. Dashed line represents
islets incubated at one-30th the concentration of
IFN.gamma./IL-1.beta.. D. TNF.alpha. levels. (E) Insulin induction
assay. (F) Streptozotocin toxicity. Each image depicts a
representative islet from one pancreas. (G) Cellular content of
islets. (H) MHC class II expression.
[0048] FIGS. 5A-5D illustrates the effect of AAT on TNF.alpha.. (A)
Islets from C57BL/6 mice were cultured (100 islets/well in
triplicate) in the presence of AAT (0.5 mg/ml) or TACE inhibitor
(10 mM) 1 hour before stimulation by IFN.gamma. (5 ng/ml) plus
IL-1.beta. (10 ng/ml). Left, mean.+-.SEM change in TNF.alpha. in
supernatants after 72 hours of incubation. Right, mean SEM fold
change in membrane TNF.alpha. on islet cells after 5 hours of
incubation, according to FACS analysis. (B) Representative FACS
analysis of membrane TNF.alpha. on stimulated islet cells in the
absence (open area) or presence (shaded area) of AAT. (C)
Streptozotocin-induced hyperglycemia.
[0049] FIGS. 6A-6D illustrates the effect of AAT on Islet allograft
transplantation. 6A illustrates the time course study after
transplantation. 6B illustrates an immune infiltrate found outside
the graft area. 6C illustrates an increase in the presence of CD4+
and a comparative decrease in monocytes and neutrophils. 6D
illustrates levels of glucose reflecting a level of tolerance with
respect to days following allografting of the same donor (left) and
a 3.sup.rd donor re-graft (right), indicating induction of specific
immune tolerance.
[0050] FIGS. 7A-7E illustrates the production of AAT by islet cell
and reflection of islet graft survival. 7A illustrates a time
course expression of mouse AAT mRNA after cytokine production
(IL-I.beta. and IFN.gamma.) (left) and at 8 hours (right). 7B
illustrates an example of islet injury during pancreatitis; the
histology of normal islets (top left), the histology of islets of
an inflamed pancreas (top right) and expression of mouse AAT in
islets obtained from the pancreata in an acute pancreatitis model
(bottom). 7C illustrates an example of samples of islet allografts
taken post grafting and the percent change in AAT mRNA levels were
assessed. 7D illustrates an example of islet protection from
cytokine injury with endogenous AAT by introducing oncostatin M (an
interleukin 6 (IL-6) family member) that induces AAT expression in
islets, oncostatin M and AAT levels (top left); nitric oxide and
viability levels assessed (top right) and nitric oxide production
representing islet viability after 4 day exposure to oncostatin M
and AAT production decreasing cytokine effects on the islets
(bottom).
[0051] FIGS. 8A-8D illustrates the effect of AAT on human islets
and the production of nitric oxide (8A), TNF-.alpha. production
(8B) IL-6 (8C) and IL-8 (8D).
[0052] FIGS. 9A-9D illustrate exemplary extended AAT monotherapy
where exposure induces strain-specific immune tolerance towards
islet allografts in mice. Islet -allograft transplantation was
performed and blood glucose was followed in mice that received
albumin (ALB, n=6) or hAAT monotherapy (n=24) for various periods
of times. (A) Islet graft survival curve. (B) Summary of
uninterrupted normoglycemic intervals achieved during and after
hAAT monotherapy ("First graft") and during a second grafting
procedure that was carried out in explanted animals in the absence
of therapy ("Second graft") (n=7). Double-underlined headings
indicate number of hAAT monotherapy and therapy-free days. The
outcome of the second grafting procedure is indicated per
individual mouse. (C) Representative Blood glucose follow-up.
Albumin (ALB)-treated animals are represented by dashed line. Day
of hAAT treatment withdrawal is indicated. Treatment-free glucose
levels were determined during the ensuing days. Graft removal by
nephrectomy, resulting in hyperglycemia, is indicated. A second
grafting without further hAAT treatment was performed with same
strain islet allograft (left) or third strain islet allograft
(right). Transplantation outcome of the second grafting is
monitored for 50 days. (D) Histology. Representative day 72
explanted graft from hAAT-treated mice 20 days after withdrawal of
hAAT treatment. H&E stain, image of entire islet graft site.
Islet mass appears flanked by a dense mononuclear cell population
(thick arrows).
[0053] FIG. 10 represent exemplary effects of hAAT monotherapy on
gene expression profile in islet allografts. RT-PCR of explanted
islet allografts from albumin (ALB)-treated control and
hAAT-treated mice. Left 4 columns, initial days after islet
transplantation into control mice. Right column, day 72 after islet
transplantation into hAAT-treated mice (see FIG. 9). Data are
representative of n=6 (ALB) and n=3 (hAAT, time points between days
30 and 72 after transplantation).
[0054] FIG. 11 represent exemplary cell-specific effects of hAAT.
Inducible IFN.gamma. levels (left) and cell proliferation (right)
assessed in Con A-primed PBMC that were stimulated with increasing
concentrations of IL-2 in the presence of 0.5 mg/ml hAAT or albumin
(CT). Data are mean.+-.SEM of three individual donors.
[0055] FIGS. 12A-12B Identification of hAAT-induced
IL-10-expressing Treg cells in non-rejecting islet allografts. (A)
RT-PCR of explanted islet allografts in albumin (ALB)-treated graft
recipients during acute allorejection (Left 4 columns, days 1-7)
and hAAT-treated graft recipient 20 days after withdrawal of hAAT
treatment (Right column, day 72, see FIG. 12). Data are
representative of n=6 (ALB) and n=3 (hAAT, representative time
point between days 30 and 72 after transplantation). (B) Intragraft
gene expression profile throughout hAAT therapy. RT-PCR of
explanted islet allografts in hAAT-treated graft recipients during
hAAT treatment. K, tissue from pole opposite to the grafting site.
G, intragraft gene profile.
[0056] FIGS. 13A-13C represent exemplary time-dependent
hAAT-induced distribution of Treg cells between DLN and allograft.
Foxp3-GFP knock-in mice (H-2b) were grafted with wild-type Balb/c
tissue (H-2d). Mice received a 10-day hAAT treatment or albumin
protocol (see FIG. 9). (A) Inguinal DLN. FACS analysis of
CD4+-sorted foxp3-GFP-positive DLN cells. Inset, RT-PCR for foxp3
mRNA transcripts in DLN. Illustrated are representative
time-points. (B) Matrigel-skin graft. Treg cells in matrigel grafts
on day 10 identified by fluorescent microscopy of unstained
material (left) plus DAPI-counter stained material (right). (C)
Islet graft. Day 14 Treg cells identified in the "cuff" site (see
FIG. 9D). Anti-GFP antibody immunostaining and DAPI
counter-staining. Representative image of three hAAT-treated
grafts. Grafts from albumin-treated mice contained no "cuff" (not
shown).
[0057] FIG. 14 represents an exemplary method illustrating of early
local and systemic effects of hAAT. Wild-type islet-matrigel grafts
containing increasing concentrations of hAAT (indicated, amount per
matrigel) were explanted 48 hours after transplantation into
hAAT-Tg recipients. Top, identification of CD14-positive cells
(RT-PCR) and identification of host-cells inside the graft
(genomic). Bottom, RT-PCR depiction of insulin and VEGF intragraft
transcripts.
[0058] FIG. 15. represents an exemplary effect of AAT on dendritic
cell migration and maturation. CD86, MHC class II and IL 10
expression in renal DLN. 72 hours after allogeneic skin grafting
under the renal capsule DLN were harvested and examined by RT-PCR.
DLN from non-grafted mice (first bar on left) is compared to
72-hour DLN gene expression from untreated (CT) and hAAT-treated
(AAT) mice. Mean.+-.SEM from three experiments. * p<0.05, **
p<0.01 between CT and AAT.
[0059] FIGS. 16A-16B represent exemplary experiments cell-specific
effects of hAAT. (A) Mouse splenocytes. Inducible IFN.gamma. levels
(top), cell proliferation (bottom) and clump formation (right) in
Con A-stimulated splenocytes in the presence indicated
concentrations of hAAT or albumin (ALB). CT, cells with no added
Con A. Photomicrographs depict an example of Con A-driven cell
clumping. The data represent mean.+-.SEM of three independent
experiments. (B) Mouse peritoneal macrophages. Inducible nitric
oxide production in peritoneal macrophages that were stimulated
with IFN.gamma. (5 ng/ml) in the presence of increasing
concentrations of hAAT. The data are mean.+-.SD. * p<0.05, **
p<0.01.
[0060] FIG. 17 represents exemplary effects of early local and
systemic effects of hAAT. hAAT-induced changes in serum cytokines.
Top-Left, Unprovoked serum cytokines after a 10-day schedule (see
FIG. 9) of hAAT-treatment (n=3) compared to albumin treated mice (n
=3). Relative cytokine levels were determined in duplicate by the
Proteome Profiler (see Methods). Results are presented as
mean.+-.SEM fold-change (all p<0.05) in hAAT-treated mice over
that observed in albumin-treated mice. Out of 36 cytokines tested,
those without statistically significant changes are not shown.
Top-Right, LPS-elicited cytokines. Following a 10-day schedule of
hAAT (n=3) or albumin treatment (n=3), mice were injected with LPS
(1 mg/kg) and after 2 hours serum was collected. Differences are
shown as mean.+-.SEM fold-change in hAAT-treated mice compared to
albumin-treated mice. Bottom, sterile-inflammation-induced serum
cytokines. Sera from hAAT-treated and saline-treated mice that were
injected intramuscularly with either turpentine or saline 24 hours
earlier. IL 10 and IL 6 levels were measured by specific ELISA.
Mean.+-.SEM from 3 experiments.
[0061] FIGS. 18A-18B represent exemplary experiment effect of AAT
on dendritic cell migration and maturation. (A) Graft-derived cell
migration into DLN. GFP-transgenic skin grafts were transplanted
into wild-type recipient mice treated with a 10-day treatment
schedule of hAAT or albumin (see FIG. 9). PCR amplification of
genomic DNA extracted from the graft tissue is shown in the left
lane and from the inguinal DLN in the remaining lanes.
Representative data from one of three independent experiments is
shown. (B) In vitro dendritic cell maturation. Bone-marrow-derived
GM-CSF-differentiated dendritic cell were cultured with no
stimulant (CT) or LPS (100 ng/ml) in the absence or presence of
hAAT (0.5 mg/ml) for 24 hours. FACS analysis of CD 11c-positive
cells for surface levels of MHCII (top) and CD86 (bottom).
Proportion of the double-positive population is depicted as percent
from total cells. Representative panels from three experiments
performed in 6-plicate.
[0062] FIG. 19 represents exemplary effects of AAT on stimulated
human islets.
[0063] FIG. 20 represents exemplary effects of AAT on stimulated
human islets. Levels of IL-6, IL-8 and TNF.alpha. (percent from
stimulated islets) and nitric oxide in supernatant are
represented.
[0064] FIG. 21 represents exemplary experiments where right after
isolation, islet cells were supplemented with AAT (or left
untreated, CT) for 24 hrs. The cells were then washed and incubated
for 72 hours with IL-1.beta. and IFN.gamma., without AAT. LDH was
measured in supernatants.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Definitions
[0065] Terms that are not otherwise defined herein are used in
accordance with their plain and ordinary meaning.
[0066] As used herein, "a" or "an" may mean one or more than one of
an item.
[0067] As used herein "analog of alpha-1-antitrypsin" may mean a
compound having alpha-1-antitrypsin-like activity. In one
embodiment, an analog of alpha-1-antitrypsin is a functional
derivative of alpha-1-antitrypsin. In a particular embodiment, an
analog of alpha-1-antitrypsin is a compound capable of
significantly reducing serine protease activity. For example, an
inhibitor of serine protease activity has the capability of
inhibiting the proteolytic activity of trypsin, elastase,
kallikrein, thrombin, cathepsin G, chymotrypsin, plasminogen
activators, plasmin and/or other serine proteases.
[0068] As used herein "immunomodulatory drugs or agents", it is
meant, e.g., agents which act on the immune system, directly or
indirectly, e.g., by stimulating or suppressing a cellular activity
of a cell in the immune system, e.g., T-cells, B-cells,
macrophages, or antigen presenting cells (APC, dendritic cells), or
by acting upon components outside the immune system which, in turn,
stimulate, suppress, or modulate the immune system, e.g. cytokines,
e.g., hormones, receptor agonists or antagonists, and
neurotransmitters; immunomodulators can be, e.g.,
immunosuppressants or immunostimulants.
[0069] It is to be understood that the terminology and phraseology
employed herein are for the purpose of description and should not
be regarded as limiting
DETAILED DESCRIPTION OF THE INVENTION
[0070] In the following sections, various exemplary compositions
and methods are described in order to detail various embodiments of
the invention. It will be obvious to one skilled in the art that
practicing the various embodiments does not require the employment
of all or even some of the specific details outlined herein, but
rather that concentrations, times and other specific details may be
modified through routine experimentation. In some cases, well known
methods or components have not been included in the
description.
[0071] Embodiments of the present invention provide for methods for
treating a subject having or in need of a transplant. In accordance
with these embodiments, a subject may be treated with a composition
capable of significantly reducing serine protease activity. In
addition, one embodiment of the present invention provides for
methods including treating a subject with a composition comprising
a compound having .alpha.-1-antitrypsin activity. In one
embodiment, the composition can include .alpha.-1-antitrypsin,
analog thereof or a serine protease inhibitor to for example,
promote transplant survival or reduce a side effect of the
transplant. Further, the administration of the composition can be
before transplantation, during transplantation, after
transplantation or combination thereof. In addition, the
composition may further include one or more additional therapies
such as immunosuppressive therapies. A transplant of the present
invention may include transplantation of an organ such as lung,
kidney, heart, liver, skin, pancreas, or bowel organ or non-organ
such bone marrow, pancreatic islet, cornea, and/or soft tissue.
[0072] Human pancreatic islet transplantation has a low 5-year
graft survival rate. The current immunosuppression protocol in this
procedure is void of anti-inflammatory corticosteroids.
Alpha-1-antitrypsin (AAT) reduces cytokine-mediated islet damage
and interferes with inflammatory processes. Certain embodiments
disclosed herein concern AAT monotherapy for allografts with
anti-inflammatory conditions that impair dendritic cell maturation
and favor development of antigen-specific T regulatory cell. Given
its established safety in patients, AAT may be considered for use
during human islet transplantation.
[0073] Islet transplantation can provide type-1 diabetes patients
with tight glycemic control that can eliminate the need for
exogenous insulin injections. In this procedure, isolated islets
are introduced into the hepatic portal circulation of a diabetic
patient. The immunosuppressive protocol used for islet
transplantation excludes diabetogenic corticosteroids and therefore
is void of anti-inflammatory activity. To date, islet loss in most
transplant patients steadily progresses and results in a low 5-year
graft survival rate.
[0074] Islets are particularly prone to injury during inflammatory
conditions. Immediately after transplantation, viable islet mass
rapidly decreases, regardless of allogeneic discrepancy. As damage
intensifies, necrotic islet beta cells secrete injurious cytokines
and chemokines while presenting allogeneic antigens to the host.
Thus, grafted islets actively participate in the inflammatory flare
and become activators, and targets, of resident macrophages.
[0075] Extent of inflammation and injury can determine the degree
of antigen presentation and closely affects the expansion of
allospecific effector cells. In addition, the favorable state of
immune tolerance can be elaborated by a shift in balance between
effector T cells and protective regulatory T (Treg) cells, a
process which requires the uninterrupted activity of IL-2. By
reducing the intensity of inflammation while allowing IL-2 activity
one may provide optimal conditions for prolonged allograft
survival.
[0076] In addition to its ability to inhibit serine proteases,
alpha-1-antitrypsin (AAT) possesses anti-inflammatory properties.
AAT has been disclosed to prevent the demise of islet beta cells
from normal mice, enabling insulin secretion in the presence of
IL-1.beta. and IFN.gamma. and reducing cytokine and chemokine
secretion, previously described. Administered to animals, AAT
reduced the susceptibility of islets to inflammation and prolonged
islet allograft survival. In addition, AAT allows for uninterrupted
IL-2 activity.
[0077] As disclosed herein, effects of extended AAT monotherapy on
islet allograft rejection were examined. In order to allow extended
therapy with hAAT, mice heterozygous for the human AAT transgene
(hAAT-Tg) were used as graft recipients. In these mice, the human
AAT sequence is preceded by a surfactant promoter, thus limiting
hAAT expression to lung epithelial cells and circulating hAAT
levels to less than 10 ng/ml. Thus, the impact of monotherapy on
the process of allograft rejection was examined in the setting of a
normal immune system. After rendered diabetic, the mice were
grafted with islets from another mouse strain and were treated with
hAAT for extended periods of time. Unexpectedly, therapy withdrawal
revealed the fervent induction of strain-specific treatment-induced
immune tolerance.
[0078] Embodiments herein provide for administration of
compositions including, but not limited to, AAT, serine protease
inhibitors, derivatives or fragments of the carboxy-terminus of AAT
that bind to the SEC receptor, or AAT derivative or composition
with AAT-like activity to a subject having or previously having had
graft surgery for example, cellular implantation, cellular
supplementation, organ implantation and/or tissue implantation. In
certain embodiments, a subject in need of increased immune
tolerance is contemplated to reduced rejection of the implanted or
grafted cells, tissue or organ. In other embodiments,
administration of AAT or AAT derivative or composition with
AAT-like activity to a subject can be prior to implantation to
reduce rejection and/or reduce immune response to the
transplantation. Subject contemplated herein in need of immune
tolerance include, but are not limited to, subjects having
undergone a transplant or subjects scheduled for a transplant.
[0079] Embodiments herein provide for administration of
compositions including, but not limited to, AAT or AAT derivative
or composition with AAT-like activity to a subject having acute
rejection from having undergone a transplant such as a kidney,
liver or other transplant. In certain embodiments, a subject may be
administered one or more infusions of an AAT composition to reduce
rejection of the transplant or prolong acceptance of the
transplanted organ or tissue in the subject. In certain examples,
the subject may have undergone a transplant recently, a month ago
or a year or more prior to administration of the AAT
composition.
[0080] In certain embodiments, compositions of AAT or AAT
derivative, or carboxy terminal fragment of AAT capable of binding
to the SEC receptor or compositions with AAT-like activity may be
administered to a subject in need thereof to induce immune
tolerance in the subject. As used herein carboxy terminus of AAT
can include the later half of the AAT molecule toward the carboxy
end of the molecule (e.g. from the predominant naturally occurring
form of AAT that would include from about AA 209 to about AA 418 of
AAT).
[0081] Yet other embodiments herein concern administering AAT
compositions or AAT derivative or compositions with AAT-like
activity to a burn patient. For example, these compositions can be
administered to a burn patient undergoing interim therapy of
cadaver skin applied to the burn regions of the patient. In
accordance with these embodiments, the patient can be administered
iv AAT compositions at periodic times (e.g. daily, weekly or
monthly) to prolong the tolerance period for the cadaver skin
permitting re-growth of the patient's own tissue.
[0082] Embodiments herein provide for administration of
compositions including, but not limited to, AAT or AAT derivative
or AAT-like activity to a subject in need of immune tolerance.
Subject contemplated herein in need of immune tolerance include,
but are not limited to, subjects in a biologically compatible
[0083] In other exemplary embodiments, a subject contemplated
herein may have early phase type 1 diabetes. This disease often
affects young children and can be called "juvenile type 1
diabetes." There is also a similar type 1 diabetes that affects
older individuals and is called "late onset" type 1 diabetes.
Embodiments herein contemplate that AAT suppresses the immune
response for example, by promoting the generation and proliferation
of T regulatory cells, as well as sustaining the activity of T
regulatory cells. Since both juvenile, as well as late onset type 1
diabetes, have an autoimmune response directed against the insulin
producing beta cells in the pancreatic islets, ATT can be used as a
treatment for both juvenile as well as late onset type 1 diabetes
via the T-regulatory cells. In addition, the anti-inflammatory
properties of AAT, contributes to protecting the beta cells from
the cytotoxic effects of pro-inflammatory cytokines and
inflammatory mediators.
[0084] In other embodiments, compositions contemplated herein can
be used to treat a subject having type 2 diabetes. For example, a
subject may be treated with periodic application of the composition
such as monthly or weekly administrations of compositions disclosed
herein.
[0085] In some embodiments, AAT can be used to treat autoimmune
diseases since these diseases reveal a low number and/or function
of Tregs. Since AAT does not affect IL-2 production or activity,
Tregs can be stimulated during AAT therapy to increase development
of Tregs. Many immunosuppressive agents suppress IL-2 production
and/or function, development of T regs is impaired. For example, in
a an autoimmune disease such a rheumatoid arthritis, the use of
methotrexate may impair the development of T-regs. Lowering a dose
of an immunosuppressive agent may be performed during AAT
treatment. Other autoimmune diseases that are treated with
immunosuppressive regimens may also be treated with AAT while
lowering the dose of immunosuppressive agents include, but are not
limited to, lupus erythematosus, Crohn's disease, ulcerative
colitis, psoriasis, biliary cirrhrosis, and thrombocytopenia.
[0086] Serine protease inhibitors, have been found in a variety of
organisms. At least nine separate, well-characterized proteins are
now identified, which share the ability to inhibit the activity of
various proteases. Several of the inhibitors have been grouped
together, such as the .alpha..sub.1-antitrypsin-proteinase
inhibitor. Serine proteases include but are not limited to
leukocyte elastase, thrombin, cathepsin G, chymotrypsin,
plasminogen activators, and plasmin.
[0087] Embodiments of the present invention provide for methods for
promoting transplantation, graft survival, reducing graft rejection
and/or reducing or preventing side-effects associated with graft
rejection. In accordance with these embodiments, side-effects may
include conditions associated with graft versus host disease
(GVHD), or graft rejection. In one example, methods disclosed
herein may be used to treat a subject undergoing bone marrow
transplantation. In another embodiment, symptoms or signs may
include but is not limited to one or more of the following,
malaise, fever, dry cough, myalgias, and chest pains, ventilatory
compromise, sweating, nausea, vomiting, fever, abdominal pain,
bloody diarrhea, mucosal ulcerations, reduced renal function
(increased creatinine, decreased urine output), reduced pulmonary
function (increased shortness of breadth, fever, cough, sputum,
hypoxemia), reduced cardiac function (shortness of breach, chest
pain, fatigue, pulmonary or peripheral edema, valvulopathy),
reduced islet function (increased glucose, diabetes mellitus),
graft versus host disease (gastrointestinal (GI) ulceration,
pulmonary failure, skin ulceration).
[0088] Embodiments of the present invention provide for methods for
treating a subject in need of an immunotolerance therapy. In
accordance with these embodiments, a subject may be treated with a
composition for inducing immune tolerance. This achieved while
reducing the risk of a dysfunctional immune responses or a
side-effect of a dysfunctional immune response in a subject as
typically encountered during standard immune suppression. For
example, a dysfunctional immune response may be an effect of graft
rejection, pneumonia, sepsis, fungal infection, cancer. In
accordance with this method the subject can be administered a
composition including a compound that is capable of significantly
reducing serine protease activity or other activity associated with
.alpha.1-antitrypsin or .alpha.1-antitrypsin analog. In certain
embodiments, a composition capable of significantly reducing serine
protease activity can include .alpha.-1-antitrypsin, an analog
thereof or a combination thereof. In accordance with these
embodiments, one example for immunotolerance therapy can include
inhibiting cytokine production.
[0089] Any of the embodiments detailed herein may further include
one or more a therapeutically effective amount of anti-microbial
drugs anti-inflammatory agent, immunomodulatory agent, or
immunosuppressive agent or combination thereof.
[0090] Non-limiting examples of anti-rejection agents/drugs may
include for example cyclosporine, azathioprine, corticosteroids,
FK506 (tacrolimus), RS61443, mycophenolate mofetil, rapamycin
(sirolimus), mizoribine, 15-deoxyspergualin, and/or leflunomide or
any combination thereof.
[0091] In addition, other combination compositions of methods
disclosed in the present invention include certain antibody-based
therapies. Non-limiting examples include, polyclonal
anti-lymphocyte antibodies, monoclonal antibodies directed at the
T-cell antigen receptor complex (OKT3, TIOB9), monoclonal
antibodies directed at additional cell surface antigens, including
interleukin-2 receptor alpha. Antibody-based therapies may be used
as induction therapy and/or anti-rejection drugs in combination
with the compositions and methods of the present invention.
[0092] Embodiments of the present invention provide for methods
treating a subject in need of an immunotolerance therapy. In
accordance with these embodiments, a subject may be treated with a
composition capable of significantly reducing serine protease
activity. In one embodiment, the composition can include
.alpha.-1-antitrypsin, analog thereof or a serine protease
inhibitor to for example, to reduce or inhibit the production of
cytokines. In accordance with these embodiments, combination
therapies are contemplated, such as combining .alpha.-1-antitrypsin
composition with an anti-inflammatory agent.
[0093] In one particular embodiment, the present inventions provide
for methods for reducing levels and activities of cytokines such as
TNF.alpha. (tumor necrosis factor alpha). For example, the
composition can include alpha-1-antitrypsin or analog thereof or
combination thereof alone or in combination with other
therapies.
[0094] In one embodiment, the reduction, prevention or inhibition
of rejection of transplantation or side effects thereof associated
with one or more of each of the above-recited conditions may be
about 10-20%, 30-40%, 50-60%, or more reduction or inhibition due
to administration of the disclosed compositions.
[0095] In one embodiment of the present invention a composition may
include compounds that engage molecules for the SEC receptor to
treat a subject undergoing a transplantation and/or in need of
immunotolerance therapy. In each of the recited methods, an
.alpha.1-antitrypsin (e.g. mammalian derived) or inhibitor of
serine protease activity substance contemplated for use within the
methods of the present invention can include a series of peptides
including carboxyterminal amino acid peptides corresponding to AAT.
These pentapeptides can be represented by a general formula (I):
I-A-B-C-D-E-F-G-H-TT (note: in the Sequence Listing F=X), wherein I
is Cys or absent; A is Ala, Gly; Val or absent; B is Ala, Gly, Val,
Ser or absent; C is Ser, Thr or absent; D is Ser, Thr, Ans, Glu,
Arg, Ile, Leu or absent; E is Ser, Thr, Asp or absent; F is Thr,
Ser, Asn, Gln, Lys, Trp or absent; G is Tyr or absent; H is Thr,
Gly, Met, Met(O), Cys, Thr or Gly; and II is Cys, an amide group,
substituted amide group, an ester group or absent, wherein the
peptides includes 4 or more consecutive amino acids and
physiologically acceptable salts thereof. Among this series of
peptides, several are equally acceptable including FVFLM (SEQ ID
NO. 1), FVFAM (SEQ. ID NO. 2), FVALM (SEQ. ID NO. 3), FVFLA (SEQ.
ID NO. 4), FLVFI (SEQ. ID NO. 5), FLMII (SEQ. ID NO. 6), FLFVL
(SEQ. ID NO. 7), FLFVV (SEQ. ID NO. 8), FLFLI (SEQ. ID NO. 9),
FLFFI (SEQ. ID NO. 10), FLMFI (SEQ. ID NO. 11), FMLLI (SEQ. ID NO.
12), FIIMI (SEQ. ID NO. 13), FLFCI (SEQ. ID NO. 14), FLFAV (SEQ. ID
NO. 15), FVYLI (SEQ. ID NO. 16), FAFLM (SEQ. ID NO. 17), AVFLM
(SEQ. ID NO. 18), and any combination thereof.
[0096] In several embodiments herein, AAT peptides contemplated for
use in the compositions and methods of the present invention are
also intended to include any and all of those specific AAT peptides
of SEQ ID NO. 1 depicted supra. Any combination of consecutive
amino acids simulating AAT or AAT-like activity may be used, such
as amino acids 209-219, amino acids 314-324, 394-404, etc. In
addition, combinations of consecutive amino acid sequences such as
5-mers or 10-mers of the carboxy terminus can also be used. For
example, any combinations of 5-mers or 10-mers from SEQ ID NO. 61
AAs 209-418 can be used in compositions contemplated herein.
Another example is SEQ ID NOs 19 through 60 below may be combined
in any composition contemplated herein.
[0097] As contemplated herein, the last amino acid is the carboxyl
terminus. In certain embodiments, the carboxyl domain of AAT going
backwards from the carboxyl terminus is defined as those amino
acids most conserved among the difference species and do not
participate in the protease binding domain of AAT. In addition, in
other embodiments, AAT protease binding domain can be mutated in
order to reduce or eliminate the protease function of the molecule
and not inhibit elastase activity; these molecules can be used in
any composition contemplated herein. In certain embodiments, a
mutated AAT can be used to protect islet cells before, after,
and/or during transplantation either supplied directly to the
islets and/or administered to a subject in need of islet cell
infusion. In other embodiments, a mutated molecule (e.g. having
reduced or essentially no protease activity) retains its an
anti-inflammatory effects and can be used as an anti-inflammatory
molecule in a subject having an autoimmune condition or undergoing
an organ or cellular transplant. It is also contemplated herein
that the carboxyl domain is the non-protease binding domain. One
skilled in the art would understand a non-protease binding domain
of AAT.
[0098] In each of the above-recited methods, .alpha.1-antitrypsin
or analogs thereof are contemplated for use in a composition
herein. These analogs may include peptides. The peptides may
include but are not limited to amino acid peptides containing
MPSSVSWGIL(SEQ ID NO:19), LLAGLCCLVP(SEQ ID NO:20), VSLAEDPQGD(SEQ
ID NO:21), AAQKTDTSHH(SEQ ID NO:22), DQDHPTFNKI(SEQ ID NO:23),
TPNLAEFAFS(SEQ ID NO:24), LYRQLAHQSN(SEQ ID NO:25), STNIFFSPVS(SEQ
ID NO:26), IATAFAMLSL(SEQ ID NO:27), GTKADTHDLI(SEQ ID NO:28),
LEGLNFNLTL(SEQ ID NO:29), IPLAQIHLGF(SEQ ID NO:30), QLLLRTLNQP(SEQ
ID NO:31), DSQLQLTTGN(SEQ ID NO:32), GLFLSLGLKL(SEQ ID NO:33),
VDKFLLDVKK(SEQ ID NO:34), LYHSLAFTVN(SEQ ID NO:35), FGDTLLAKKQ(SEQ
ID NO:36), INDYVLKGTQ(SEQ ID NO:37), GKIVDLVKLL(SEQ ID NO:38),
DRDTVFALVN(SEQ ID NO:39), YIFFKGKWER(SEQ ID NO:40), PFLVKDTLLL(SEQ
ID NO:41), DFHVDQVTTV(SEQ ID NO:42), KVPMMKRLGM(SEQ ID NO:43),
FNIQHCKKLS(SEQ ID NO:44), SWVLLMKYLG(SEQ ID NO:45), NATAIFFLPD(SEQ
ID NO:46), LGKLQHLLNL(SEQ ID NO:47), LTHDIITKFL(SEQ ID NO:48),
LNLDRRSASL(SEQ ID NO:49), HLPKLSITGT(SEQ ID NO:50), YDLKSVLGQL(SEQ
ID NO:51), GITKVFSNGA(SEQ ID NO:52), DLSGVTLLAP(SEQ ID NO:53),
LKLSKAVHKA(SEQ ID NO:54), VLTIDLKGTL(SEQ ID NO:55), AAGAMFLLAI(SEQ
ID NO:56), PMSIPPLVKF(SEQ ID NO:57), NKPFVFLMIL(SEQ ID NO:58),
QNTKSPLFMG(SEQ ID NO:59), KVVNPTQK(SEQ ID NO:60), or any
combination thereof.
[0099] In accordance with embodiments of the present invention, the
peptide can be protected or derivitized in by any means known in
the art for example, N-terminal acylation, C-terminal amidation,
cyclization, etc. In a specific embodiment, the N-terminus of the
peptide is acetylated.
Pharmaceutical Compositions
[0100] Embodiments herein provide for administration of
compositions to subjects in a biologically compatible form suitable
for pharmaceutical administration in vivo. By "biologically
compatible form suitable for administration in vivo" is meant a
form of the active agent (i.e. pharmaceutical chemical, protein,
gene, antibody etc of the embodiments) to be administered in which
any toxic effects are outweighed by the therapeutic effects of the
active agent. Administration of a therapeutically active amount of
the therapeutic compositions is defined as an amount effective, at
dosages and for periods of time necessary to achieve the desired
result. For example, a therapeutically active amount of a compound
may vary according to factors such as the disease state, age, sex,
and weight of the individual, and the ability of antibody to elicit
a desired response in the individual. Dosage regima may be adjusted
to provide the optimum therapeutic response.
[0101] In one embodiment, the compound (i.e. pharmaceutical
chemical, protein, peptide etc. of the embodiments) may be
administered in a convenient manner such as subcutaneous,
intravenous, by oral administration, inhalation, transdermal
application, intravaginal application, topical application,
intranasal or rectal administration. Depending on the route of
administration, the active compound may be coated in a material to
protect the compound from the degradation by enzymes, acids and
other natural conditions that may inactivate the compound. In a
preferred embodiment, the compound may be orally administered. In
another preferred embodiment, the compound may be administered
intravenously. In one particular embodiment, the compound may be
administered intranasally, such as inhalation.
[0102] A compound may be administered to a subject in an
appropriate carrier or diluent, co-administered with enzyme
inhibitors or in an appropriate carrier such as liposomes. The term
"pharmaceutically acceptable carrier" as used herein is intended to
include diluents such as saline and aqueous buffer solutions. It
may be necessary to coat the compound with, or co-administer the
compound with, a material to prevent its inactivation. The active
agent may also be administered parenterally or intraperitoneally.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations may contain a
preservative to prevent the growth of microorganisms.
[0103] Pharmaceutical compositions suitable for injectable use may
be administered by means known in the art. For example, sterile
aqueous solutions (where water soluble) or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersion may be used. In all cases, the composition
cant be sterile and can be fluid to the extent that easy
syringability exists. It might be stable under the conditions of
manufacture and storage and may be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The pharmaceutically acceptable carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyetheylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of microorganisms can be achieved by heating, exposing the agent to
detergent, irradiation or adding various antibacterial or
antifungal agents.
[0104] Sterile injectable solutions can be prepared by
incorporating active compound (e.g. a compound that reduces serine
protease activity) in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization.
[0105] Aqueous compositions can include an effective amount of a
therapeutic compound, peptide, epitopic core region, stimulator,
inhibitor, and the like, dissolved or dispersed in a
pharmaceutically acceptable carrier or aqueous medium. Compounds
and biological materials disclosed herein can be purified by means
known in the art.
[0106] Solutions of the active compounds as free-base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0107] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above. It is contemplated that slow release
capsules, timed-release microparticles, and the like can also be
employed. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration.
[0108] The active therapeutic agents may be formulated within a
mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001
to 0.1 milligrams, or about 0.1 to 1.0 or even about 1 to 10 gram
per dose. Single dose or multiple doses can also be administered on
an appropriate schedule for a predetermined condition.
[0109] In another embodiment, nasal solutions or sprays, aerosols
or inhalants may be used to deliver the compound of interest.
Additional formulations that are suitable for other modes of
administration include suppositories and pessaries. A rectal
pessary or suppository may also be used. In general, for
suppositories, traditional binders and carriers may include, for
example, polyalkylene glycols or triglycerides; such suppositories
may be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, preferably 1%-2%.
[0110] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. In certain defined embodiments, oral
pharmaceutical compositions will comprise an inert diluent or
assimilable edible carrier, or they may be enclosed in hard or soft
shell gelatin capsule, or they may be compressed into tablets, or
they may be incorporated directly with the food of the diet. For
oral therapeutic administration, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
should contain at least 0.1% of active compound. The percentage of
the compositions and preparations may, of course, be varied and may
conveniently be between about 2 to about 75% of the weight of the
unit, or preferably between 25-60%. The amount of active compounds
in such therapeutically useful compositions is such that a suitable
dosage will be obtained.
[0111] A pharmaceutical composition may be prepared with carriers
that protect active ingredients against rapid elimination from the
body, such as time-release formulations or coatings. Such carriers
include controlled release formulations, such as, but not limited
to, microencapsulated delivery systems, and biodegradable,
biocompatible polymers, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid
and others are known.
[0112] Pharmaceutical compositions are administered in an amount,
and with a frequency, that is effective to inhibit or alleviate
side effects of a transplant and/or to reduce or prevent rejection.
The precise dosage and duration of treatment may be determined
empirically using known testing protocols or by testing the
compositions in model systems known in the art and extrapolating
therefrom. Dosages may also vary with the severity of the
condition. A pharmaceutical composition is generally formulated and
administered to exert a therapeutically useful effect while
minimizing undesirable side effects. In general, an oral dose
ranges from about 200 mg to about 1000 mg, which may be
administered for example 1 to 3 times per day.
[0113] It will be apparent that, for any particular subject,
specific dosage regimens may be adjusted over time according to the
individual need. The preferred doses for administration can be
anywhere in a range between about 0.01 mg and about 100 mg per ml
of biologic fluid of treated patient. In one particular embodiment,
the range can be between 1 and 100 mg/kg which can be administered
daily, every other day, biweekly, weekly, monthly etc. In another
particular embodiment, the range can be between 10 and 75 mg/kg
introduced weekly to a subject. The therapeutically effective
amount of .alpha.1-antitrypsin, peptides, or drugs that have
similar activities as .alpha.1-antitrypsin or peptides can be also
measured in molar concentrations and can range between about 1 nM
to about 2 mM.
[0114] The tablets, troches, pills, capsules and the like may also
contain the following: a binder, as gum tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent.
[0115] Liposomes can be used as a therapeutic delivery system and
can be prepared in accordance with known laboratory techniques. In
addition, dried lipids or lyophilized liposomes prepared as
previously described may be reconstituted in a solution of active
agent (e.g. nucleic acid, peptide, protein or chemical agent), and
the solution diluted to an appropriate concentration with a
suitable solvent known to those skilled in the art. The amount of
active agent encapsulated can be determined in accordance with
standard methods.
[0116] In a preferred embodiment, a nucleic acid (e.g.
.alpha.1-antitrypsin or analogs thereof) and the lipid
dioleoylphosphatidylcholine may be employed. For example,
nuclease-resistant oligonucleotides may be mixed with lipids in the
presence of excess t-butanol to generate liposomal-oligonucleotides
for administration.
[0117] The pharmaceutical compositions containing the
.alpha.1-antitrypsin, analog thereof, or inhibitor of serine
protease activity or a functional derivative thereof may be
administered to individuals, particularly humans, for example by
subcutaneously, intramuscularly, intranasally, orally, topically,
transdermally, parenterally, gastrointestinally, transbronchially
and transalveolarly. Topical administration is accomplished via a
topically applied cream, gel, rinse, etc. containing
therapeutically effective amounts of inhibitors of serine
proteases. Transdermal administration is accomplished by
application of a cream, rinse, gel, etc. capable of allowing the
inhibitors of serine proteases to penetrate the skin and enter the
blood stream. In addition, osmotic pumps may be used for
administration. The necessary dosage will vary with the particular
condition being treated, method of administration and rate of
clearance of the molecule from the body.
[0118] In each of the aforementioned compositions and methods, a
compound having serine protease inhibitor activity and/or having
.alpha.1-antitrypsin activity or analog thereof may be used in a
single therapeutic dose, acute manner or a chronic manner to treat
episodes or prolonged bouts, respectively, in promoting graft
survival, treating graft rejection and/or associated graft
rejection-induced side-effects.
[0119] In certain embodiments of the methods of the present
invention, the subject may be a mammal such as a human or a
veterinary and/or a domesticated animal.
Therapeutic Methods
[0120] In one embodiment of the present invention, methods provide
for treating a subject in need of or undergoing a transplant. For
example, treatments for reducing graft rejection, promoting graft
survival, and promoting prolonged graft function by administering
to a subject in need thereof a therapeutically effective amount of
a composition. The composition can include a compound capable of
inhibiting at least one serine protease for example,
.alpha.1-antitrypsin, or analog thereof.
Preserving the Graft during Transplant before Engraftment
[0121] According to the methods of the present invention,
transplantation complications can be reduced or inhibited to obtain
important therapeutic benefits. Therefore, administration of a
therapeutic composition contemplated by embodiments of the
invention, i.e., .alpha.1-antitrypsin, derivative or analog
thereof, can be beneficial for the treatment of transplantation
complications or conditions.
[0122] Another beneficial effect of use of the compositions and
methods of the present invention include reducing negative effects
on an organ or non-organ during explant, isolation, transport
and/or prior to implantation. For example, the composition can
reduce apoptosis, reduce production of cytokines, reduce production
of NO, or combination thereof in an organ for transplant. In one
particular embodiment, a composition can include a compound that
includes alpha-1-antitrypsin, an analog thereof, a serine protease
inhibitor, serine protease inhibitor-like activity, analog thereof
or a combination thereof. The transplant organ or non-organ can
include but is not limited to, lung, kidney, heart, liver, soft
tissue, skin, pancreas, intestine, soft tissue cornea, bone marrow,
stem cell, pancreatic islet, and combination thereof.
[0123] In a further embodiment, the methods and compositions of the
invention are useful in the therapeutic treatment of graft
rejection associated side effects. In a yet further embodiment,
graft rejection associated side effects can be prevented by the
timely administration of the agent of the invention as a
prophylactic, prior to onset of one or more symptoms, or one or
more signs, or prior to onset of one or more severe symptoms or one
or more signs of a graft rejection associated disease. Thus, a
patient at risk for a particular graft rejection or graft
rejection-associated disease or clinical indication can be treated
with serine protease inhibitors, for example,
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-Trifluoromethylbenzyl)-1,2,4-ox-
adiazolyl)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide; as a
prophylactic measure.
[0124] It is contemplated herein that the present compositions and
methods of the present invention can be used to treat patients with
one or more grafts who require chronic therapy to maintain graft
integrity, and such patients will therefore benefit from indefinite
or chronic use of the rejection repressive therapy of the methods
of the present invention. Yet another embodiment can be used to
treat flairs of acute rejection so as to minimize the effects of
acute clinical rejection, organ failure, and/or eventual
destruction of the graft.
[0125] Desirable blood levels may be maintained by continuous
infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent
infusions containing about 0.4-20 mg/kg of the active
ingredient(s). Buffers, preservatives, antioxidants and the like
can be incorporated as required. It is intended herein that the
ranges recited also include all those specific percentage amounts
between the recited range. For example, the range of about 0.4 to
20 mg/kg also encompasses 0.5 to 19.9%, 0.6 to 19.8%, etc, without
actually reciting each specific range therewith,
Serine Protease Inhibitors
[0126] It is to be understood that the present invention is not
limited to the examples described herein, and other serine
proteases known in the art can be used within the limitations of
the invention. For example, one skilled in the art can easily adopt
inhibitors as described in WO 98/24806, which discloses substituted
oxadiazole, thiadiazole and triazole as serine protease inhibitors.
U.S. Pat. No. 5,874,585 discloses substituted heterocyclic
compounds useful as inhibitors of serine proteases for example,
(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-trifluoromethylbenzyl)-1,2,4-ox-
a diazolyl)carbonyl)-2-(S)-methylpropyl]-L-prolinamide
benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(2-phenylethyl)-1,2,4-oxadiazolyl)c-
arbonyl)-2-(S)-methylpropyl]-L-prolinamide; and
(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(2-methoxybenzyl)-1,2,4-oxadiazoly-
l)carbonyl)-2-(S)-methylpropyl]-L-prolinamide.
[0127] .alpha.1-antitrypsin is a glycoprotein of MW 51,000 with 417
amino acids and 3 oligosaccharide side chains. Human
.alpha.1-antitrypsin is a single polypeptide chain with no internal
disulfide bonds and only a single cysteine residue normally
intermolecularly disulfide-linked to either cysteine or
glutathione. The reactive site of .alpha.1-antitrypsin contains a
methionine residue, which is labile to oxidation upon exposure to
tobacco smoke or other oxidizing pollutants. Such oxidation reduces
the elastase-inhibiting activity of .alpha.1-antitrypsin; therefore
substitution of another amino acid at that position, i.e. alanine,
valine, glycine, phenylalanine, arginine or lysine, produces a form
of .alpha.1-antitrypsin which is more stable. .alpha.1-antitrypsin
can be represented by the following formula: SEQ ID 61:
mpssvswgil llaglcclvp vslaedpqgd aaqktdtshh dqdhptftiki tpnlaefafs
60 lyrqlahqsn stniffspvs iatafamlsl gtkadthdei leglnfnlte
ipeaqihegf 120 qellrtlnqp dsqlqlttgn glflseglkl vdkfledvkk
lyhseaftvn fgdteeakkq 180 indyvekgtq gkivdlvkel drdtvfalvn
yiffkgkwer pfevkdteee dflhvdqvttv 240 kvpmmkrlgm fniiqhckkls
swvllmkylg nataifflpd egklqhlene lthdiitkfl 300 enedrrsasl
hlpklsitgt ydlksvlgql gitkvfsnga dlsgvteeap lklskavhka 360
vltidekgte aagamfleai pmsippevkf nkpfvflmie qntksplfing kvvnptqk
418
[0128] One important amino acid sequence near the carboxyterminal
end of .alpha.1-antitrypsin is shown in bold and underlined and is
pertinent to this invention (details of the sequence can be found
for example in U.S. Pat. No. 5,470,970, as incorporated by
reference).
[0129] Extrahepatic sites of AAT production include neutrophils,
monocytes and macrophages, and the expression of AAT is inducible
in response to LPS, TNF.alpha., IL-1 and IL-6 in various cell
types. Deficiency in AAT is associated with immune dysfunctional
conditions such as rheumatoid arthritis and systemic lupus
erythematosus.
[0130] Other serine protease inhibitor molecules, which may be used
in any of the disclosed compositions may include compounds
disclosed in the following: WO 98/20034 disclosing serine protease
inhibitors from fleas; WO98/23565 disclosing aminoguanidine and
alkoxyguanidine compounds useful for inhibiting serine proteases;
WO98/50342 disclosing bis-aminomethylcarbonyl compounds useful for
treating cysteine and serine protease disorders; WO98/50420 cyclic
and other amino acid derivatives useful for thrombin-related
diseases; WO 97/21690 disclosing D-amino acid containing
derivatives; WO 97/10231 disclosing ketomethylene group-containing
inhibitors of serine and cysteine proteases; WO 97/03679 disclosing
phosphorous containing inhibitors of serine and cysteine proteases;
WO 98/21186 benzothiazo and related heterocyclic inhibitors of
serine proteases; WO 98/22619 disclosing a combination of
inhibitors binding to P site of serine proteases with chelating
site of divalent cations; WO 98/22098 disclosing a composition
which inhibits conversion of pro-enzyme CPP32 subfamily including
caspase 3 (CPP32/Yama/Apopain); WO 97/48706 disclosing
pyrrolo-pyrazine-diones; and WO 97/33996 disclosing human placental
bikunin (recombinant) as serine protease inhibitor.
[0131] Other compounds having serine protease inhibitory activity
are equally suitable and effective for use in the methods of the
present invention, including but not limited to: tetrazole
derivatives as disclosed in WO 97/24339; guanidinobenzoic acid
derivatives as disclosed in WO 97/37969 and in a number of U.S.
Pat. Nos. 4,283,418; 4,843,094; 4,310,533; 4,283,418; 4,224,342;
4,021,472; 5,376,655; 5,247,084; and 5,077,428; phenylsulfonylamide
derivatives represented by general formula in WO 97/45402; novel
sulfide, sulfoxide and sulfone derivatives represented by general
formula in WO 97/49679; novel amidino derivatives represented by
general formula in WO 99/41231; other amidinophenol derivatives as
disclosed in U.S. Pat. Nos. 5,432,178; 5,622,984; 5,614,555;
5,514,713; 5,110,602; 5,004,612; and 4,889,723 among many
others.
Graft Rejection and Graft Survival-Side-Effects and Conditions
[0132] One of the beneficial effects of use of the compositions and
methods of the present invention include, for example, and not by
way of limitation, reduced infiltration of graft with cells or
serum factors (including but not limited to, complement, anti graft
antibody that generate inflammation and graft rejection), reduced
cytokines, reduced nitric oxide, reduced apoptosis, and reduced
specific immune response against the graft or any combination
thereof.
Management of Graft Rejection
[0133] By preventing or reducing the side effects or conditions
associated with graft survival or graft rejection using this novel
approach, several advantages are obtained compared to alternative
approaches, for example, and not by way of limitation:
[0134] 1. Reduced infiltration of graft with cells or serum factors
(for example, and not by way of limitation, complement, anti graft
antibody that generate inflammation and graft rejection); reduced
production of cytokines or nitric oxide (NO) that can induce
inflammation or apoptosis; inhibits apoptosis; inhibits immune
activation, inhibits CMV or any combination thereof.
[0135] 2. Synthetic inhibitors of serine proteases (AAT-like mimics
or analogs) can and have been developed by means known in the art.
Such a pharmaceutical agent may be formulated as for example, a
cream to treat graft rejection and/or promote graft survival.
[0136] 3. Commercially available agents already approved for
different use in humans will work as a treatment for graft
rejection and/or promote graft survival. These agents are currently
used for indications other than graft rejection and/or to promote
graft survival, and include injectible AAT, plasma preparations,
aprotinin and others (American J. of Resp Critical Care Med 1998,
VII 158: 49-59, incorporated herein by reference in its entirety).
In one embodiment, serine protease inhibitors may be delivered by
inhalation. An inhaled agent (natural AAT or a synthetic AAT-like
mimic/or other inhibitor of serine protease) may be especially
useful due to elevated local concentrations, ease of drug delivery,
and lack of side effects (since administration is not systemic).
This mode of focused drug delivery may augment serine protease
inhibitor activity within the lung tissues and associated
lymphatics, which are two of the principal sites where diseases
and/or clinical conditions associated with graft rejection and/or
promotion of graft survival develop.
[0137] 4. By promoting graft survival and/or treating graft
rejection, the direct cause of the side effect is disrupted in
affected individuals. This invention specifically contemplates
inhibiting host cell serine proteases or induce the SEC receptor or
combination thereof as a method of treating graft rejection and/or
promoting graft survival in a mammal in need thereof in conjunction
with administration of one or more anti-rejection and/or
anti-microbial.
[0138] 5. There is an extensive clinical experience using
injectible AAT to treat patients with genetic AAT deficiency. No
long-term negative effects have been detected to date (American J.
of Resp Critical Care Med 1998, VII 158: 49-59; Wencker et al.
Chest 2001 119:737-744). Moreover, a small molecule inhibitor of
host serine protease has been administered to patients with
Kawasaki's Disease (Ulinistatin, Ono pharmaceuticals).
Isolated Proteins For Use In The Compositions And Methods Of The
Invention
[0139] One aspect of the invention pertains to proteins, and
portions thereof, as well as polypeptide fragments suitable for use
as immunogens to raise antibodies directed against a polypeptide of
the invention. In one embodiment, the native polypeptide can be
isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, polypeptides of the invention are produced
by recombinant DNA techniques. Alternative to recombinant
expression, a polypeptide of the invention can be synthesized
chemically using standard peptide synthesis techniques.
[0140] Recombinant unmodified and mutant variants of .alpha.sub.
1-antitrypsin produced by genetic engineering methods are also
known (see U.S. Pat. No. 4,711,848). The nucleotide sequence of
human alpha.sub. 1-antitrypsin and other human
alpha.sub.1-antitrypsin variants has been disclosed in
international published application No. WO 86/00,337, the entire
contents of which are incorporated herein by reference. This
nucleotide sequence may be used as starting material to generate
all of the AAT amino acid variants and amino acid fragments
depicted herein, using recombinant DNA techniques and methods known
to those of skill in the art.
[0141] An isolated and/or purified or partially purified protein or
biologically active portion thereof may be used in any embodiment
of the invention. A protein that is substantially free of cellular
material includes preparations of protein having less than about
30%, 20%, 10%, or 5% (by dry weight) of heterologous protein. When
the protein or biologically active portion thereof is recombinantly
produced, it can also be substantially free of culture medium. When
the protein is produced by chemical synthesis, it is preferably
substantially free of chemical precursors or other chemicals.
Accordingly, such preparations of the protein have less than about
30%, 20%, 10%, and 5% (by dry weight) of chemical precursors or
compounds other than the polypeptide of interest.
[0142] Biologically active portions of a polypeptide of the
invention include polypeptides including amino acid sequences
sufficiently identical to or derived from the amino acid sequence
of the protein (e.g., the amino acid sequence shown in any of SEQ
ID Nos: 1 to 60, which exhibit at least one activity of the
corresponding full-length protein). A biologically active portion
of a protein of the invention can be a polypeptide, which is, for
example, 5, 10, 25, 50, 100 or more amino acids in length.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of the native form of a polypeptide of the
invention.
[0143] Preferred polypeptides have the amino acid sequence of SEQ
ID Nos: 1 to 60. Other useful proteins are substantially identical
(e.g., at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or
99%) to any of SEQ ID NOs: 1 to 60, and retain the functional
activity of the protein of the corresponding naturally-occurring
protein yet differ in amino acid sequence due to natural allelic
variation or mutagenesis.
[0144] The compounds of the present invention can be used as
therapeutic agents in the treatment of a physiological (especially
pathological) condition caused in whole or part, by excessive
serine protease activity. In addition, a physiological (especially
pathological) condition can be inhibited in whole or part. Peptides
contemplated herein may be administered as free peptides or
pharmaceutically acceptable salts thereof. The peptides should be
administered to individuals as a pharmaceutical composition, which,
in most cases, will include the peptide and/or pharmaceutical salts
thereof with a pharmaceutically acceptable carrier.
[0145] When utilizing BLAST, Gapped BLAST, and PSI-Blast programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
[0146] The present invention also pertains to variants of the
polypeptides of the invention. Such variants have an altered amino
acid sequence which can function as either agonists (mimetics) or
as antagonists. Variants can be generated by mutagenesis, e.g.,
discrete point mutation or truncation. An agonist can retain
substantially the same, or a subset, of the biological activities
of the naturally occurring form of the protein. An antagonist of a
protein can inhibit one or more of the activities of the naturally
occurring form of the protein by, for example, competitively
binding to a downstream or upstream member of a cellular signaling
cascade which includes the protein of interest. Thus, specific
biological effects can be elicited by treatment with a variant of
limited function. Treatment of a subject with a variant having a
subset of the biological activities of the naturally occurring form
of the protein can have fewer side effects in a subject relative to
treatment with the naturally occurring form of the protein.
[0147] Variants of a protein of the invention which function as
either agonists (mimetics) or as antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the protein of the invention for agonist or antagonist
activity.
Fusion Polypeptides
[0148] In other embodiments, compounds having serine protease
inhibitor activity such as .alpha.1-antitrypsin and/or analog
thereof, may be part of a fusion polypeptide. In one example, a
fusion polypeptide may include .alpha.1-antitrypsin (e.g. mammalian
.alpha.1-antitrypsin) or an analog thereof and a different amino
acid sequence that may be heterologous to the .alpha.1-antitrypsin
or analog substance.
[0149] In yet other embodiments, the fusion polypeptide
contemplated for use in the methods of the present invention can
additionally include an amino acid sequence that is useful for
identifying, tracking or purifying the fusion polypeptide, e.g., a
FLAG or HIS tag sequence. The fusion polypeptide can include a
proteolytic cleavage site that can remove the heterologous amino
acid sequence from the compound capable of serine protease
inhibition, such as mammalian .alpha.1-antitrypsin or analog
thereof.
[0150] In one embodiment, fusion polypeptides of the invention are
produced by recombinant DNA techniques. Alternative to recombinant
expression, a fusion polypeptide of the invention can be
synthesized chemically using standard peptide synthesis techniques.
The present invention also provides compositions that comprise a
fusion polypeptide of the invention and a pharmaceutically
acceptable carrier, excipient or diluent.
[0151] In particular, in one embodiment the fusion protein
comprises a heterologous sequence that is a sequence derived from a
member of the immunoglobulin protein family, for example, comprise
an immunoglobulin constant region, e.g., a human immunoglobulin
constant region such as a human IgG1 constant region. The fusion
protein can, for example, include a portion of
.alpha.1-antitrypsin, analog thereof or inhibitor of serine
protease activity polypeptide fused with the amino-terminus or the
carboxyl-terminus of an immunoglobulin constant region, as
disclosed, e.g., in U.S. Pat. No. 5,714,147, and U.S. Pat. No.
5,116,964. In accordance with these embodiments, the FcR region of
the immunoglobulin may be either wild-type or mutated. In certain
embodiments, it is desirable to utilize an immunoglobulin fusion
protein that does not interact with an Fc receptor and does not
initiate ADCC reactions. In such instances, the immunoglobulin
heterologous sequence of the fusion protein can be mutated to
inhibit such reactions. See, e.g., U.S. Pat. No. 5,985,279 and WO
98/06248.
[0152] In yet another embodiment, .alpha.1-antitrypsin, analog
thereof, or inhibitor of serine protease activity polypeptide
fusion protein comprises a GST fusion protein in which is fused to
the C-terminus of GST sequences. Fusion expression vectors and
purification and detection means are known in the art.
[0153] Expression vectors can routinely be designed for expression
of a fusion polypeptide of the invention in prokaryotic (e.g., E.
coli) or eukaryotic cells (e.g., insect cells (using baculovirus
expression vectors), yeast cells or mammalian cells) by means known
in the art.
[0154] Expression of proteins in prokaryotes may be carried out by
means known in the art. Such fusion vectors typically serve three
purposes: 1) to increase expression of recombinant protein; 2) to
increase the solubility of the recombinant protein; and 3) to aid
in the purification of the recombinant protein by acting as a
ligand in affinity purification.
[0155] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression vector
as described in the art. In another embodiment, the recombinant
mammalian expression vector is capable of directing expression of
the nucleic acid preferentially in a particular cell type (e.g.,
tissue-specific regulatory elements are used to express the nucleic
acid) such as pancreas-specific promoters (Edlund et al. (1985)
Science 230:912-916), and mammary gland-specific promoters (e.g.,
milk whey promoter; U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). A host cell can be any
prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells,
yeast or mammalian cells). Vector DNA can be introduced into
prokaryotic or eukaryotic cells via conventional transformation or
transfection techniques.
Combination Therapies
[0156] In each of the aforementioned methods of the present
invention, the use of a compound capable of inhibiting serine
protease or .alpha.1-antitrypsin or analog thereof alone or in
combination with standard immunosuppressive agents enables
transplantation of grafts into immunosuppressed or
immunocompromised recipients. This combination therapy will expand
the eligible patient population able to receive this form of
treatment.
[0157] In each of the aforementioned aspects and embodiments of the
invention, combination therapies other than those already
enumerated above are also specifically contemplated herein. In
particular, the compositions of the present invention may be
admininistered with one or more macrolide or non-macrolide
antibiotics, anti-bacterial agents, anti-fungals, anti-viral
agents, and anti-parasitic agents. Examples of macrolide
antibiotics that may be used in combination with the composition of
the present invention include but are not limited to synthetic,
semi-synthetic or naturally occurring macrolidic antibiotic
compounds: methymycin, neomethymycin, YC-17, litorin, TMP-SSX,
erythromycin A to F, and oleandomycin. Examples of preferred
erythromycin and erythromycin-like compounds include: erythromycin,
clarithromycin, azithromycin, and troleandomycin.
[0158] Examples of anti-bacterial agents include, but are not
limited to, penicillins, quinolonses, aminoglycosides, vancomycin,
monobactams, cephalosporins, carbacephems, cephamycins,
carbapenems, and monobactams and their various salts, acids, bases,
and other derivatives.
[0159] Anti-fungal agents include, but are not limited to,
caspofungin, terbinafine hydrochloride, nystatin, and selenium
sulfide.
[0160] Anti-viral agents include, but are not limited to,
gancyclovir, acyclovir, valacylocir, amantadine hydrochloride,
rimantadin and edoxudine
[0161] Examples of macrolide antibiotics that may be used in
combination with the composition of the present invention include
but are not limited to synthetic, semi-synthetic or naturally
occurring macrolidic antibiotic compounds: methymycin,
neomethymycin, YC-17, litorin, TMP-SSX, erythromycin A to F, and
oleandomycin. Examples of preferred erythromycin and
erythromycin-like compounds include: erythromycin, clarithromycin,
azithromycin, and troleandomycin.
[0162] Anti-parasitic agents include, but are not limited to,
pirethrins/piperonyl butoxide, permethrin, iodoquinol,
metronidazole, co-trimoxazole (sulfamethoxazole/trimethoprim), and
pentamidine isethionate.
[0163] In another aspect, in the method of the present invention,
one may, for example, supplement the composition by administration
of a therapeutically effective amount of one or more an
anti-inflammatory or immunomodulatory drugs or agents. By
"anti-inflammatory drugs", it is meant, e.g., agents which treat
inflammatory responses, i.e., a tissue reaction to injury, e.g.,
agents which treat the immune, vascular, or lymphatic systems.
[0164] Anti-inflammatory or immunomodulatory drugs or agents
suitable for use in this invention include, but are not limited to,
interferon derivatives, (e.g., betaseron); prostane derivatives,
(e.g., compounds disclosed in PCT/DE93/0013, iloprost, cortisol,
dexamethasone; immunsuppressives, (e.g., cyclosporine A, FK-506
(mycophenylate mofetil); lipoxygenase inhibitors, (e.g., zileutone,
MK-886, WY-50295); leukotriene antagonists, (e.g., compounds
disclosed in DE 40091171 German patent application P 42 42 390.2);
and analogs; peptide derivatives, (e.g., ACTH and analogs); soluble
TNF-receptors; TNF-antibodies; soluble receptors of interleukins,
other cytokines, T-cell-proteins; antibodies against receptors of
interleukins, other cytokines, and T-cell-proteins.
Kits
[0165] In still further embodiments, the present invention concerns
kits for use with the methods described above. Small molecules,
proteins or peptides may be employed for use in any of the
disclosed methods. In addition, other agents such as anti-bacterial
agents, immunosuppressive agents, anti-inflammatory agents may be
provided in the kit. The kits will thus can include, in suitable
container means, a protein or a peptide or analog agent, and
optionally one or more additional agents.
[0166] The kits may further include a suitably aliquoted
composition of the encoded protein or polypeptide antigen, whether
labeled or unlabeled, as may be used to prepare a standard curve
for a detection assay.
[0167] The container means of the kits will generally include at
least one vial, test tube, flask, bottle, syringe or other
container means, into which the antibody or antigen may be placed,
and preferably, suitably aliquoted. Where a second or third binding
ligand or additional component is provided, the kit will also
generally contain a second, third or other additional container
into which this ligand or component may be placed. The kits of the
present invention will also typically include a means for
containing the antibody, antigen, and any other reagent containers
in close confinement for commercial sale. Such containers may
include injection or blow-molded plastic containers into which the
desired vials are retained.
EXAMPLES
[0168] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Alpha-1-Antitrypsin Prolongs Graft Islet Graft Survival in Mice
[0169] FIG. 1A-1D. Islets from DBA/2 mice (H-2d) were transplanted
under the renal capsule of streptozotocin-induced hyperglycemic
C57BL/6 mice (H-2b). (A) Glucose levels from days 6-18. Control
consists of mice that were untreated (n=3) or treated from day -1
every 3 days with human albumin (ALB, 6 mg, n=3). Prolonged islet
graft survival is observed in mice treated from day-1 every 3 days
with human AAT (2 mg, n=10). * P<0.05, ** P<0.01, ***
P<0.001 between glucose levels on same day. (B) Treatment
protocols. Control and full AAT treatment are described in panel A.
Early AAT treatment consists of treatment on days -1, 1 and 3 (2
mg, n=3). Late AAT treatment consists of treatment from day 2 and
on every 2 days (2 mg, n=3). Rejection indicates the day that
glucose levels exceed 300 mg/dl. (C) Effect of mouse anti-human-AAT
antibodies. Dashed line indicates post transplantation glucose
levels of a mouse under full AAT treatment protocol (see A, B) that
was immunized by multiple administrations of human AAT prior to
transplantation (1 representative, n=3). Solid line indicates
glucose levels of a non-immunized mouse treated under full AAT
treatment protocol (1 representative, n=10). Arrow indicates
detection of treatment-induced, anti-human-AAT antibodies in the
non-immunized representative mouse. (D) Comparison of day 15
post-transplantation glucose levels in mice that were under full
treatment protocol with ALB (n=3) or AAT (non-immunized n=10,
immunized n=3). Of the AAT-treated group, antibodies were detected
on day 15 in 3/3 immunized mice and in 6/10 non-immunized mice. **
P=0.005 between mice that produced antibodies (n=6) and mice that
did not produce antibodies (n=4).
[0170] Treatment with human albumin (6 mg) resulted in graft
rejection comparable to that of untreated recipient mice. In
contrast, recipient mice that received AAT (2 mg) exhibited
prolonged graft function. As depicted in FIG. 1b, neither of the
partial treatment protocols, i.e., days -1, 1 and 3 (`early
treatment`) or days 2 and beyond (`late treatment`) prolonged
allograft survival.
[0171] AAT-treated mice developed anti-human-AAT antibodies (FIGS.
1C and D). Individual mice exhibited anti-human-AAT antibodies at
various time points (data not shown). To ascertain that the
antibodies reduce the protective effect of AAT, a group of mice was
pre-exposed ("immunized") to human AAT two months before being
rendered hyperglycemic and transplanted with allogeneic islets.
These graft recipients were treated with the full AAT protocol,
despite exhibiting high titers of specific antibodies before
engraftment, and displayed rapid graft rejection (FIG. 1C). Day 15
was chosen to depict an association between antibody formation and
loss of AAT protective activity; at this time point AAT-treated
mice were divided into positive and negative producers of
anti-human-AAT antibodies. As shown in FIG. 1D, on day 15 all
antibody-positive mice were hyperglycemic and all antibody-negative
mice were normoglycemic.
Example 2
[0172] FIG. 2A-2D illustrates an exemplary method of the effect of
AAT on thioglycolate-elicited peritoneal cellular infiltrates. Mice
were administered intraperitoneal 0.1 ml saline, ALB, AAT or
oxidized-AAT followed by 1 ml of saline or thioglycolate (ThG, 3%
w/v, n=3 per group). Peritoneal lavage was performed on separate
groups after 24 and 48 hours. (A) Total cell population of lavaged
cells of (open bars) saline or (closed bars) AAT-treated (5 mg)
thioglycolate-injected mice. ** P<0.05. (B) Percent cell
population from saline-treated mice at 48 hours. ** P<0.05. (C)
Oxidation of AAT. AAT was subjected to oxidative radicals (see
Methods). Loss of serine protease activity of oxidized AAT was
assessed in an elastase assay. Activity of elastase in the absence
of native AAT was set at 100% and the percentage of activity in the
presence of native and oxidized AAT was calculated (n=3). ***
P<0.001. In FIG. 2D, elicited macrophages and neutrophils are
identified. Peritoneal infiltrates from 48 hour lavages of ALB (6
mg) and AAT-treated (6 mg), thioglycolate-injected mice were
stained for FACS analysis by specific antibodies. Macrophages and
neutrophils were identified on the basis of F4/80 and GR1 versus
side scatter flow cytometry profiles. Top, FACS analysis
representative graphs (n=3). Quantified FACS results (n=3) are
depicted in the bottom.
AAT Inhibits Cellular Infiltration
[0173] To address the possibility that AAT affects effector cell
infiltration, two models of cell emigration were examined:
thioglycolate (ThG)-elicited peritoneal infiltration, and cellular
infiltration due to intraperitoneal injection of MHC-incompatible
fibroblasts.
[0174] As shown in FIG. 2A, there was a progressive increase in
total cell count at 24 and 48 hours in mice injected with ThG,
whereas no significant increase was observed in mice injected with
AAT and ThG. At 48 hours, total cell count in peritoneal lavage of
AAT-treated mice was 50% of that of control (FIG. 2B). Total cell
count in mice that received albumin control was similar to that of
saline-treated mice. There was a dose-dependent effect of AAT in
that one-sixth the dose was found to reduce cell count to a lesser
extent in a significant manner. Oxidized AAT, which had lost its in
vitro anti-elastase activity (FIG. 2C), did not affect cellular
infiltrate at 1 mg (FIG. 2B).
[0175] The decrease in total cell count is primarily attributed to
a decrease in the number of neutrophils (FIG. 2D), identified by
their GR-1high/intermediate side-scatter (SSC) profile. No major
difference was observed with the infiltration of macrophages,
identified by their F4/80int, GR-1int, intermediate SSC
profile.sup.12, which is distinct from the F4/80very high, GR-1low,
high SSC profile of resident macrophages.sup.12 (data not
shown).
Example 3
[0176] FIG. 3A-3C illustrates an exemplary method of the effect of
AAT on MHC-incompatible, NIH-3T3-fibroblast-elicited peritoneal
cellular infiltrates. Mice (C57BL/6; H-2b) were injected i.p. 0.1
ml saline or AAT (1 mg) followed by 1 ml NIH-3T3 cells (1 '107
cells in saline; H-2d). Peritoneal lavage was performed daily on
days 1-5 and cell subpopulations were identified by FACS analysis.
(n=3 per treatment). (A) Cell numbers. The number of cells in each
subpopulation was calculated from the percentages obtained by FACS
analysis, and total number of cells in the infiltrate. * P<0.05,
** P<0.01 between cell numbers on the same day. (B)
Representative FACS analysis. (C) Effect of AAT on intensity and
function of infiltrate elicited by islet allograft. Left,
Hematoxilyn and Eosin (H&E) staining of day 7 islet allografts.
A section of AAT-treated islet graft (white frame) is compared to a
similar section of ALB-treated diabetic recipient mouse (full
treatment protocol, see FIG. 1A). Arrow points at border between
islet and surrounding infiltrate. Right, Immunohistochemistry (1HC)
with anti-insulin antibodies of day 15 islet grafts. A section of
autologous islet graft (white frame) is compared to similar
sections of allografts of AAT- and ALB-treated recipient mice. R,
renal parenchyma, G, graft, C, renal capsule.
[0177] As illustrated in FIG. 3A, introduction of allogeneic cells
evoked a cellular infiltrate that consisted of early appearing
neutrophils and activated macrophages, and late appearing CD3+ and
NK cells (FIG. 3B). AAT-treated mice exhibited a reduction in
neutrophils, CD3+ and NK cells, dark color is insulin staining.
[0178] To evaluate the level of cellular infiltration into grafted
islets, grafts from AAT- and ALB-treated recipient mice were
removed on day 7, fixed in paraformaldehyde and stained with
Hematoxilin and Eosin. As depicted in FIG. 3C (left), a cellular
infiltrate is demonstrable regardless of AAT treatment, and
includes neutrophils and lymphocytes. However, the infiltrates
evoked by grafts of ALB-treated recipient mice were more massive
and cause the disruption of islet borders, compared to intact
islets of AAT-treated recipient mice. To evaluate islet function,
grafts from AAT- and ALB-treated recipient mice were removed on day
15, and immunohistochemistry was performed with anti-insulin
antibodies, dark color is insulin staining. As depicted in FIG. 3C
(right), insulin production is preserved on day 15 in islets of
AAT-treated recipients.
Example 4
[0179] FIG. 4A-4H illustrates an exemplary method of the effect of
AAT on islet responses. (A-D) Islets from C57BL/6 mice were
cultured at 100 islets/well, in duplicate. AAT was incubated at the
indicated concentrations for 1 hour before the addition of
IFN.gamma. (5 ng/ml) plus IL-1.beta. (10 ng/ml). 72 hours later,
supernatants were collected and islet viability was assessed. Islet
cells responses in the absence of AAT were set at 100%. Data are
combined from 3 individual experiments, in duplicate. ** P<0.01,
*** P<0.001 between AAT-treated and untreated islets.
Mean.+-.SEM of a. nitrite levels, b. Cell viability and c.
MIP-1.alpha. levels. Dashed line represents islets incubated at
one-30th the concentration of IFN.gamma./IL-1.beta.. d. TNF.alpha.
levels. (E) Insulin induction assay. Islets were incubated in
triplicate (20 islets/well) in the presence of AAT (0.5 mg/ml) or
ALB (0.5 mg/ml) 1 hour before addition of IFN.gamma. (5 ng/ml) plus
IL-1 (10 ng/ml). 24 hours later, islets were transferred to a 3 mM
or 20 mM glucose solution for 30 minutes and insulin levels were
measured. Vertical axis depicts the ratio between insulin levels at
both glucose concentrations. * P<0.05 between AAT-treated and
ALB-treated islets. (F) Streptozotocin toxicity. C57BL/6 mice were
injected i.p. with AAT (5 mg) or saline, one day before, on same
day and one day after injection of streptozotocin (225 mg/kg) or
saline (n=3 per group). 48 hours later, pancreata were removed and
insulin-containing cells were identified by immunohistochemistry.
Each image depicts a representative islet form one pancreas. Graph,
mean.+-.SEM percent change of insulin-containing cells as
determined manually from images of 2 islets per pancreas (n=6 per
treatment group). * P<0.05. (G) Cellular content of islets.
Freshly isolated islets (100 islets in triplicate) and residual
non-islet pancreatic debris were dissociated into single cell
suspensions and stained for FACS analysis with anti-CD45-APC or
isotype control antibody. Shaded area, islets. Open area, debris.
(H) MHC class II expression. Islets from C57BL/6 mice were cultured
(100 islets/well in duplicate) in the presence of AAT (0.5 mg/ml) 1
hour before the addition of IFN.gamma. (5 ng/ml) plus IL-1 (10
ng/ml). 24 hours later, islets were dissociated into single cell
suspensions and double-stained for FACS analysis with anti-CD45-APC
and anti-MHCII-PE, or isotype control antibodies. Left, Mean.+-.SEM
percent change from control (CT) unstimulated islets. * P<0.05
between AAT-treated and untreated islets. Right, Representative
FACS analysis; Shaded area, AAT-treated islets. Open area,
stimulated islets. Events are gated for CD45+.
AAT Modifies Islet Response to Proinflammatory Mediators
[0180] Various islet responses to IL-1.beta./IFN.gamma. were
examined in vitro. Islets exposed to IL-IL-1 .beta./IFN.gamma. for
72 hours produce nitric oxide (NO) in a concentration-dependent
manner and exhibit NO-dependent loss of viability. As shown in
FIGS. 4A and B, in the presence of AAT, less NO was produced and
greater islet viability was obtained. The production of
MIP-1.alpha. was decreased in the presence of AAT, particularly
when stimulated by low concentrations of IL-1.beta./IFN.gamma.
(FIG. 4C). Notably, TNF.alpha. level in supernatants was markedly
diminished by AAT (FIG. 4D). Insulin induction was inhibited by
IL-1.beta./IFN.gamma., but was intact in the presence of
IL-1.beta./IFN.gamma. plus AAT (FIG. 4E). To test the effect of AAT
on islets in vivo, STZ toxicity was evaluated. AAT (2 mg) was
administered one day before, on the same day and a day after STZ
injection. Immunohistochemistry of pancreata with anti-insulin
antibodies at 48 hours after STZ injection reveals more
insulin-producing cells in islets of AAT- than ALB-treated mice
(26.3%.+-.2.6 and 12.8%.+-.2.3 insulin-producing cells per islet,
respectively, FIG. 4f). White cell content of freshly isolated
islets was evaluated by FACS analysis. Islets contain CD45+ cells
(FIG. 4G) that are also positive for the monocytic/granulocytic
markers GR1 and F4/80 (data not shown). This cell population
responded to AAT with decreased surface MHC class II (FIG. 4H).
Example 5
[0181] FIG. 5A-5D illustrates the effect of AAT on TNF-.alpha.. (A)
Islets from C57BL/6 mice were cultured (100 islets/well in
triplicate) in the presence of AAT (0.5 mg/ml) or TACE inhibitor
(10 mM) 1 hour before stimulation by IFN.gamma. (5 ng/ml) plus
IL-1.beta. (10 ng/ml). Left, mean.+-.SEM change in TNF.alpha. in
supernatants after 72 hours of incubation. Right, mean SEM fold
change in membrane TNF.alpha. on islet cells after 5 hours of
incubation, according to FACS analysis. *** P<0.001 compared
control (CT) levels in the absence of AAT. (B) Representative FACS
analysis of membrane TNF.alpha. on stimulated islet cells in the
absence (open area) or presence (shaded area) of AAT. Events are
gated for CD45+. (C) Streptozotocin-induced hyperglycemia. C57BL/6
mice were injected i.p. with saline (n=3), AAT (5 mg, n=3) or
TNF.alpha. (1 mg/kg, n=3) or administered p.o. with TACE inhibitor
(TACEi, 60 mg/kg, n=6) one day before injection of STZ (225 mg/kg,
i.p.). Subsequently, AAT and TNF.alpha. were injected daily; TACE
inhibitor was administered twice a day. At 48 hours, mean.+-.SEM
glucose levels are compared to those of normal littermates (n=3). *
P<0.05, ** P<0.01 compared to saline-treated, STZ-injected
mice.
AAT Inhibits Release of Membrane TNF.alpha.
[0182] Proteolytic cleavage of membrane TNF.alpha. releases soluble
TNF.alpha. from activated cells by the action of
TNF.alpha.-converting-enzyme (TACE). The inventors examined the
levels of membrane TNF.alpha. on stimulated islets in the presence
of AAT. The effect of AAT was compared to that of a TACE inhibitor.
Both AAT and TACE inhibitor decreased TNF.alpha. levels in
supernatants of islets exposed to IL-1.beta./IFN.gamma. (FIG. 5A,
left). Under these conditions, membrane TNF.alpha. accumulated on
the cell surface of CD45+ islet cells (FIG. 5A, right).
[0183] To assess the possibility that islet protection occurs via
inhibition of release of membrane TNF.alpha. in vivo, TACE
inhibitor, p75 TNF receptor (TNF BP) or AAT were introduced to mice
prior to STZ injection. Although all mice developed hyperglycemia
after day 4, the progression of .beta.-cell toxicity was
significantly affected by treatments. As shown in FIG. 5C, the
effect of STZ at 48 hours was decreased in the presence of AAT (a
decrease of 23.2%.+-.2.3 in fasting glucose levels compared to
STZ/saline injected mice). The effect of TACE inhibitor and p75 TNF
receptor was not as profound. Similarly, TACE inhibitor prolonged
islet graft survival to a lesser extent than AAT (preliminary data
not shown).
[0184] Splenocytes that were harvested 48 hours after ThG injection
produced TNF.alpha. in culture (FIG. 5D). AAT administered prior to
thioglycolate decreased TNF.alpha. release from cultured
splenocytes. A similar trend was found with IFN.gamma. (data not
shown), signifying that the response to ThG had effects that extend
beyond the peritoneal compartment and that pretreatment with AAT
reduced these effects.
Example 6
[0185] FIG. 6A-6D illustrates the effect of AAT on Islet allograft
transplantation. 6A illustrates the time course study after
transplantation of islet cells. This example indicates that treated
mice maintain normoglycemia over a 60 day period (n=4), after the
AAT therapy was withdrawn. After withdraw of the therapy, the
normoglycemia lasted another 20 days. 6A illustrates the glucose
follow-up. Positive insulin staining in a day-85 treated islet
graft was also demonstrated (data not shown). 6B illustrates an
immune infiltrate found outside the graft area. 6C illustrates an
increase in the presence of CD4+ and a comparative decrease in
monocytes and neutrophils. It was also shown that massive
vascularization was evident inside the graft (data not shown). It
has been observed that long-lasting accepted islet grafts can be
spared from an immune alloresponse even after therapy removal,
whether the therapy had evoked an immune tolerance specific for the
strain of donor islets was evaluated. For this, grafts were
explanted by nephrectomy and the now-hyperglycemic original
recipients were re-transplanted with either the same strain of
islets as before (n=2), or a 3.sup.rd strain which they had never
encountered before (n=2). In accordance with established strain
specific immune tolerance, mice accepted grafts from original
donors, but had acutely rejected 3.sup.rd-strain grafts (6D); the
same donor (left) and a 3.sup.rd donor re-graft (right).
Example 7
[0186] FIG. 7A-7E illustrates the production of AAT by islet cell
and reflection of islet graft survival. 7A illustrates a time corse
expression of mouse AAT mRNA after cytokine production (IL-1.beta.
and IFN.gamma.) (left) and at 8 hours (right). To demonstrate the
relevance of endogenous alpha-1-antitrypsin in physiological
conditions, the issue of islet injury during pancreatitis was
addressed. In mouse model of acute pancreatitis, isolated islets of
pancreata that are inflamed express inducible alpha-1-antitrypsin.
7B illustrates an example of islet injury during pancreatitis; the
histology of normal islets (top left), the histology of islets of
an inflamed pancreas (top right) and expression of mouse AAT in
islets obtained from the pancreata in an acute pancreatitis model
(bottom). Alpha-1-antitrypsin levels during pancreatitis (caerulein
model for acute pancreatitis). Top, histology of an islet in a
normal pancreas (left) and an islet in an inflamed pancreas
(right), representative of n=3. Bottom, expression of mouse
alpha-1-antitrypsin in islets obtained from pancreata in acute
pancreatitis model. Treatment of mice with exogenous
alpha-1-antitrypsin resulted in down-regulation of endogenous
alpha-1-antitrypsin expression, as well as decrease in serum
TNF.alpha. levels (not shown).
[0187] To demonstrate the relevance of endogenous
alpha-1-antitrypsin in islet transplantation, islet allografts from
untreated transplanted mice on days 1 through 7 after
transplantation (n=3) were excised. These were examined for
alpha-1-antitrypsin expression and reveal a pattern which may fit
inflammation phase (days 1-3) followed by loss of islet mass (days
4-7). 7C illustrates an example of samples of islet allografts
taken post grafting and percent change in AAT mRNA levels were also
assessed. Total RNA was extracted and mRNA for alpha-1-antitrypsin
evaluated by RT-PCR.
[0188] Islet protection from cytokine injury was examined using
endogenous alpha-1-antitrypsin by introducing oncostatin M, a
member of IL-6 family that induces alpha-1-antitrypsin expression
in islets without causing islet death. After 4 days that human
islets were incubated with oncostatin M, for the purpose of
accumulation of sufficient alpha-1-antitrypsin, islets were added
the .beta.-cell-toxic combination of IL-1.beta./IFN.gamma..
Pretreated islets that had excess alpha-1-antitrypsin were
protected from injury, supporting the concept that islet-derived
alpha-1-antitrypsin may participate in islet protection during
inflammation. 7D illustrates an example of islet protection from
cytokine injury with endogenous AAT by introducing oncostatin M (an
interleukin 6 (IL-6) family member) that induces AAT expression in
islets, oncostatin M and AAT levels (top left); nitric oxide and
viability levels assessed (top right). Bottom, human islets exposed
to oncostatin M for 4 days produce enough alpha-1-antitrypsin to
diminish the effects of IL-1.beta./IFN.gamma. added for an
additional 48 hours.
Example 8
[0189] In one exemplary study, alpha-1-antitrypsin on human islets
was examined. FIG. 8A-8D illustrates the effect of AAT on human
islets. The production of nitric oxide (8A), TNF-.alpha. production
(8B) IL-6 (8C) and IL-8 (8D) was examined. 100 human islets per
well were seeded in triplicates and added alpha-1-antitrypsin (AAT)
2 hours before stimuli. Supernatants were assayed 72 hours later.
3A, nitric oxide; 3B, TNF.alpha.; 3C, IL-6; 3D, IL-8. Results are
mean.+-.SEM and are representative of separate islet isolations
from three human donors.
Methods
[0190] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition 1989, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.; Animal Cell Culture, R. I. Freshney, ed.,
1986).
[0191] Mice. C57BL/6 and DBA/2 females were purchased from Jackson
Laboratories.
[0192] Induction of hyperglycemia by streptozotocin, islet
isolation and islet transplantation. In one exemplary method, 5-6
weeks old C57BL/6 mice were treated intraperitoneally (i.p.) with
225 mg/kg Streptozotocin (STZ) (Sigma). Mice with established
hyperglycemia were used at least 5 days after STZ administration.
Islets were isolated from DBA/2 mice on day of transplantation, or
24 hours before in vitro assays, by enzymatic digestion of
pancreata, by means known in the art, with minor modifications.
Briefly, mice were anesthetized with i.p. ketamine (50 mg/kg, Vedco
Inc.) and xylazine (10 mg/kg, Vedco Inc.). Each pancreas was
inflated with 3.5 ml cold collagenase (1 mg/ml, type XI, Sigma),
excised and immersed for 40 minutes at 37.degree. C. in water bath.
Pancreata were gently vortexed and filtered through 500-micron
metal sieve. The pellet was washed twice in cold HBSS containing
0.5% BSA (Sigma) and reconstituted in RPMI-1640 (Cellgro,
Mediatech) supplemented with 10% FCS (Cellgro), 50 IU/ml Penicillin
(Cellgro) and 50 .mu.g/ml streptomycin (Cellgro). Islets were
collected on a 100-micron nylon cell strainer (BD Falcon), released
into a petri dish by rinsing with HBSS (Cellgro, Mediatech) and
0.5% BSA (Sigma) and hand picked under a stereomicroscope. For
transplantation, 450 islets were thoroughly washed from residual
FCS in HBSS and 0.5% BSA and mounted on 0.2 ml tip for immediate
transplantation. For in vitro assays islets were left to incubate
for 24 hours at 37.degree. C. Islet transplantation was performed
into the left renal subcapsular space. Recipient mice were
anesthetized, as described above. An abdominal wall incision was
made over the left kidney. Islets were released into the
subcapsular space through a puncture and the opening was sealed by
means known in the art. Blood glucose follow-up was performed 3
times a week from end-tail blood drop using glucosticks (Roche).
(Nanji, S. A. & Shapiro, A. M. Islet transplantation in
patients with diabetes mellitus: choice of immunosuppression.
BioDrugs 18, 315-28 (2004).)
[0193] Development of anti-human-AAT antibodies in mice. In another
exemplary method, in order to evoke specific antibody production
against human AAT, mice were injected i.p. with 10 mg human AAT per
20-gram mouse for four times in intervals of 1 week. Mice were used
in experiments 2 months after last administration. Antibody
production was evaluated before transplantation experiments were
carried out.
[0194] In one example, assaying for anti-human-AAT antibody levels
was performed as described in the art. Briefly, mouse sera were
kept at -70.degree. C. until assayed for anti-human-AAT levels.
Plates were coated with human AAT or albumin (2 .mu.g/ml) in PBS at
4.degree. C. overnight, then washed and blocked for 1 hour at
25.degree. C. as described. Negative control serum was used in
addition to test serum. Bound anti-AAT antibody using standard TMB
substrate solution was measured (Sigma).
[0195] Cells. NIH-3T3 cell line (e.g. ATCC) were cultured. On day
of peritoneal inoculation, 1.times.10.sup.7 cells were freshly
collected by trypsinization and washed with cold PBS. Pellet was
resuspended in 1 ml cold PBS for immediate injection.
[0196] Infiltration experiments. Peritoneal infiltration was
elicited by i.p. injection of 1 ml autoclaved thioglycolate (3%
w/v, Sigma) or allogeneic cells (NIH-3T3), together with 0.1 ml
saline, human albumin, human AAT or oxidized AAT. Peritoneal lavage
was performed at 24 and 48 hours (thioglycolate) or on days 1-5
(allogeneic cells). For lavage, mice were anesthetized by
isoflurane inhalation and injected immediately with 5.5 ml cold PBS
containing 5% FCS and 5 U/ml heparin into the peritoneal cavity.
After massaging the abdomen, peritoneal fluid was recovered. Red
blood cells were lysed (RBC lysing buffer, BD PharMingen) and cell
counts were performed with a hemocytometer. Cells were then
isolated. Cells (about 1.times.10.sup.6/polypropylene vial) were
incubated with Fc.gamma.RIII/II receptor block antibodies (Table 1)
for 10 min. Cells were then divided into two groups and incubated
with mAbs for leukocytes and either CD3/NK cells or
neutrophil/monocytes/macrophages (Table 1) for 30 min. Cells were
washed and fixed. The number of cells expressing a particular
marker was calculated by multiplying percentages obtained from
flow-cytometry by the concentration of cells in lavage fluid.
TABLE-US-00001 TABLE I Rat Anti-Mouse mAbs Used for Flow Cytometry
Purpose mAb (1) Specificity (2) Source Blocking 2.4G2
Fc.gamma.RIII/II BD PharMingen Leukocytes 30-F11 (APC) CD45
(leukocytes) BD PharMingen Macrophages and F4/80 (PE) F4/80
(macrophages/monocytes) eBiosciences Neutrophils RB6-8C5 (FITC) GR1
(neutrophils/monocytes) BD PharMingen CD3 DX5 (PE) Pan-NK cells
Miltenyi Biotec NK cells 17A2 (FITC) CD3 BD PharMingen TNF.alpha.
MP6-XT22 (PE) Mouse TNFa eBiosciences MHC class II M5/114.15.2 (PE)
I-A.sup.b/d, I-E.sup.d BD PharMingen Isotype control Rat IgG1 (PE)
eBiosciences
[0197] An insulin assay and immunohistochemistry were performed by
means known in the art (Nanji, S. A. & Shapiro, A. M. Islet
transplantation in patients with diabetes mellitus: choice of
immunosuppression. BioDrugs 18, 315-28 (2004)).
[0198] AAT oxidation by myeloperoxidase (MPO) system. In one
example, AAT (4 mg/ml) was incubated at 37.degree. C. for 45
minutes with MPO (1 U/ml, Sigma), H.sub.2O.sub.2 (80 .mu.M, Sigma)
and NaCl (2.5 mM) in PBS, pH 7.4, by means known in the art.
Reaction was terminated by boiling for 1 hour followed by
filter-centrifugation of the system products. In this example,
boiling was needed for the inactivation of MPO but this did not
inactivate AAT (data not shown). Loss of activity of oxidized AAT
was confirmed by elastase activity assay.
[0199] Elastase activity assay. In another exemplary method,
inhibition of a the serine protease elastase was evaluated 30
minutes after co-incubation of AAT or oxidized AAT with porcine
elastase (Sigma) in triplicate, by known methods. The ability of
elastase to liberate 4-nitroaniline (A.sub.410) from
SucAla.sub.3-PNA was determined by kinetic measurement of light
absorbance at 410 nm. Activity in the absence of inhibitors was set
as 100% at the linear range of the assay.
[0200] Cytokine assays. An electrochemiluminescence (ECL) assay as
known in the art was used for the measurement of mouse TNF.alpha.
and MIP-1.alpha.. Briefly, cytokine-specific goat anti-mouse
affinity purified antibodies were labeled with ruthenium (e.g.
BioVeris) according to manufacturer's instructions. Biotinylated
polyclonal anti-mouse antibodies (e.g. R&D Systems) were used.
The amount of TNF.alpha. and MIP-1.alpha. chemiluminescence was
determined using an Origen Analyzer (BioVeris).
[0201] Membrane TNF.alpha.. Membrane TNF.alpha. on islet cells was
detected by modification of a method for the evaluation of membrane
TNF.alpha. on human PBMC. Briefly, single-cell suspension of islets
was incubated with anti-mTNF.alpha.-PE mAb (Table 1). Cells were
washed with FACS buffer and resuspended in 0.5 ml 2% EM-grade
formaldehyde.
[0202] Nitric oxide assay. Nitrite levels in supernatants were
determined using Griess reagent (Promega), as previously described
(Chan, E. D. & Riches, D. W. Am J Physiol Cell Physiol 280,
C441-50 (2001).
[0203] Apoptosis Assay. The protective effect of AAT on islets may
address one of the major obstacles in islet transplantation today,
namely the inadequacy of islet mass and post-isolation islet
viability. Freshly isolated human islets activate stress signaling
pathways and exhibit high rate of apoptosis due to the process of
isolation, necessitating the use of more than one islet donor per
diabetic patient (Nanji, (2004); Abdelli, S. et al. Intracellular
stress signaling pathways activated during human islet preparation
and following acute cytokine exposure. Diabetes 53, 2815-23
(2004)).
[0204] In this example, apoptosis that follows islet isolation is
diminished when islets are cultured with AAT (data not shown) and
demonstrate that islets that are cultured with AAT for 24 hours
prior to transplantation are able to normalize serum glucose levels
of diabetic mice when transplanted autologously at an otherwise
sub-functional mass (data not shown).
[0205] AAT dosage. Normal human plasma contains 0.8-2.4 mg/ml AAT,
with a half life of 5-6 days.sup.1. In gene transfer studies in
C57BL/6 mice, plasma levels of 0.8-1.0 mg/ml were achieved and
provided protection from type I diabetes in NOD mice (Song, S. et
al Gene Therapy 11, 181-6 (2004)). AAT administered
intraperitoneally at 0.3-1.0 mg per mouse protected from
TNF.alpha.-induced lethal response, and 0.8 mg AAT protected from
D-galactosamine/LPS induced hepatic injury. Libert, C., et al., J
Immunol 157, 5126-9 (1996).
[0206] Since AAT levels rise 3- to 4-fold during the acute phase
response 1, 2 mg per mouse results in plasma levels that do not
exceed physiological levels.
[0207] Statistical analysis. Comparisons between groups were
analyzed by two-sided t-test or ANOVA for experiments with more
than two subgroups. Results are presented as mean.+-.SEM.
[0208] Prolongation of Islet Graft Survival by AAT.
[0209] In the present study, administration of clinical grade AAT
to mice transplanted with allogeneic islets prolonged graft
survival. In addition, AAT reduced migration of neutrophils and the
subsequent infiltration of lymphocytes and NK cells in models of
peritonitis. AAT also decreased secretion of TNF.alpha. and
MIP-1.alpha. from islets and inhibited surface MHC class II
expression on CD45+ islet cells in vitro. AAT was protective in a
model of streptozotocin (STZ)-induced .beta.-cell toxicity. Thus,
it appears that AAT monotherapy targets several aspects of an
activated inflammatory immune system, culminating in prolongation
of islet allograft survival.
[0210] Effect of AAT on Cell Infiltration.
[0211] AAT diminished neutrophil migration into the peritoneum of
mice injected with either thioglycolate or MHC-incompatible
fibroblast cells. Other studies demonstrate that AAT inhibits
neutrophil infiltration into kidneys during ischemia/reperfusion
injury and into lungs following intratracheal administration of
silica. In the present study AAT decreased islet production of
MIP-1.alpha. and TNF.alpha., resulting in islets deficient in
chemotactic capabilities and therefore less immunogenic. The
detrimental effect of neutrophils recruited to islets has been
clearly demonstrated.
[0212] The involvement of macrophages in islet destruction is
critical; their presence precedes insulitis in NOD mice and in
prediabetic BB rat, and their depletion is protective during islet
transplantation in rats. Islets are potent recruiters of
macrophages; of the 51 gene products identified in freshly isolated
human islets by cDNA array, expression of MCP-1 was found to be
high. In mice, blockade of MCP-1 prolongs islet allograft survival
when combined with a short subtherapeutic course of rapamycin.
Islet allograft rejection is associated with a steady increase in
intragraft expression of MCP-2, MCP-5, CCL5, CXCL-10 and CXCL9, and
the chemokine receptors CCR2, CCR5, CCR1 and CXCR337. Accordingly,
CCR2-/- mice and CXCR3-/- mice exhibit prolongation of islet
allograft survival. In transplant settings, cytokines that are
produced locally, as TNF.alpha. and IL-1.beta., cause damage to
proximal cells independent of antigen recognition, and complement
activation is critical for graft survival independent of
allospecific immunity. The relevance of macrophages during early
events in islet graft rejection is strengthened by the
identification of CD45, F4/80 and Gr1 positive cells that express
MHC class II in freshly isolated islets. In the presence of AAT,
MHC class II levels were decreased below those of
IL-1.beta./IFN.gamma.-stimulated and unstimulated islets,
supporting the idea that the process of islet isolation is
sufficient to provoke activation of inflammatory pathways in islet
cells. In light of the involvement of neutrophils and macrophages
in graft rejection, interference with their functions by AAT
provides an unusually non-inflammatory environment for the survival
and recovery of engrafted islets.
[0213] As shown in the present study and elsewhere intraperitoneal
injection of allogeneic NIH-3T3 cells evokes infiltration of
macrophage and neutrophil on days 1-2 and of CD3+ and NK cells on
days 4-5. The intensity of the latter infiltration was decreased by
administration of AAT prior to allogeneic cell-line injection, but
not by administration of AAT on day 3 (data not shown). In
transplant settings, early non-specific factors contribute to
subsequent specific immune response. It is therefore possible that
the decrease in CD3+ and NK cell infiltration in the present study
is secondary to the functional failure of the early innate
response. However, regardless of AAT treatment, histological
examination of islet grafts demonstrated that the infiltrate evoked
by allogeneic islets consists of neutrophils and lymphocytes.
Nevertheless, day 7 infiltrate was diminished in AAT-treated
recipients, and, according to day 15 insulin immunohistochemistry,
the infiltrate caused less islet destruction.
AAT Inhibits Release of TNF.alpha..
[0214] Supernatants of IL-1.beta./IFN.gamma.-stimulated islets
contained strikingly less TNF.alpha. when incubated with AAT
(induction of 100.0%.+-.22.0 mean.+-.SEM at 0 mg/ml AAT;
10.2%.+-.11.2 at 0.5 mg/ml and 0.8%.+-.0.1 at 1.0 mg/ml). In
stimulated human PBMC, AAT was shown to diminish TNF.alpha. release
without affecting TNF.alpha.-mRNA levels. In mice, accordingly,
serum TNF.alpha. levels are decreased in LPS-injected AAT-treated
mice. Importantly, treatment of mice with AAT blocks
TNF.alpha.-mediated LPS-induced, but not TNF.alpha.-induced
lethality in mice. In the present study, cultured mouse splenocytes
isolated from thioglycolate-injected mice secreted less TNF.alpha.,
48 hours after injection of AAT.
[0215] In the presence of AAT, membrane TNF.alpha. accumulated in
IL-11/IFN.gamma.-stimulated CD45+ islet cells. TNF.alpha. is
released from the cell surface of macrophages by the action of
TNF.alpha. converting enzyme (TACE), a metalloproteinase that
cleaves membrane TNF.alpha. into the soluble form of TNF.alpha..
Inhibitors of TACE reduce TNF.alpha. release and increase the
levels of membrane TNF.alpha., as demonstrated by FACS analysis.
Although the regulation of TACE activity is unclear, there is
evidence to suggest that extracellular proteases are involved: TACE
does not require its cytoplasmic domain for its activation, its
activity does not depend on the amount of TACE on the cell surface,
co-expression of TACE and transmembrane TNF.alpha. is not
sufficient for processing of TNF.alpha. and the enzyme is expressed
constitutively in various cells. Serpins, such as serpin PN-152,
are suggested to possess extracellular regulatory effects on
various surface proteins.
[0216] TACE is likely to be relevant for graft rejection since TACE
inhibitor decreased injury parameters in a rat model of
post-transplant lung injury. In addition to a decrease in
TNF.alpha. levels, the study shows lower expression of MCP-1 and
ICAM-1, and a reduction in neutrophil infiltration. Similar
findings were obtained with both AAT and a broad spectrum
metalloproteinase inhibitor in a model of silica induced neutrophil
influx into lungs. However, TACE inhibitor only partially
reproduced the protective effect of AAT on islet graft survival
(preliminary data). Similarly, AAT protection from STZ-induced
hyperglycemia was only partially reproduced by TACE inhibition and
by recombinant p75-TNF-receptor. Despite the fact that locally
secreted TNF.alpha. is detrimental to islet graft function, there
is, to our knowledge, no report that describes protection of islet
grafts by neutralization of TNF.alpha. activity. This distinction
between AAT and TACE inhibition supports the possibility that AAT
affects multiple aspects of the immune system, including not only
TNF.alpha. release but also events that are downstream to
TNF.alpha. activities.
[0217] In one embodiment, it is contemplated that a composition of
the present invention may include AAT, an analog thereof, a serine
protease, TACE inhibitor (TACEi) or any combination thereof. These
compositions may be administered to a subject having or in need of
a transplant and or in need of immunotolerance therapy.
Transplanted Islets are Stimulated by the Process of Isolation.
[0218] The process of islet isolation initiates in the islets an
inflammatory cascade of cytokines and chemokines. Thus, isolated
islets contain an intrinsic proinflammatory potential that may
affect local host immune responses. The mechanism of
cytokine-induced islet toxicity is believed to involve expression
of inducible nitric oxide synthase and subsequent production of
nitric oxide (NO) by non-.beta.-cells. In the present study, AAT
decreased NO production in IL-1.beta./IFN.gamma.-treated islets.
Accordingly, islet viability was increased in a low NO environment,
as attained by either incubation with a low concentration of
stimulators (data not shown) or by introduction of AAT. Insulin
induction, which is typically incomplete in the presence of
cytokines, was intact in the presence of AAT and cytokines. In
vivo, AAT protected islets in mice injected with STZ, as concluded
by lower serum glucose levels. The portion of viable .beta.-cells
was visually assessed by insulin immunohistochemistry and was
proportional to the decrease in serum glucose levels. The
protection of AAT was limited to the initial days that follow STZ
administration, suggesting that AAT interferes with NO production
and immune activation and not with intracellular DNA alkylation.
Freshly isolated non-stimulated CD45+ islet cells expressed MHC
class II, which is involved in immune responses against islets. The
levels of MHC class II were elevated in the presence of
IL-1.beta./IFN.gamma. and decreased in the presence of AAT.
Interestingly, MHCII expression was unaffected by the presence of
TACE (TNF alpha converting enzyme) inhibitor (data not shown),
confirming that AAT activities extend beyond those of TACE
inhibition.
[0219] According to the present study, the activities of AAT are
directed against multiple components of the innate immune system,
culminating in a protective effect on islet graft destruction.
Islets in particular exhibited a high degree of protection from
inflammatory processes in the presence of AAT. Pretreatment with
AAT prior to islet transplantation may reduce both islet loss and
the immunological response against the graft.
[0220] The terms and expressions that have been employed are used
as terms of description and not of limitation, and there is no
intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed. Thus, it
should be understood that although the present invention has been
specifically disclosed herein, optional features, modification and
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this invention
as defined by the appended claims. In addition, where features or
aspects of the invention are described in terms of Markush groups,
those skilled in the art will recognize that the invention is also
thereby described in terms of any individual member or subgroup of
members of the Markush group and that other members of the
described groups are included but may not be listed.
[0221] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
Example 9
Prolonged Administration of hAAT to Diabetic Islet Allograft
Recipient Mice Results in Strain-Specific Immune Tolerance
[0222] In one exemplary method, to examine the outcome of islet
allograft transplantation during extended monotherapy with hAAT,
mice heterozygous for tissue-specific hAAT (hAAT-Tg) that exhibit
levels of circulating hAAT that are below detection were used as
graft recipients. hAAT-Tg mice (H-2b) were rendered diabetic,
transplanted with allogeneic islets (H-2d) and treated with serial
doses of hAAT (n=24) or albumin (n=6). As shown in FIG. 9A, control
albumin-treated mice rejected allografts by day 12. In contrast,
all hAAT-treated mice that were treated for the various durations
indicated exhibited extended normoglycemia. The shortest 14-day
course of hAAT therapy resulted in delayed loss of function in 50%
of the transplants and in graft acceptance in the remainder. A
21-day course resulted in a single delayed graft failure event out
of the six transplanted islet grafts. All twelve mice that received
hAAT treatment for 30 days or more achieved graft acceptance
(treatment duration 30 days n=8, 41 days n=1, 52 days n=2, 60 days
n=1).
[0223] Removal of grafts by nephrectomy restored hyperglycemia. In
another exemplary method, as illustrated in FIG. 9C left and right,
by day 12 after transplantation the albumin-treated mouse (broken
line) had mounted an acute allograft rejection response and
developed overt hyperglycemia, whereas the hAAT-treated mice
maintained normoglycemia for the duration of therapy. After
withdrawal of hAAT, continued graft-derived insulin production was
observed (FIG. 9B second column and FIG. 9C days 52-72), raising
the possibility that allospecific immune tolerance was achieved. To
examine this, grafts were removed and a second grafting procedure
was undertaken in the subcapsular space of the remaining kidney
without further treatment, using the same strain of islets that had
been originally transplanted (n=4, H-2d, FIG. 9B and illustrated in
an example in Fib. 9C left). In each case, following
re-engraftment, recipient mice remained normoglycemic for over 50
days. To ascertain that antigen-specific immune tolerance had been
induced, islets from a third strain (H-2k) were used as the source
of the second graft in three hyperglycemic mice without further
hAAT treatment (FIG. 9B and FIG. 9C right). As shown, all three
mice exhibited acute rejection of third-strain allografts.
[0224] According to histology, a "cuff" of mononuclear cells
surrounded the entire islet mass in all explanted grafts (FIG. 9D).
The mononuclear cells were located at the intersection between the
renal parenchyma and capsule, flanking an intact islet graft mass.
By staining for several cell-specific markers we found the near
absence of activated macrophages (CD11b, not shown), and a
predominance of CD4 or CD8-positive cells, interspersed with
CD25-positive cells (not shown).
Example 10
Inflammatory and Anti-Inflammatory Gene Expression in Islet
Allografts
[0225] In another example, in light of the sensitivity of islet
beta cells to inflammatory mediators, expression of
inflammation-related genes in explanted islet grafts was examined.
FIG. 10 represents a comparison between steady-state mRNA patterns
present early after transplantation in albumin-treated mice (days
1, 3, 5 and 7) and in long-lasting hAAT-treated islet grafts
(representative day 72). As illustrated, transcripts of genes
coding for islet-injurious ligands were low in grafts from
hAAT-treated mice. These include the beta cell toxic IL-1.beta., in
addition to CD 14, a marker for invading macrophages, IL 2, carried
by invading T cells and ICAM-1, which represents a pivotal adhesion
molecule typically essential for cell migration. In addition, mRNA
transcripts that encode for the pro-neutrophilic CXC chemokines, KC
and macrophage inflammatory protein (MIP)-2, were undetectable in
long-lasting islet allografts. Islet allograft explants from
hAAT-treated mice also exhibited elevated expression of IL 1
receptor antagonist (IL 1Ra) and isoforms of IL-18 binding protein
(IL 18BP), both reported to protect islet allografts. In contrast,
explants of albumin-treated mice exhibited either low or
undetectable expression of IL 1Ra and IL-18BP (FIG. 10, days 1, 3,
5 and 7). The intensity of the cellular infiltration can be
appreciated by the progressive increase in GAPDH-mRNA levels in
grafts from albumin-treated mice. The identification of insulin
transcripts confirms the presence of beta cells in the
explants.
Example 11
Some Cell-Specific Effects of AAT
[0226] In another example, expression of anti-inflammatory
molecules observed here belong to grafts that were explanted
several weeks after withdrawal of hAAT treatment (see FIG. 9). It
is therefore likely that the intragraft anti-inflammatory gene
expression profile reflects the acquired cellular components that
have progressively accumulated in the antigen-rich site. In order
to examine the effects of hAAT on major cell subpopulations, in
vitro assays were performed for lymphocytic and non-lymphocytic
responses. As illustrated in FIG. 11, IL-2-stimulated human
peripheral blood mononuclear cells (PBMC) were able to produce
IFN.gamma. and proliferate, as expected, in the presence of hAAT.
Similarly, mouse splenocytes responded to Con A with secretion of
IFN.gamma., as well as increased cell proliferation and cell
clumping, each response unaffected by hAAT (FIG. 16A). In contrast
to lymphocytic responses, peritoneal macrophages responded to hAAT
by secreting significantly less IFN.gamma.-induced nitric oxide in
a concentration-dependent manner (FIG. 16B).
Example 12
Treg-Related Gene Expression in hAAT-Treated Islet Allografts
[0227] In the unique set of genes expressed within grafts of
hAAT-treated recipient mice, we also observed the expression of
genes indicative of Treg cells (FIG. 12). As shown, grafts from
hAAT-treated mice (FIG. 12A representative day 72) exhibit a
significantly elevated expression of foxp3, TGF.beta. and CTLA-4,
representing the expected phenotype of Treg cells. In contrast, the
expression of these genes was either below detection or terminated
early in grafts from albumin-treated mice (days 1, 3, 5 and 7). As
depicted in FIG. 4b, the presence of foxp3-positive cells was
observed as early as day-14 of hAAT therapy in sections that
contained the graft site (FIG. 4b, G). Notably, in renal tissue
from kidney portions that did not contain the grafted islets (FIGS.
12B, K), foxp3-positive cells were also observed. CTLA-4 expression
was only present inside the graft (G). Of particular importance,
IL-10 transcript levels were closely associated with
foxp3-expression, suggesting that the identified Treg cells are
also producers of IL-10.
Example 13
Time-Dependent Distribution of Treg Cells between Draining Lymph
Nodes (DLN) and Allograft
[0228] In another example, in order to examine the effect of hAAT
on Treg cell development during transplantation, foxp3-GFP knock-in
mice were used as graft recipients (C57BL/6 background, H-2b). A
vigorous allo-recognition response was evoked by implanting
wild-type skin grafts (H-2d) under the surface of both left and
right thighs. Animals were treated with hAAT (n=13) or albumin
(n=13) using the same dosing schedule employed in the islet
transplantation protocol (see FIG. 1). Inguinal DLN were removed on
various days after grafting and CD4+-sorted cells were examined by
FACS and by RT-PCR for foxp3-positive cells. As shown in FIG. 13A,
between transplantation and 3 days after engraftment of islets the
number of foxp3-positive cells in the DLN unvaryingly decreases in
both the albumin-control and hAAT-treated graft recipient mice.
However, between days 4 and 9 DLN from hAAT-treated mice had more
foxp3-positive cells. In the days that followed, the gap in the
size of the Treg population was restored. Gene expression analysis
corroborated FACS findings (FIG. 13A inset). By using foxp3-GFP
knock-in mice as matrigel-skin graft recipients, we were able to
observe Treg cells infiltrating into allografts by day 10 after
transplantation. This model offers a particular advantage as
invading fluorescent cells can be directly identified in
freshly-obtained, unstained specimens using fluorescent microscopy.
As shown in FIG. 13B, invading foxp3-positive cells localized to
grafts in hAAT-treated animals (bottom). In this technique,
autofluorescent fur can be observed. Total intensity of
infiltrating cells can be appreciated by DAPI counter-staining.
Similarly, as shown in FIG. 13C, islet allografts that had been
transplanted into foxp3-GFP knock-in recipient mice also contained
foxp3-positive cells in the "cuff" site. The proportion of
foxp3-positive cells approximated that found with CD4/CD25
co-staining (not shown). From these findings, it appears that hAAT
treatment promotes early accumulation of Treg cells in the draining
lymph nodes and a progressive migration into the alloantigen-rich
site.
Example 14
Early Local and Systemic Effects of hAAT
[0229] In another exemplary method, events that might precede the
changes observed in the DLN were studies. Islets embedded into
matrigel offer a model for examination of islet-driven cellular
invasion during the first 48 hours of provocation. Allogeneic
islets were introduced into hAAT-containing matrigel plugs
(12.5-100 .mu.g hAAT per graft) and implanted subcutaneously into
mice. Grafts were retrieved 48 hours after transplantation and
intragraft steady-state mRNA levels were assessed. As shown in FIG.
14, a dose-dependent decrease in CD14 mRNA levels had occurred,
reflecting hAAT-dependent inhibition of macrophage invasion.
Distinctively, the recipient mice carry the hAAT genomic insert and
invading host cells can thus be identified. The amount of invading
cells was decreased in the presence of hAAT, as corroborated by
histological examination of the explanted matrigel at 48 hours (not
shown). Copies of insulin mRNA transcripts correlated with the
amount of added hAAT, representing improved hAAT-mediated beta cell
viability. hAAT treatment also resulted in a dose-dependent
increase in VEGF mRNA levels. VEGF mRNA in the matrigel-islet graft
is likely to be of islet-cell origin since VEGF mRNA copies
coincided with near absence of host genomic DNA (FIG. 14).
[0230] In another method, in addition to local events that reflect
an inflammation-dampened antigen presentation environment, more
comprehensive changes that may support the generation of Tregs were
examined. Mice were subject to 10 days of hAAT treatment in order
to reproduce the circulating cytokine environment of a treated
islet graft recipient. Control mice received albumin. Serum levels
of cytokines were then measured. As shown in FIG. 17, serum levels
of Th17-related cytokines, IL-17 and IL-23, were 3-fold lower
compared to levels in control mice. Serum IL-6 and MCP-1 were also
decreased. On the other hand, serum IL-10 levels increased 2-fold
and the levels of I-309, a chemoattractant for Treg cells,
increased 2-fold. To examine the circulating cytokines evoked
during a vigorous inflammatory response, hAAT or albumin-treated
mice (10 days) were challenged with LPS, and serum cytokines were
assessed after 2 hours (FIG. 17). Once more, I-309 and MIP-2 levels
exhibited the favorable changes observed in hAAT-treated
non-challenged mice. Most strikingly, circulating IL-1Ra levels
increased 3-fold. The effect of hAAT treatment on serum IL-6 and
IL-10 levels were also studied during a sterile inflammatory
response (FIG. 17). In this procedure the inflammatory response
results in increased levels of IL 13.beta.-dependent IL-6. However,
mice treated with hAAT exhibited a 30% decrease in serum IL-6
protein levels and a 27% increase in serum IL-10 protein
levels.
Effect of AAT on Dendritic Cell Migration, Maturation and
Function
[0231] To investigate the implications of a dampened antigen
presentation process during transplantation, we studied dendritic
cell activation in vitro and in vivo. Using transgenic GFP-positive
donor skin grafts and subsequent PCR amplification of DNA isolated
from DLN, we evaluate the migration of graft-derived cells towards
DLN in vivo (FIG. 18A). Mice received hAAT one day before grafting,
as during islet allograft transplantation. Graft-derived DNA was
present in DLN after transplantation in both control and
hAAT-treated mice.
Example 15
[0232] Effects of hAAT on the transcript levels of CD86, CD3 and
IL-10 in renal DLN of mice receiving skin grafts into the renal
subcapsular space were examined(FIG. 15). For background gene
expression, DLN from non-transplanted mice were examined.
Seventy-two hours after transplantation, CD86 mRNA transcript
levels were reduced by hAAT treatment 2-fold. At the same time
point, DLN contained 2-fold less total CD3 mRNA transcripts.
Notably, a 2.5-fold rise in IL-10 gene expression was observed in
DLN from hAAT-treated grafted mice.
[0233] In another example, to examine the direct effect of hAAT on
dendritic cell activation and maturation, dendritic cells were
cultured in vitro with LPS in the absence and presence of hAAT
(FIG. 18B). According to FACS analysis, LPS stimulation in the
presence of hAAT resulted in a marked decrease in the levels of
inducible surface MHC class II and CD86.
[0234] To address the possibility that hAAT treatment had
specifically induced IL-10 production in Treg cells in vivo, hAAT
or albumin were administered 3 days before LPS challenge to
foxp3-GFP knock-in mice. 16 hours later spleens were harvested and
splenocytes were isolated to examine IL-10 release in a cytometric
secretion assay. LPS administration alone resulted in
foxp3-positive cells that released IL-10 (6.1.+-.0.1%, compared to
0.2.+-.0.1% without LPS). The number of IL-10-secreting Treg cells
increased in hAAT-treated mice to 10.6.+-.1.2% (mean.+-.SEM,
p=0.0167).
[0235] These data support the possibility that hAAT monotherapy
modifies the antigen presentation process towards the generation of
a migrating, yet immature and tolerogenic dendritic cell phenotype,
culminating in the expansion of functioning, IL-10-producing
Tregs.
Methods
[0236] Mice. hAAT-Tg mice, background strain C57BL/6, were
engineered as described previously and studied as detailed in
Supplementary Methods. Circulating levels of hAAT in heterozygote
hAAT-Tg mice were determined by a specific ELISA for human AAT, as
described previously. Serum levels were below the limit of
detection (10 ng/ml). Wild-type Balb/c, CBA/Ca and DBA/2 mice were
purchased from Jackson Laboratories.
[0237] Islet allograft transplantation. Renal subcapsular islet
transplantation was performed as described previously. Briefly,
hAAT-Tg heterozygote mice weighing 25-30 g were rendered diabetic
by a single i.p. streptozotocin injection (225 mg/kg, Sigma). Donor
islets were isolated and collected on 100-micron cell strainer (BD
Falcon, Franklin Lakes, N.J.), as described previously. 450
hand-picked isolated islets from DBA/2, Balb/c, C57BL/6 or CBA/Ca
donor mice were grafted under the renal subcapsular space. hAAT
treatment was initiated one day before transplantation and every
third day (2 mg per mouse, Aralast, Baxter, Westlake Village,
Calif.). Control hAAT-Tg mice received the same amount of human
serum albumin (Abbott, North Chicago, Ill.). In the experiments in
which monotherapy exceeded 14 days the amount of hAAT was increased
by 0.5 mg every third day until a 6 mg maintenance dose was
reached. Islet allograft rejection was defined as the day blood
glucose exceeded 300 mg/dl after a period of at least 3 days of
normoglycemia.
[0238] Skin allografts. 1 mm.sup.3 freshly-prepared skin derived
from shaved avascular portion of the abdominal midline was used as
donor tissue. A graft was inserted into the subcutaneous space of
each thigh in foxp3-GFP knock-in mice through a 1 mm-long incision.
Incision site was sealed with a 3-0 suture.
[0239] Immunocyte responses in vitro. Peripheral blood mononuclear
cells (PBMC) were isolated from healthy individuals, as described
previously. Studies of human blood were approved by the Colorado
Multiple Institutional Review Board. Splenocytes and resident
peritoneal macrophages were obtained from C57BL/6 mice, as
described previously. Response assays, see Supplementary
Methods.
[0240] Statistical analysis. Comparisons between groups were
performed by two-sided t-test.
[0241] hAAT transgenic Mice. Experiments were performed with
heterozygote hAAT-Tg mice, obtained by mating of hAAT-Tg mice with
wild-type C57BL/6 mice (Jackson Laboratories, Bar Harbor, Me.).
Litters were screened for the presence of the human AAT gene by
standard tail DNA extraction (XNAT2 Extraction Kit, Sigma, St.
Louis, Mo.) followed by two-step nested PCR amplification using the
following primers: outer sequence (450 bp): forward
5'-ACTCCTCCGTACCCTCAACC-3' (SEQ. ID NO. 62) and reverse
5'-GCATTGCCCAGGTATTTCAT-3'(SEQ. ID NO. 63), and inner sequence (249
bp): forward 5'-ACTGTCAACTTCGGGGACAC-3' (SEQ. ID NO. 64) and
reverse 5'-CATGCCTAAACGCTTCATCA-3'(SEQ. ID NO. 65).
[0242] Assessment of explanted renal allografts. Subcapsular renal
grafts were removed by nephrectomy between first and second
grafting procedures under anesthesia by ligation of renal vessels
and severing of the kidney together with the islet graft. Incision
site was sealed with a 3-0 suture and mice were allowed to recover.
Upon graft explantation, tissue was maintained on ice and islet
graft sites were identified macroscopically on the surface of the
kidneys. For RT-PCR, the region containing the graft was removed
with a number 11 blade and immediately transferred to liquid
nitrogen. An equivalent size of tissue was removed from the
opposite renal pole to control for background gene expression of
non-graft tissue. For histology, samples were fixed in 10%
formalin.
[0243] Histology. Kidneys or matrigel explants were fixed in
buffered formalin and 24 hours later cut into two equal portions
through the center of the graft for embodiment in paraffin. Blocks
were sliced serially for multiple staining with either H&E,
DAPI or with the following antibodies: Insulin (as previously
described), VWF, CD4, CD8, CD11b and GFP (eBiosciences, San Diego,
Calif.). Immunostaining methods previously described.
[0244] RT-PCR. Total RNA was extracted (Qiagen) and reverse
transcription followed (Invitrogen, Carlsbad, Calif.). Primers:
mouse IL-1.beta. forward '5-CTCCATGAGCTTTGTACAAGG-3' (SEQ. ID NO.
66) and reverse '5-TGCTGATGTACCAGTTGGGG-3' (SEQ. ID NO. 67), CD14
forward '5-CATTTGCATCCTCCTGGTTTCTGA-3' (SEQ. ID NO. 68) and reverse
'5-GAGTGAGTTTTCCCCTTCCGTGTG-3' (SEQ. ID NO. 69), IL-2 forward
'5-TTCAAGCTCCACTTCAAGCTCTACAGCGGAAG-3' (SEQ. ID NO. 70) and reverse
'5-GACAGAAGGCTATCCATCTCCTCAGAAAGTCC-3'(SEQ. ID NO. 71), IL-10
forward 5'-TGTGAAAATAAGAGCAAGGCAGTG-3' (SEQ. ID NO. 72) and reverse
'5-CATTCATGGCCTTGTAGACACC-3' (SEQ. ID NO. 73), CD3.epsilon. forward
'5-GCCTCAGAAGCATGATAAGC-3' (SEQ. ID NO. 74) and reverse
'5-CCCAGAGTGATACAGATGTC-3' (SEQ. ID NO. 75), CD86 forward
'5-TCCAGAACTTACGGAAGCACCCACG-3'(SEQ. ID NO. 76) and reverse
'5-CAGGTTCACTGAAGTTGGCGATCAC-3'(SEQ. ID NO. 77), ICAM-1 forward
'5-AGGGCTGGCATTGTTCTCTA-3' (SEQ. ID NO. 78) and reverse
'5-CTTCAGAGGCAGGAAACAGG-3' (SEQ. ID NO. 79), KC forward
'5-CGCTCGCTTCTCTGTGCA-3' (SEQ. ID NO. 80) and reverse
'5-ATTTTCTGAACCAAGGGAGCT-3' (SEQ. ID NO. 81), MIP-2 forward
'5-TGCCGGCTCCTCAGTGCTG-3' (SEQ. ID NO. 82) and reverse
'5-AAACTTTTTGACCGCCCTTGA-3' (SEQ. ID NO. 83), GAPDH forward
'5-ATTGACCACTACCTGGGCAA-3' (SEQ. ID NO. 84) and reverse
'5-GAGATACACTTCAACACTTTGACCT-3' (SEQ. ID NO. 85), insulin forward
'5-CAGAAACCATCAGCAAGCAGG -3' (SEQ. ID NO. 86) and reverse
'5-TTGACAAAAGCCTGGGTGGG -3' (SEQ. ID NO. 87), IL-1Ra forward
'5-GACCCTGCAAGATGCAAGCC-3'(SEQ. ID NO. 88) and reverse
'5-GAGCGGATGAAGGTAAAGCG-3'(SEQ. ID NO. 89), IL-18BP forward
'5-CCCACCCTACGAAGTACCAA-3' (SEQ. ID NO. 90) and reverse
'5-CTGGTCAAGGTCATGGTGTG-3'(SEQ. ID NO. 91), foxp3 forward
'5-CCCACCTACAGGCCCTTCTC-3' (SEQ. ID NO. 92) and reverse
'5-GGCATGGGCATCCACAGT-3' (SEQ. ID NO. 93), TGF.beta.1 forward
'5-GAACAAAAAGGTACATGGCCCCTGA-3' (SEQ. ID NO. 94) and reverse
'5-CCTTCTGTTCCCTCTTCAGTGAGGTA-3' (SEQ. ID NO. 95), TGF.beta.2
forward '5-ATGCCCATCGTGCACAGGGACCTCA-3' (SEQ. ID NO. 96) and
reverse '5-CGTTCTGCCACACTGGGCTGTGA-3'(SEQ. ID NO. 97), CTLA-4
forward '5-GTAGCCCTGCTCACTCTTCTT-3' (SEQ. ID NO. 98) and reverse
'5-AGGTACAGTCCCGTGTCAAC-3' (SEQ. ID NO. 99), VEGF forward
'5-GGAGATCCTTCGAGGAGCAGCACTT-3' (SEQ. ID NO. 100) and reverse
'5-GGCGATTTAGCAGCAGATATAAGAA-3' (SEQ. ID NO. 101).
[0245] FACS analysis. Analyses were conducted using a flow
cytometer (FACS Calibur, Becton Dickinson, Mountain View, Calif.).
Fluorescence data were analyzed by the Cell Quest program. At least
50,000 cells were analyzed per sample. Foxp3-GFP-positive cell
analysis was performed on unstained CD4.sup.+-sorted lymphocytes
(1.times.10.sup.6 per sample, see `DLN analysis`). Dendritic cells
(1.times.10.sup.6 per sample) were double-stained with CD11c-APC
and anti-CD86-PE, or anti-CD11c-APC and anti-MHCII-PE (all
antibodies obtained from eBioscience). Antibodies were diluted to
recommended concentrations according to the manufacturer's
instructions. Nonspecific binding of antibodies was assessed with
cells labeled with matching isotype control antibodies. Nonspecific
Fc staining was excluded by the addition of Fc-blocking antibodies
(eBioscience).
[0246] IL-10 cytometric secretion assay. The assay was performed
according to manufacturer's instructions (Miltenyi Biotech,
Bergisch Gladbach, Germany). Briefly, an anti-CD45-pan-leukocytic
chimeric antibody that also specifically binds to IL-10 was added
to the freshly isolated spleen cells. During a short incubation,
IL-10 that is released from activated cells is captured by the
chimeric antibody and is bound to the surface of the secreting
cell. Culture conditions preclude cell-to-cell association. The
assay was performed on cells from foxp3-GFP knock-in mice. As such,
the foxp3-positive cell subpopulation was identified by GFP and
surface bound IL-10 was identified by using anti-IL-10-PE in the
same cell preparation (Miltenyi Biotech).
[0247] Lymphocytic response assays. PBMC were cultured in RPMI
supplemented with 10% FCS, 50 U/ml penicillin and 50 .mu.g/ml
streptomycin (Cellgro, Hemdon, Va.). Cells (5.times.10.sup.5 per
well) were primed for 72 hours with concanavalin A (Con A, 1
.mu.g/ml, Sigma). PBMC were then washed, resuspended
(5.times.10.sup.5 per well) and activated with human IL-2
(Peprotech, Rocky Hill, N.J.) in the presence of hAAT or human
serum albumin. Splenocytes (5.times.10.sup.5 per well) were
stimulated with Con A (1 .mu.g/ml) for 24 hours in the presence of
hAAT or albumin. Clumping was examined microscopically. Peritoneal
macrophages Cells (5.times.10.sup.5 per well) were stimulated for
24 hours with murine IFN.gamma. (Peprotech) in the presence of hAAT
or human serum albumin. Bone-marrow derived dendritic cells were
obtained by growing bone marrow stem cells from wild-type mouse
femurs in Dulbecco's Modified Eagle's Medium (DMEM) (Biological
Industries, Bet Haemek, Israel) supplemented with 10% FCS, 50 .mu.M
.beta.-mercaptoethanol (Sigma), 100 U/ml penicillin and 100
.mu.g/ml streptomycin (Biological Industries). Cells were cultured
in the presence of 10 ng/ml granulocyte-macrophage
colony-stimulating factor (Biological Industries) both in the
initial seeding step and again after 5 days of culture. For
stimulation with LPS, the dendritic cells from 10-day cultures were
mechanically removed from the culture wells with a rubber spatula,
washed, and then 3.times.10.sup.6 cells were incubated for 18 h
with 100 ng/ml LPS (Sigma).
[0248] DLN analysis. Depending on the graft site, renal or inguinal
DLN were harvested on indicated days. To examine gene expression,
the lymphoid tissue was snap-frozen, total RNA extracted,
quantified and normalized, and RT-PCR performed as described below.
To examine the proportion of foxp3-positive Treg cells in DLN by
FACS analysis, lymphoid tissue from foxp3-GFP knock-in mice was
mechanically dissociated immediately after lymph node removal and
CD4-positive cells sorted by magnetic-bead enrichment, according to
manufacturer's instructions (EasySep MuCD4, Stem Cell Technologies,
Vancouver, Canada). FACS analysis was carried out to measure
percent of GFP-positive cells in the population. In migration
experiments, DLN were harvested from mice that were grafted with
GFP-positive allogeneic skin and the specific presence of
graft-derived cells was assessed by DNA isolation and PCR
amplification of the transgenic sequence as described.
[0249] Matrigel-islet grafts. Growth factor reduced matrigel (BD
Pharmingen, Erembodegen, Belgium) remains at a liquid state at
4.degree. C. and at a semi-solid state when introduced to mice
subcutaneously. Fluid phase matrigel (0.3 ml) was mixed with 100
freshly isolated allogeneic islets from wild-type mice together
with albumin or hAAT. Immediately after mixing, the matrigel-islet
allografts were injected subcutaneously into the scruff region of
the neck of anesthesized hAAT-Tg mice through a 21G needle.
Explantation of matrigel plugs was performed under anesthesia. One
section was fixed in 10% formalin and processed for histology, the
other section was immersed at 37.degree. C. in constant stirring
with dispase (BD Pharmingen) for 2 hours for gentle digestion of
the matrigel and release of inhabitant and invading cells. The
cells recovered from the digested matrigel were washed three times
with PBS and divided into two equal parts: one part was processed
for RNA extraction (Qiagen, Inc., Valencia, Calif.) followed by
RT-PCR (see below). The second part was processed for genomic DNA
extraction (GenScript, Piscataway, N.J.) followed by PCR for hAAT
(see `Mice`).
[0250] Matrigel-skin grafts. A single 1 mm.sup.3 freshly-prepared
skin section (see `Skin allograft` above) was mixed with matrigel
and grafted subcutaneously into the scruff region of the neck of
foxp3-GFP knock-in mice. Upon matrigel harvest, one section was
placed immediately on a cover slip and analyzed by fluorescent
microscopy for GFP-positive cells. DAPI mounting medium was added
in order to evaluate total cell content. An equal size portion of
the matrigel was processed for RT-PCR analysis (see `Matrigel-islet
grafts`).
[0251] Cytokine measurements. Murine IFN.gamma. was measured by
ELISA (BD Pharmingen) and human IFN.gamma. was measured by
electrochemiluminescence (ECL) assay using the Origen Analyzer
(BioVeris, Gaithersburg, Md.), as previously described. Murine IL-6
and IL-10 levels were determined by specific ELISA (eBioscience).
Murine serum cytokine levels were also measured using cytokine
Proteome Profiler.TM. blotting according to manufacturer's
instructions (R&D Systems, Minneapolis, Minn.). Nitric oxide
levels were determined by Griess reaction (Promega, Madison,
Wis.).
TABLE-US-00002 TABLE 2 Normoglycemia after islet transplantation in
diabetic recipient mice during and after hAAT treatment, after
graft removal and following second allografting. First graft.sup.1
Second graft.sup.2 hAAT treatment No hAAT treatment No hAAT
treatment Days between Days between Days from transplant and
withdrawal of hAAT second allograft, withdrawal of hAAT and
nephrectomy donor strain 8 52 19 >50, DBA/2 1368 30 22 >54,
DBA/2 1439 30 24 >52, DBA/2 1350 50 22 Died during surgery 959
52 19 11, CBA/Ca, H-2.sup.k 981 52 26 11, CBA/Ca, H-2.sup.k
.sup.1Donor strain: DBA/2, H-2.sup.d, recipient strain: hAAT-Tg,
H-2.sup.b .sup.2Nephrectomized hyperglycemic mice grafted with
islets in contralateral renal subcapsular space
Example 16
[0252] Human islets, as well as mouse islets, are extremely
sensitive to inflammatory mediators. In the presence of certain
cytokines, islets will loose their rounded dense morphology and
become injured; critical islet mass will be lost in the first 48
hours after transplantation, requiring more than one donor per
diabetic human recipient of an islet graft. In one exemplary
method, FIG. 19 represents the effects of AAT on stimulated human
islets. Human islets were cultured in the presence of IL-1.beta.
plus IFN.gamma. for 72 hours with indicated concentrations of AAT.
As illustrated in FIG. 19, there are morphological indications in
islets incubated with AAT compared to control islets from the same
donor without AAT.
[0253] The multicellular islet contains cells capable of secreting
important inflammatory agents. Upon transplantation, these
contribute to loss of graft. AAT reduced the amounts of critical
cytokines secreted by islets in response to inflammation, and
lowered nitric oxide levels. Islets were cultured in the presence
of IL-1.beta. plus IFN.gamma. for 72 hours with indicated
concentrations of AAT (micrograms/mL)) As shown in FIG. 20, levels
of IL-6, IL-8 and TNF.alpha. (percent from stimulated islets) and
nitric oxide were determined in supernatants. Data are mean levels
5 islet donors.
[0254] When added immediately after isolation, a procedure possible
only in a center where human islet cells are isolated, islet cell
death driven by exogenous cytokines (as would occur during
transplantation) is abolished. The effect of AAT inhibition in the
experiment is represented in FIG. 21. As shown, CT (control) are
islets incubated without AAT and the LDH levels (indicator of cell
death) are elevated when exposed to the combination of IL-1.beta.
plus IFN.gamma.. On the right, islets were exposed to the
combination of IL-1.beta. plus IFN.gamma. in the presence of AAT
(0.5 mg/mL) and LDH levels were the same as in islets without
IL-1.beta. plus IFN.gamma..
Example 17
[0255] In another exemplary method, a subject scheduled for a
transplant is treated with an AAT composition intravenously. In
this example, a patient is identified in need of transplant
surgery. The patient is administered an infusion via iv of an AAT
composition (e.g. 60 mg/kg of Aralast AAT). Then the patient is
sent to the fluoroscopy room where a catheter is implanted in a
portal vein under fluoroscopy and islet cells are infused directly
into the subject (e.g. where the cells then lodge in the liver).
Later, for example 2 days after transplant of the islet cells, a
patient is treated with another iv infusion of an AAT composition
(e.g. 60 mg/kg of Aralast AAT). In addition, the patient may be
treated in another several days, for example, about 5 days later
with another iv infusion of an AAT composition (e.g. 60 mg/kg of
Aralast AAT). In order to assess whether periodic administration of
an AAT composition is needed, blood glucose levels of the patient
can be measured to assess immune tolerance of the transplanted
cells and administration of additional AAT composition infusions
can be determined and delivered as needed. In addition, the patient
can be monitored for anti-inflammatory levels using for example
drawing one or more blood samples from a patient and assessing the
level of anti-inflammatory compounds in the blood (e.g. cytokine
levels in the blood). In this example, depending on patient need,
AAT compositions can be administered, before, during and/or after
islet cell transplantation.
[0256] All of the COMPOSITIONS and METHODS disclosed and claimed
herein may be made and executed without undue experimentation in
light of the present disclosure. While the COMPOSITIONS and METHODS
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variation may be applied
to the COMPOSITIONS and METHODS and in the steps or in the sequence
of steps of the METHODS described herein without departing from the
concept, spirit and scope of the invention. More specifically, it
will be apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
Sequence CWU 1
1
10115PRTArtificialsynthetic peptide 1Phe Val Phe Leu Met1
525PRTArtificialsynthetic peptide 2Phe Val Phe Ala Met1
535PRTArtificialsynthetic peptide 3Phe Val Ala Leu Met1
545PRTArtificialsynthetic peptide 4Phe Val Phe Leu Ala1
555PRTArtificialsynthetic peptide 5Phe Leu Val Phe Ile1
565PRTArtificialsynthetic peptide 6Phe Leu Met Ile Ile1
575PRTArtificialsynthetic peptide 7Phe Leu Phe Val Leu1
585PRTArtificialsynthetic peptide 8Phe Leu Phe Val Val1
595PRTArtificialsynthetic peptide 9Phe Leu Phe Leu Ile1
5105PRTArtificialsynthetic peptide 10Phe Leu Phe Phe Ile1
5115PRTArtificialsynthetic peptide 11Phe Leu Met Phe Ile1
5125PRTArtificialsynthetic peptide 12Phe Met Leu Leu Ile1
5135PRTArtificialsynthetic peptide 13Phe Ile Ile Met Ile1
5145PRTArtificialsynthetic peptide 14Phe Leu Phe Cys Ile1
5155PRTArtificialsynthetic peptide 15Phe Leu Phe Ala Val1
5165PRTArtificialsynthetic peptide 16Phe Val Tyr Leu Ile1
5175PRTArtificialsynthetic peptide 17Phe Ala Phe Leu Met1
5185PRTArtificialsynthetic peptide 18Ala Val Phe Leu Met1
51910PRTHomo sapiens 19Met Pro Ser Ser Val Ser Trp Gly Ile Leu1 5
102010PRTHomo sapiens 20Leu Leu Ala Gly Leu Cys Cys Leu Val Pro1 5
102110PRTHomo sapiens 21Val Ser Leu Ala Glu Asp Pro Gln Gly Asp1 5
102210PRTHomo sapiens 22Ala Ala Gln Lys Thr Asp Thr Ser His His1 5
102310PRTHomo sapiens 23Asp Gln Asp His Pro Thr Phe Asn Lys Ile1 5
102410PRTHomo sapiens 24Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser1 5
102510PRTHomo sapiens 25Leu Tyr Arg Gln Leu Ala His Gln Ser Asn1 5
102610PRTHomo sapiens 26Ser Thr Asn Ile Phe Phe Ser Pro Val Ser1 5
102710PRTHomo sapiens 27Ile Ala Thr Ala Phe Ala Met Leu Ser Leu1 5
102810PRTHomo sapiens 28Gly Thr Lys Ala Asp Thr His Asp Glu Ile1 5
102910PRTHomo sapiens 29Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu1 5
103010PRTHomo sapiens 30Ile Pro Glu Ala Gln Ile His Glu Gly Phe1 5
103110PRTHomo sapiens 31Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro1 5
103210PRTHomo sapiens 32Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn1 5
103310PRTHomo sapiens 33Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu1 5
103410PRTHomo sapiens 34Val Asp Lys Phe Leu Glu Asp Val Lys Lys1 5
103510PRTHomo sapiens 35Leu Tyr His Ser Glu Ala Phe Thr Val Asn1 5
103610PRTHomo sapiens 36Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln1 5
103710PRTHomo sapiens 37Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln1 5
103810PRTHomo sapiens 38Gly Lys Ile Val Asp Leu Val Lys Glu Leu1 5
103910PRTHomo sapiens 39Asp Arg Asp Thr Val Phe Ala Leu Val Asn1 5
104010PRTHomo sapiens 40Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg1 5
104110PRTHomo sapiens 41Pro Phe Glu Val Lys Asp Thr Glu Glu Glu1 5
104210PRTHomo sapiens 42Asp Phe His Val Asp Gln Val Thr Thr Val1 5
104310PRTHomo sapiens 43Lys Val Pro Met Met Lys Arg Leu Gly Met1 5
104410PRTHomo sapiens 44Phe Asn Ile Gln His Cys Lys Lys Leu Ser1 5
104510PRTHomo sapiens 45Ser Trp Val Leu Leu Met Lys Tyr Leu Gly1 5
104610PRTHomo sapiens 46Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp1 5
104710PRTHomo sapiens 47Glu Gly Lys Leu Gln His Leu Glu Asn Glu1 5
104810PRTHomo sapiens 48Leu Thr His Asp Ile Ile Thr Lys Phe Leu1 5
104910PRTHomo sapiens 49Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu1 5
105010PRTHomo sapiens 50His Leu Pro Lys Leu Ser Ile Thr Gly Thr1 5
105110PRTHomo sapiens 51Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu1 5
105210PRTHomo sapiens 52Gly Ile Thr Lys Val Phe Ser Asn Gly Ala1 5
105310PRTHomo sapiens 53Asp Leu Ser Gly Val Thr Glu Glu Ala Pro1 5
105410PRTHomo sapiens 54Leu Lys Leu Ser Lys Ala Val His Lys Ala1 5
105510PRTHomo sapiens 55Val Leu Thr Ile Asp Glu Lys Gly Thr Glu1 5
105610PRTHomo sapiens 56Ala Ala Gly Ala Met Phe Leu Glu Ala Ile1 5
105710PRTHomo sapiens 57Pro Met Ser Ile Pro Pro Glu Val Lys Phe1 5
105810PRTHomo sapiens 58Asn Lys Pro Phe Val Phe Leu Met Ile Glu1 5
105910PRTHomo sapiens 59Gln Asn Thr Lys Ser Pro Leu Phe Met Gly1 5
10608PRTHomo sapiens 60Lys Val Val Asn Pro Thr Gln Lys1
561418PRTHomo sapiens 61Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu
Leu Ala Gly Leu Cys1 5 10 15Cys Leu Val Pro Val Ser Leu Ala Glu Asp
Pro Gln Gly Asp Ala Ala 20 25 30Gln Lys Thr Asp Thr Ser His His Asp
Gln Asp His Pro Thr Phe Asn 35 40 45Lys Ile Thr Pro Asn Leu Ala Glu
Phe Ala Phe Ser Leu Tyr Arg Gln 50 55 60Leu Ala His Gln Ser Asn Ser
Thr Asn Ile Phe Phe Ser Pro Val Ser65 70 75 80Ile Ala Thr Ala Phe
Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr 85 90 95His Asp Glu Ile
Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro 100 105 110Glu Ala
Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn 115 120
125Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu
130 135 140Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val
Lys Lys145 150 155 160Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe
Gly Asp Thr Glu Glu 165 170 175Ala Lys Lys Gln Ile Asn Asp Tyr Val
Glu Lys Gly Thr Gln Gly Lys 180 185 190Ile Val Asp Leu Val Lys Glu
Leu Asp Arg Asp Thr Val Phe Ala Leu 195 200 205Val Asn Tyr Ile Phe
Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 210 215 220Lys Asp Thr
Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val225 230 235
240Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys
245 250 255Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly
Asn Ala 260 265 270Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu
Gln His Leu Glu 275 280 285Asn Glu Leu Thr His Asp Ile Ile Thr Lys
Phe Leu Glu Asn Glu Asp 290 295 300Arg Arg Ser Ala Ser Leu His Leu
Pro Lys Leu Ser Ile Thr Gly Thr305 310 315 320Tyr Asp Leu Lys Ser
Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe 325 330 335Ser Asn Gly
Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys 340 345 350Leu
Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly 355 360
365Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile
370 375 380Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met
Ile Glu385 390 395 400Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys
Val Val Asn Pro Thr 405 410 415Gln Lys6220PRTmouse 62Ala Cys Thr
Cys Cys Thr Cys Cys Gly Thr Ala Cys Cys Cys Thr Cys1 5 10 15Ala Ala
Cys Cys 206320PRTmouse 63Gly Cys Ala Thr Thr Gly Cys Cys Cys Ala
Gly Gly Thr Ala Thr Thr1 5 10 15Thr Cys Ala Thr 206420PRTmouse
64Ala Cys Thr Gly Thr Cys Ala Ala Cys Thr Thr Cys Gly Gly Gly Gly1
5 10 15Ala Cys Ala Cys 206520PRTmouse 65Cys Ala Thr Gly Cys Cys Thr
Ala Ala Ala Cys Gly Cys Thr Thr Cys1 5 10 15Ala Thr Cys Ala
206621PRTmouse 66Cys Thr Cys Cys Ala Thr Gly Ala Gly Cys Thr Thr
Thr Gly Thr Ala1 5 10 15Cys Ala Ala Gly Gly 206720PRTmouse 67Thr
Gly Cys Thr Gly Ala Thr Gly Thr Ala Cys Cys Ala Gly Thr Thr1 5 10
15Gly Gly Gly Gly 206824PRTmouse 68Cys Ala Thr Thr Thr Gly Cys Ala
Thr Cys Cys Thr Cys Cys Thr Gly1 5 10 15Gly Thr Thr Thr Cys Thr Gly
Ala 206924PRTmouse 69Gly Ala Gly Thr Gly Ala Gly Thr Thr Thr Thr
Cys Cys Cys Cys Thr1 5 10 15Thr Cys Cys Gly Thr Gly Thr Gly
207032PRTmouse 70Thr Thr Cys Ala Ala Gly Cys Thr Cys Cys Ala Cys
Thr Thr Cys Ala1 5 10 15Ala Gly Cys Thr Cys Thr Ala Cys Ala Gly Cys
Gly Gly Ala Ala Gly 20 25 307132PRTmouse 71Gly Ala Cys Ala Gly Ala
Ala Gly Gly Cys Thr Ala Thr Cys Cys Ala1 5 10 15Thr Cys Thr Cys Cys
Thr Cys Ala Gly Ala Ala Ala Gly Thr Cys Cys 20 25 307224PRTmouse
72Thr Gly Thr Gly Ala Ala Ala Ala Thr Ala Ala Gly Ala Gly Cys Ala1
5 10 15Ala Gly Gly Cys Ala Gly Thr Gly 207322PRTmouse 73Cys Ala Thr
Thr Cys Ala Thr Gly Gly Cys Cys Thr Thr Gly Thr Ala1 5 10 15Gly Ala
Cys Ala Cys Cys 207420PRTmouse 74Gly Cys Cys Thr Cys Ala Gly Ala
Ala Gly Cys Ala Thr Gly Ala Thr1 5 10 15Ala Ala Gly Cys
207520PRTmouse 75Cys Cys Cys Ala Gly Ala Gly Thr Gly Ala Thr Ala
Cys Ala Gly Ala1 5 10 15Thr Gly Thr Cys 207625PRTmouse 76Thr Cys
Cys Ala Gly Ala Ala Cys Thr Thr Ala Cys Gly Gly Ala Ala1 5 10 15Gly
Cys Ala Cys Cys Cys Ala Cys Gly 20 257725PRTmouse 77Cys Ala Gly Gly
Thr Thr Cys Ala Cys Thr Gly Ala Ala Gly Thr Thr1 5 10 15Gly Gly Cys
Gly Ala Thr Cys Ala Cys 20 257820PRTmouse 78Ala Gly Gly Gly Cys Thr
Gly Gly Cys Ala Thr Thr Gly Thr Thr Cys1 5 10 15Thr Cys Thr Ala
207920PRTmouse 79Cys Thr Thr Cys Ala Gly Ala Gly Gly Cys Ala Gly
Gly Ala Ala Ala1 5 10 15Cys Ala Gly Gly 208018PRTmouse 80Cys Gly
Cys Thr Cys Gly Cys Thr Thr Cys Thr Cys Thr Gly Thr Gly1 5 10 15Cys
Ala8121PRTmouse 81Ala Thr Thr Thr Thr Cys Thr Gly Ala Ala Cys Cys
Ala Ala Gly Gly1 5 10 15Gly Ala Gly Cys Thr 208219PRTmouse 82Thr
Gly Cys Cys Gly Gly Cys Thr Cys Cys Thr Cys Ala Gly Thr Gly1 5 10
15Cys Thr Gly8321PRTmouse 83Ala Ala Ala Cys Thr Thr Thr Thr Thr Gly
Ala Cys Cys Gly Cys Cys1 5 10 15Cys Thr Thr Gly Ala 208420PRTmouse
84Ala Thr Thr Gly Ala Cys Cys Ala Cys Thr Ala Cys Cys Thr Gly Gly1
5 10 15Gly Cys Ala Ala 208525PRTmouse 85Gly Ala Gly Ala Thr Ala Cys
Ala Cys Thr Thr Cys Ala Ala Cys Ala1 5 10 15Cys Thr Thr Thr Gly Ala
Cys Cys Thr 20 258621PRTmouse 86Cys Ala Gly Ala Ala Ala Cys Cys Ala
Thr Cys Ala Gly Cys Ala Ala1 5 10 15Gly Cys Ala Gly Gly
208720PRTmouse 87Thr Thr Gly Ala Cys Ala Ala Ala Ala Gly Cys Cys
Thr Gly Gly Gly1 5 10 15Thr Gly Gly Gly 208820PRTmouse 88Gly Ala
Cys Cys Cys Thr Gly Cys Ala Ala Gly Ala Thr Gly Cys Ala1 5 10 15Ala
Gly Cys Cys 208920PRTmouse 89Gly Ala Gly Cys Gly Gly Ala Thr Gly
Ala Ala Gly Gly Thr Ala Ala1 5 10 15Ala Gly Cys Gly 209020PRTmouse
90Cys Cys Cys Ala Cys Cys Cys Thr Ala Cys Gly Ala Ala Gly Thr Ala1
5 10 15Cys Cys Ala Ala 209120PRTmouse 91Cys Thr Gly Gly Thr Cys Ala
Ala Gly Gly Thr Cys Ala Thr Gly Gly1 5 10 15Thr Gly Thr Gly
209220PRTmouse 92Cys Cys Cys Ala Cys Cys Thr Ala Cys Ala Gly Gly
Cys Cys Cys Thr1 5 10 15Thr Cys Thr Cys 209318PRTmouse 93Gly Gly
Cys Ala Thr Gly Gly Gly Cys Ala Thr Cys Cys Ala Cys Ala1 5 10 15Gly
Thr9425PRTmouse 94Gly Ala Ala Cys Ala Ala Ala Ala Ala Gly Gly Thr
Ala Cys Ala Thr1 5 10 15Gly Gly Cys Cys Cys Cys Thr Gly Ala 20
259526PRTmouse 95Cys Cys Thr Thr Cys Thr Gly Thr Thr Cys Cys Cys
Thr Cys Thr Thr1 5 10 15Cys Ala Gly Thr Gly Ala Gly Gly Thr Ala 20
259625PRTmouse 96Ala Thr Gly Cys Cys Cys Ala Thr Cys Gly Thr Gly
Cys Ala Cys Ala1 5 10 15Gly Gly Gly Ala Cys Cys Thr Cys Ala 20
259723PRTmouse 97Cys Gly Thr Thr Cys Thr Gly Cys Cys Ala Cys Ala
Cys Thr Gly Gly1 5 10 15Gly Cys Thr Gly Thr Gly Ala 209821PRTmouse
98Gly Thr Ala Gly Cys Cys Cys Thr Gly Cys Thr Cys Ala Cys Thr Cys1
5 10 15Thr Thr Cys Thr Thr 209920PRTmouse 99Ala Gly Gly Thr Ala Cys
Ala Gly Thr Cys Cys Cys Gly Thr Gly Thr1 5 10 15Cys Ala Ala Cys
2010025PRTmouse 100Gly Gly Ala Gly Ala Thr Cys Cys Thr Thr Cys Gly
Ala Gly Gly Ala1 5 10 15Gly Cys Ala Gly Cys Ala Cys Thr Thr 20
2510125PRTmouse 101Gly Gly Cys Gly Ala Thr Thr Thr Ala Gly Cys Ala
Gly Cys Ala Gly1 5 10 15Ala Thr Ala Thr Ala Ala Gly Ala Ala 20
25
* * * * *
References