U.S. patent application number 13/092823 was filed with the patent office on 2012-02-23 for methods and compositions for islet cell preservation.
Invention is credited to Charles A. Dinarello, Eli C. Lewis, Leland Shapiro.
Application Number | 20120045417 13/092823 |
Document ID | / |
Family ID | 37499146 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120045417 |
Kind Code |
A1 |
Lewis; Eli C. ; et
al. |
February 23, 2012 |
METHODS AND COMPOSITIONS FOR ISLET CELL PRESERVATION
Abstract
Embodiments of the present invention illustrate methods of
treating and preventing transplantation and side effects associated
with transplantation. In particular, the present invention relates
to compositions and methods for inhibition of graft rejection and
promotion of graft survival. Thus, the invention relates to
modulation of cellular activities, including graft rejection,
promotion of graft survival, graft versus host rejection and
conditions commonly associated with graft rejection. More
particularly, the present invention relates to the inhibitory
compounds comprising naturally occurring and man-made inhibitors of
serine protease and inducers of other alpha 1-antitrypsin
activities.
Inventors: |
Lewis; Eli C.; (Be'er Sheva,
IL) ; Dinarello; Charles A.; (Boulder, CO) ;
Shapiro; Leland; (Denver, CO) |
Family ID: |
37499146 |
Appl. No.: |
13/092823 |
Filed: |
April 22, 2011 |
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/US2006/022436 |
Jun 7, 2006 |
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13092823 |
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60687850 |
Jun 7, 2005 |
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Current U.S.
Class: |
424/93.7 ;
435/374; 435/375 |
Current CPC
Class: |
A61P 17/02 20180101;
C07K 16/40 20130101; A61P 1/18 20180101; A61K 35/39 20130101; A61K
2039/505 20130101; A61K 38/191 20130101; A61K 38/57 20130101; A61K
45/06 20130101; A61P 37/06 20180101; A61P 37/02 20180101; A61P 3/10
20180101; A61P 29/00 20180101; A61K 38/217 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: |
424/93.7 ;
435/375; 435/374 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61P 1/18 20060101 A61P001/18; A61P 29/00 20060101
A61P029/00; C12N 5/02 20060101 C12N005/02; A61P 3/10 20060101
A61P003/10 |
Claims
1. A method for preserving or modulating islet cell functions
comprising, alpha 1-antitrypsin or peptide derivative thereof
provided to an islet cell and preserving or modulating islet cell
functions.
2. The method of claim 1, wherein the composition is administered
to a subject having Type 1 diabetes, wherein the composition
preserves the subject's islet cell functions.
3. The method of claim 1, wherein the composition is administered
to a subject having Type 2 diabetes, wherein the composition
preserves the subject islet cell functions.
4. The method of claim 1, wherein the composition is administered
to a subject undergoing an islet cell transplant or islet cell
infusion.
5. The method of claim 1, wherein the composition is administered
to a subject having loss of islet cell function related to systemic
or pancreatic inflammation.
6. The method of claim 1, wherein preserving or modulating islet
cell functions comprise preserving or modulating production of
insulin; islet cell viability; inhibition of production, release,
levels, half-life, activity or combination thereof of chemokines;
inhibition of nitric oxide levels; and inhibition of inflammatory
cytokine production, release, levels, half-life, activity or
combination thereof.
7. The method of claim 6, wherein modulating production of insulin
comprises increasing production of insulin in islet cells.
8. The method of claim 6, wherein modulating production of insulin
comprises increasing secretion of insulin from islet cells.
9. The method of claim 1, wherein a subject is administered the
composition and the subject is a mammalian subject.
10. The method of claim 9, wherein the mammalian subject is an
adult or a juvenile.
11. The method of claim 1, wherein the composition further
comprises one or more anti-transplant rejection agents,
anti-inflammatory agents, immunosuppressive agents,
immunomodulatory agents, anti-microbial agents, or a combination
thereof.
12. The method of claim 1, wherein the composition comprises a
composition of one or more carboxyterminal peptides derivative of
alpha-1 antitrypsin.
13. The method of claim 6, wherein inhibition of inflammatory
cytokine production release, levels, half-life, activity or
combination thereof comprises inhibition of TNF.alpha. (tumor
necrosis factor alpha), IL-1 (interleukin-1.alpha. and
interleukin-1.beta.), IL-12 (interleukin-12), IL-17
(interleukin-17), IL-18 (interleukin-18), IL-23 (interleukin-23),
IFN.gamma. (interferon gamma) and a combination thereof.
14. The method of claim 6, wherein the composition reduces the
level of macrophage inflammatory protein 1 expressed by the islet
cells.
15. A method of treating a subject having compromised or reduced
islet cell function compared to a control subject comprising
administering to the subject a therapeutically effective amount of
a composition comprising alpha 1-antitrypsin or peptide derivative
thereof to the subject and improving islet cell functions compared
to a control not having such a composition.
16. The method of claim 15, wherein the subject has diabetes.
17. The method of claim 15, wherein the subject is at risk of
developing diabetes.
18. The method of claim 15, wherein the subject has Type 1
diabetes.
19. The method of claim 15, wherein the subject has Type 2
diabetes.
20. The method of claim 15, wherein the composition comprises a
composition of one or more carboxyterminal peptide derivatives of
alpha-1 antitrypsin.
21. The method of claim 15, further comprising one or more
additional cytokine inhibitory agents.
22. The method of claim 21, wherein the cytokine inhibitory agents
further inhibit inflammatory cytokines.
23. The method of claim 22, wherein the inflammatory cytokines
comprise IL-1, TNF.alpha. or a combination thereof.
Description
PRIORITY
[0001] This Application is a continuation of U.S. application Ser.
No. 11,916,521 filed Dec. 4, 2007, which is a national stage
application of PCT Application No. PCT/US2006/22436, filed Jun. 7,
2006, which claims priority to U.S. Provisional Application No.
60/687,850 filed Jun. 7, 2005. All prior applications are
incorporated herein in their entirety by reference for all
purposes.
FIELD
[0002] 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 alpha 1-antitrypsin (.alpha.1-antitrypsin)
and agents with .alpha.1-antitrypsin-like activity and/or
compositions and uses of serine protease inhibitors.
BACKGROUND
Serine Proteases
[0003] 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.
[0004] 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.
[0005] 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.
[0006] .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
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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
[0012] 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.
[0013] 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.
[0014] 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)).
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] In yet another embodiment, the present invention may include
combination therapies including compositions exhibiting
.alpha.1-antitrypsin, an analog thereof, 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.
[0022] 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.
[0023] 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.
[0024] 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).
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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
[0029] 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.
[0030] FIG. 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.
[0031] FIG. 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 ( )
AAT-treated (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.
[0032] FIG. 3A-3C illustrates an exemplary method of the effect of
AAT on MIIC-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.
[0033] FIG. 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.
[0034] 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-.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.
[0035] FIG. 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.
[0036] FIG. 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-1.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).
[0037] FIG. 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).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Definitions
[0038] Terms that are not otherwise defined herein are used in
accordance with their plain and ordinary meaning.
[0039] As used herein, "a" or "an" may mean one or more than one of
an item.
[0040] As used herein "analog of alpha-1-antitrypsin" may mean a
compound having alpha-1-antitrypsin-ike 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.
[0041] 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.
[0042] 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
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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).
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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-II (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.
[0055] 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 2-12, amino acids 3-14, 4-16, etc.
[0056] 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); LAGLCCLVPV (SEQ. ID NO. 20) SLAEDPQGDA
(SEQ. ID NO. 21); AQKTDTSHHD (SEQ. ID NO. 22) QDHPTFNKIT (SEQ. ID
NO. 23); PNLAEFAFSL (SEQ. ID NO. 24); YRQLAHQSNS (SEQ. ID NO. 25);
TNIFFSPVSI (SEQ. ID NO. 26); ATAFAMLSLG (SEQ. ID NO. 27);
TKADTHDEIL (SEQ. ID NO. 28); EGLNFNLTEI (SEQ. ID NO. 29);
PEAQIHEGFQ (SEQ. ID) NO. 30); ELLRTLNQPD (SEQ. ID NO. 31);
SQLQLTTGNG (SEQ. ID NO. 32); LFLSEGLKLV (SEQ. ID NO. 33);
DKFLEDVKKL (SEQ. ID NO. 34); YHSEAFTVNF (SEQ. ID NO. 35);
GDHEEAKKQI (SEQ. ID NO. 36); NDYVEKGTQG (SEQ. ID NO. 37);
KIVDLVKELD (SEQ. ID NO. 38); RDTVFALVNY (SEQ. 1D NO. 39);
IFFKGKWERP (SEQ. ID NO. 40); FEVKDTEDED (SEQ. ID NO. 41);
FHVDQVTTVK (SEQ. ID NO. 42); VPMMKRLGMF (SEQ. ID NO. 43);
NIQHCKKLSS (SEQ. ID NO. 44); WVLLMKYLGN (SEQ. ID NO. 45);
ATAIFFLPDE (SEQ. ID NO. 46); GKLQHLENEL (SEQ. ID NO. 47);
THDIITKFLE (SEQ. ED NO. 48); NEDRRSASLH (SEQ. ID NO. 49);
LPKLSITGTY (SEQ. ID NO. 50); DLKSVLGQLG (SEQ. ID NO. 51);
ITKVFSNGAD (SEQ. ID NO. 52); LSGVTEEAPL (SEQ. ID NO. 53);
KLSKAVHKAV (SEQ. ID NO. 54); LTIDEKGTEA (SEQ. ID NO. 55);
AGAMFLEAIP (SEQ. ID NO. 56); MSIPPEVKFN (SEQ. ID NO. 57);
KPFVFLMIEQ (SEQ. ID NO. 58); NTKSPLFMGK (SEQ. ID NO. 59); VVNPTQK
(SEQ. ID NO. 60), or any combination thereof.
[0057] 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
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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%.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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
[0078] 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
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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
[0084] 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.
[0085] .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:
TABLE-US-00001 MPSSVSWGIL LAGLCCLVPV SLAEDPQGDA AQKTDTSHHD 100
QDHPTFNKITPNLAEFAFSL YRQLAHQSNS TNIFFSPVSI ATAFAMLSLG TKADTHDEIL
EGLNFNLTEI PEAQIHEGFQ ELLRTLNQPD SQLQLTTGNG 200
LFLSEGLKLVDKFLEDVKKL YHSEAFTVNF GDHEEAKKQI NDYVEKGTQG KIVDLVKELD
RDTVFALVNY IFFKGKWERP FEVKDTEDED FHVDQVTTVK 300 VPMMKRLGMF
NIQHCKKLSS WVLLMKYLGN ATAIFFLPDE GKLQHLENEL THDIITKFLE NEDRRSASLH
LPKLSITGTY DLKSVLGQLG ITKVFSNGAD 400 LSGVTEEAPL KLSKAVHKAV
LTIDEKGTEA AGAMFLEAIP MSIPPEVKFN KPFVFLMIEQ NTKSPLFMGK VVNPTQK
417
[0086] 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).
[0087] 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.
[0088] 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.
[0089] 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.
[0090] Graft Rejection and Graft Survival-Side-Effects and
Conditions
[0091] 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
[0092] 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:
[0093] 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.
[0094] 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.
[0095] 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,
V11 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.
[0096] 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.
[0097] 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, V11 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
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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
[0115] 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.
[0116] 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.
[0117] 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.
[0118] Anti-fungal agents include, but are not limited to,
caspofungin, terbinafine hydrochloride, nystatin, and selenium
sulfide.
[0119] Anti-viral agents include, but are not limited to,
gancyclovir, acyclovir, valacylocir, amantadine hydrochloride,
rimantadin and edoxudine
[0120] 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.
[0121] Anti-parasitic agents include, but are not limited to,
pirethrins/piperonyl butoxide, permethrin, iodoquinol,
metronidazole, co-trimoxazole (sulfamethoxazole/trimethoprim), and
pentamidine isethionate.
[0122] 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.
[0123] 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
[0124] 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.
[0125] 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.
[0126] 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
[0127] 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
[0128] 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).
[0129] 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.
[0130] 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
[0131] 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
[0132] 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.
[0133] 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).
[0134] The decrease in total cell count is primarily attributed to
a decrease in the number of neutrophils (FIG. 2D), identified by
their GR-1 high/intermediate side-scatter (SSC) profile. No major
difference was observed with the infiltration of macrophages,
identified by their F4/80int, GR-1 int, intermediate SSC
profile.sup.12, which is distinct from the F4/80very high, GR-1
low, high SSC profile of resident macrophages.sup.12 (data not
shown).
Example 3
[0135] 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 (IHC)
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.
[0136] 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.
[0137] 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
[0138] 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.beta. (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
from 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.beta. (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
[0139] 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
[0140] 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.
[0141] 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).
[0142] 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).
[0143] 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
[0144] 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 3r.sup.d donor re-graft (right).
Example 7
[0145] FIG. 7A-7E illustrates the production of AAT by islet cell
and reflection of islet graft survival. 7 A 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).
[0146] 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.
[0147] 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
[0148] 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
[0149] 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).
[0150] Mice. C57BL/6 and DBA/2 females were purchased from Jackson
Laboratories.
[0151] 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).)
[0152] 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.
[0153] 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).
[0154] 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.
[0155] 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 I)
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 I) 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-00002 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 F4/80 (PE) F4/80
(macrophages/ eBiosciences and monocytes) Neutrophils RB6-8C5 GR1
(neutrophils/ BD PharMingen (FITC) monocytes) CD3 DX5 (PE) Pan-NK
cells Miltenyi Biotec NK cells 17A2 (FITC) CD3 BD PharMingen
TNF.alpha. MP6-XT22 Mouse TNF.alpha. eBiosciences (PE) MHC class
M5/114.15.2 I-A.sup.b/d, I-E.sup.d BD PharMingen II (PE) Isotype
Rat IgG1 (PE) eBiosciences control
[0156] 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)).
[0157] 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.
[0158] 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.
[0159] 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. Bio
Veris) 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 (Bio Veris).
[0160] 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 I). Cells were
washed with FACS buffer and resuspended in 0.5 ml 2% EM-grade
formaldehyde.
[0161] 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).
[0162] 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)).
[0163] 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).
[0164] 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).
[0165] Since AAT levels rise 3- to 4-fold during the acute phase
responsel, 2 mg per mouse results in plasma levels that do not
exceed physiological levels.
[0166] 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.
[0167] Prolongation of Islet Graft Survival by AAT.
[0168] 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.
[0169] Effect of AAT on Cell Infiltration.
[0170] 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.
[0171] 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, CCLS, 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.
[0172] 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..
[0173] 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.
[0174] In the presence of AAT, membrane TNF.alpha. accumulated in
IL-1.beta./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-I52,
are suggested to possess extracellular regulatory effects on
various surface proteins.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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.
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
6315PRTArtificialsynthetic 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 Ala Gly Leu Cys Cys Leu Val Pro Val1 5
102110PRTHomo sapiens 21Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala1 5
102210PRTHomo sapiens 22Ala Gln Lys Thr Asp Thr Ser His His Asp1 5
102310PRTHomo sapiens 23Gln Asp His Pro Thr Phe Asn Lys Ile Thr1 5
102410PRTHomo sapiens 24Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu1 5
102510PRTHomo sapiens 25Tyr Arg Gln Leu Ala His Gln Ser Asn Ser1 5
102610PRTHomo sapiens 26Thr Asn Ile Phe Phe Ser Pro Val Ser Ile1 5
102710PRTHomo sapiens 27Ala Thr Ala Phe Ala Met Leu Ser Leu Gly1 5
102810PRTHomo sapiens 28Thr Lys Ala Asp Thr His Asp Glu Ile Leu1 5
102910PRTHomo sapiens 29Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile1 5
103010PRTHomo sapiens 30Pro Glu Ala Gln Ile His Glu Gly Phe Gln1 5
103110PRTHomo sapiens 31Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp1 5
103210PRTHomo sapiens 32Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly1 5
103310PRTHomo sapiens 33Leu Phe Leu Ser Glu Gly Leu Lys Leu Val1 5
103410PRTHomo sapiens 34Asp Lys Phe Leu Glu Asp Val Lys Lys Leu1 5
103510PRTHomo sapiens 35Tyr His Ser Glu Ala Phe Thr Val Asn Phe1 5
103610PRTHomo sapiens 36Gly Asp His Glu Glu Ala Lys Lys Gln Ile1 5
103710PRTHomo sapiens 37Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly1 5
103810PRTHomo sapiens 38Lys Ile Val Asp Leu Val Lys Glu Leu Asp1 5
103910PRTHomo sapiens 39Arg Asp Thr Val Phe Ala Leu Val Asn Tyr1 5
104010PRTHomo sapiens 40Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro1 5
104110PRTHomo sapiens 41Phe Glu Val Lys Asp Thr Glu Asp Glu Asp1 5
104210PRTHomo sapiens 42Phe His Val Asp Gln Val Thr Thr Val Lys1 5
104310PRTHomo sapiens 43Val Pro Met Met Lys Arg Leu Gly Met Phe1 5
104410PRTHomo sapiens 44Asn Ile Gln His Cys Lys Lys Leu Ser Ser1 5
104510PRTHomo sapiens 45Trp Val Leu Leu Met Lys Tyr Leu Gly Asn1 5
104610PRTHomo sapiens 46Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu1 5
104710PRTHomo sapiens 47Gly Lys Leu Gln His Leu Glu Asn Glu Leu1 5
104810PRTHomo sapiens 48Thr His Asp Ile Ile Thr Lys Phe Leu Glu1 5
104910PRTHomo sapiens 49Asn Glu Asp Arg Arg Ser Ala Ser Leu His1 5
105010PRTHomo sapiens 50Leu Pro Lys Leu Ser Ile Thr Gly Thr Tyr1 5
105110PRTHomo sapiens 51Asp Leu Lys Ser Val Leu Gly Gln Leu Gly1 5
105210PRTHomo sapiens 52Ile Thr Lys Val Phe Ser Asn Gly Ala Asp1 5
105310PRTHomo sapiens 53Leu Ser Gly Val Thr Glu Glu Ala Pro Leu1 5
105410PRTHomo sapiens 54Lys Leu Ser Lys Ala Val His Lys Ala Val1 5
105510PRTHomo sapiens 55Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala1 5
105610PRTHomo sapiens 56Ala Gly Ala Met Phe Leu Glu Ala Ile Pro1 5
105710PRTHomo sapiens 57Met Ser Ile Pro Pro Glu Val Lys Phe Asn1 5
105810PRTHomo sapiens 58Lys Pro Phe Val Phe Leu Met Ile Glu Gln1 5
105910PRTHomo sapiens 59Asn Thr Lys Ser Pro Leu Phe Met Gly Lys1 5
10607PRTHomo sapiens 60Val Val Asn Pro Thr Gln Lys1 561417PRTHomo
sapiens 61Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Ala Gly Leu
Cys Cys1 5 10 15Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp
Ala Ala Gln 20 25 30Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro
Thr Phe Asn Lys 35 40 45Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser
Leu Tyr Arg Gln Leu 50 55 60Ala His Gln Ser Asn Ser Thr Asn Ile Phe
Phe Ser Pro Val Ser Ile65 70 75 80Ala Thr Ala Phe Ala Met Leu Ser
Leu Gly Thr Lys Ala Asp Thr His 85 90 95Asp Glu Ile Leu Glu Gly Leu
Asn Phe Asn Leu Thr Glu Ile Pro Glu 100 105 110Ala Gln Ile His Glu
Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn Gln 115 120 125Pro Asp Ser
Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu Ser 130 135 140Glu
Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys Leu145 150
155 160Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp His Glu Glu
Ala 165 170 175Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln
Gly Lys Ile 180 185 190Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr
Val Phe Ala Leu Val 195 200 205Asn Tyr Ile Phe Phe Lys Gly Lys Trp
Glu Arg Pro Phe Glu Val Lys 210 215 220Asp Thr Glu Asp Glu Asp Phe
His Val Asp Gln Val Thr Thr Val Lys225 230 235 240Val Pro Met Met
Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys Lys 245 250 255Lys Leu
Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala Thr 260 265
270Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu Asn
275 280 285Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu
Asp Arg 290 295 300Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile
Thr Gly Thr Tyr305 310 315 320Asp Leu Lys Ser Val Leu Gly Gln Leu
Gly Ile Thr Lys Val Phe Ser 325 330 335Asn Gly Ala Asp Leu Ser Gly
Val Thr Glu Glu Ala Pro Leu Lys Leu 340 345 350Ser Lys Ala Val His
Lys Ala Val Leu Thr Ile Asp Glu Lys Gly Thr 355 360 365Glu Ala Ala
Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile Pro 370 375 380Pro
Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu Gln385 390
395 400Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr
Gln 405 410 415Lys62394PRTHomo
sapiensMISC_FEATURE(355)..(358)native sequence 62Glu Asp Pro Gln
Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His1 5 10 15Asp Gln Asp
His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30Phe Ala
Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45Asn
Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55
60Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu65
70 75 80Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly
Phe 85 90 95Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu
Gln Leu 100 105 110Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu
Lys Leu Val Asp 115 120 125Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr
His Ser Glu Ala Phe Thr 130 135 140Val Asn Phe Gly Asp Thr Glu Glu
Ala Lys Lys Gln Ile Asn Asp Tyr145 150 155 160Val Glu Lys Gly Thr
Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175Asp Arg Asp
Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190Lys
Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200
205His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu
210 215 220Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp
Val Leu225 230 235 240Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile
Phe Phe Leu Pro Asp 245 250 255Glu Gly Lys Leu Gln His Leu Glu Asn
Glu Leu Thr His Asp Ile Ile 260 265 270Thr Lys Phe Leu Glu Asn Glu
Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285Pro Lys Leu Ser Ile
Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300Gln Leu Gly
Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly305 310 315
320Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala
325 330 335Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala
Met Phe 340 345 350Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val
Lys Phe Asn Lys 355 360 365Pro Phe Val Phe Leu Met Ile Glu Gln Asn
Thr Lys Ser Pro Leu Phe 370 375 380Met Gly Lys Val Val Asn Pro Thr
Gln Lys385 39063394PRTHomo sapiensMISC_FEATURE(355)..(358)novel
sequence 63Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser
His His1 5 10 15Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn
Leu Ala Glu 20 25 30Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln
Ser Asn Ser Thr 35 40 45Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr
Ala Phe Ala Met Leu 50 55 60Ser Leu Gly Thr Lys Ala Asp Thr His Asp
Glu Ile Leu Glu Gly Leu65 70 75 80Asn Phe Asn Leu Thr Glu Ile Pro
Glu Ala Gln Ile His Glu Gly Phe 85 90 95Gln Glu Leu Leu Arg Thr Leu
Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110Thr Thr Gly Asn Gly
Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125Lys Phe Leu
Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140Val
Asn Phe Gly Asp His Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr145 150
155 160Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu
Leu 165 170 175Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe
Phe Lys Gly 180 185 190Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr
Glu Asp Glu Asp Phe 195 200 205His Val Asp Gln Val Thr Thr Val Lys
Val Pro Met Met Lys Arg Leu 210 215 220Gly Met Phe Asn Ile Gln His
Cys Lys Lys Leu Ser Ser Trp Val Leu225 230 235 240Leu Met Lys Tyr
Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255Glu Gly
Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265
270Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu
275 280 285Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val
Leu Gly 290 295 300Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala
Asp Leu Ser Gly305 310 315 320Val Thr Glu Glu Ala Pro Leu Lys Leu
Ser Lys Ala Val His Lys Ala 325 330 335Val Leu Thr Ile Asp Glu Lys
Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350Leu Glu Arg Xaa Xaa
Arg Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365Pro Phe Val
Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380Met
Gly Lys Val Val Asn Pro Thr Gln Lys385 390
* * * * *
References