U.S. patent application number 14/221887 was filed with the patent office on 2014-08-07 for hsp60, hsp60 peptides and t cell vaccines for immunomodulation.
This patent application is currently assigned to Yeda Research and Development Co. Ltd.. The applicant listed for this patent is Yeda Research and Development Co. Ltd.. Invention is credited to Irun R. Cohen, Ofer Lider, Meirav Pisner, Francisco Quintana, Guy Tal, Alexandra Zanin-Zhorov.
Application Number | 20140219979 14/221887 |
Document ID | / |
Family ID | 36647863 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140219979 |
Kind Code |
A1 |
Cohen; Irun R. ; et
al. |
August 7, 2014 |
HSP60, HSP60 PEPTIDES AND T CELL VACCINES FOR IMMUNOMODULATION
Abstract
The present invention provides novel uses for peptide
p277--positions 437-460 of human heat shock protein 60 (HSP60)--in
modulation of immune responses and inflammatory diseases. The
invention further provides novel uses for HSP60 and p277 in the
treatment or prevention of hepatic disorders. The invention
discloses methods for treating, preventing or ameliorating the
symptoms of T cell mediated inflammatory and autoimmune disorders,
including hepatic disorders, which comprise administering to a
subject in need thereof a composition comprising as an active
ingredient an effective quantity of a molecule selected from:
HSP60, p277, fragments, analogs, homologs and derivatives thereof,
and nucleic acids encoding same. Also disclosed are T cell
vaccination methods for treating or preventing T cell mediated
disorders.
Inventors: |
Cohen; Irun R.; (Rehovot,
IL) ; Zanin-Zhorov; Alexandra; (Rishon le Zion,
IL) ; Tal; Guy; (Tel-Aviv, IL) ; Quintana;
Francisco; (Buenos Aires, AR) ; Pisner; Meirav;
(Karmiel-Yosef, IL) ; Lider; Ofer; (Kfar Bilu B,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yeda Research and Development Co. Ltd.
Yeda Research and Development Co. Ltd. |
Rehovot
Rehovot |
|
IL
IL |
|
|
Assignee: |
Yeda Research and Development Co.
Ltd.
Rohovot
IL
|
Family ID: |
36647863 |
Appl. No.: |
14/221887 |
Filed: |
March 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11813333 |
Mar 4, 2009 |
8691772 |
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PCT/IL2006/000014 |
Jan 4, 2006 |
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14221887 |
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60679647 |
May 11, 2005 |
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60641075 |
Jan 4, 2005 |
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Current U.S.
Class: |
424/93.71 ;
514/21.4 |
Current CPC
Class: |
A61K 39/0008 20130101;
A61K 2039/5158 20130101; C12N 2501/07 20130101; A61K 2035/124
20130101; A61K 2039/53 20130101; A61K 35/17 20130101; C12N 5/0636
20130101; A61K 38/1709 20130101; A61P 1/16 20180101; A61P 31/12
20180101 |
Class at
Publication: |
424/93.71 ;
514/21.4 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 35/14 20060101 A61K035/14 |
Claims
1. (canceled)
2. A pharmaceutical composition comprising a first population of T
cells cultured ex vivo in the presence of a second population of T
cells, the second population of T cells being attenuated activated
histocompatible T cells exposed ex vivo to a compound selected from
a group consisting of: p277 (SEQ ID NO:1),
p277(Val.sup.6Val.sup.11) (SEQ ID NO:2), p277(Ser.sup.6Ser.sup.11)
(SEQ ID NO:3), analogs, variants, derivatives and salts
thereof.
3-8. (canceled)
9. A method of treating hepatitis comprising administering to a
subject in need thereof a pharmaceutical composition comprising as
an active ingredient a therapeutically effective amount of an HSP60
fragment analog having an amino acid sequence set forth in SEQ ID
NO: 2, and a pharmaceutically acceptable carrier or diluent.
10-14. (canceled)
15. The method of claim 9, wherein the compound is administered in
the form of a pharmaceutical composition further comprising a
pharmaceutically acceptable carrier or diluent.
16. The method of claim 15, wherein the composition comprises a
sustained release formulation.
17. A method of treating or preventing liver damage comprising
administering to a subject in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising as an
active ingredient a therapeutically effective amount of an HSP60
fragment analog having an amino acid sequence set forth in SEQ ID
NO: 2, and a pharmaceutically acceptable carrier or diluent.
18-22. (canceled)
23. The method of claim 17, wherein the subject in need thereof is
afflicted with a disease selected from: (i) viral hepatitis (ii)
parasitic hepatitis (iii) autoimmune hepatitis (iv) primary biliary
cirrhosis (v) alcoholic liver disease.
24. The method of claim 17, wherein the compound is administered in
the form of a pharmaceutical composition further comprising a
pharmaceutically acceptable carrier or diluent.
25. The method of claim 24, wherein the composition comprises a
sustained release formulation.
26-56. (canceled)
57. A method of treating or preventing a T cell mediated pathology
in a subject in need thereof, comprising administering the
pharmaceutical composition according to claim 2 to the subject in
an amount sufficient to induce an anti-ergotypic response in said
subject.
58. The method of claim 57, wherein the first population of T cells
was isolated from the subject or from a donor histocompatible with
said subject.
59. (canceled)
60. The method of claim 57, wherein the T cell mediated pathology
is selected from the group consisting of: inflammatory diseases,
autoimmune diseases, allergic diseases and Th1 mediated
diseases.
61-62. (canceled)
63. The method of claim 57 wherein the T cell mediated pathology is
hepatitis or liver damage associated therewith.
64. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention relates to the use of heat shock protein 60
(HSP60) and the HSP60-derived peptide p277, fragments, analogs,
derivatives and salts thereof, nucleic acids encoding same and T
cell vaccines thereof, in modulation of immune responses and T cell
mediated inflammatory diseases.
BACKGROUND OF THE INVENTION
[0002] While the normal immune system is closely regulated,
aberrations in immune response are not uncommon. A wide variety of
medical treatments thus require regulation of the immune response
in a patient. For example, T cell-mediated inflammatory diseases
are known, in which an inappropriate T cell response is a component
of the disease. These include both diseases mediated directly by T
cells, and diseases in which an inappropriate T cell response
contributes to the production of abnormal antibodies. In some
instances, the immune system functions inappropriately and reacts
to a component of the host as if it were, in fact, foreign. Such a
response results in an autoimmune disease, in which the host's
immune system attacks the host's own tissue. T cells, as the
primary regulators of the immune system, directly or indirectly
affect such autoimmune pathologies.
[0003] Heat Shock Proteins and Immunity
[0004] During their migration into inflammatory sites, T cells
interact with tissue components and encounter a variety of
immuno-modulators, including cytokines, chemokines, acute phase
proteins, and heat shock proteins (HSP). In addition to serving as
a chaperone inside the cell, HSP60 is expressed by cells exposed to
stress (Wallin et al., 2002, Kol et al., 1999) or immune activation
(van Eden et al., 1988), and is present in the blood and tissues
during inflammation (Yokota et al., 2000; Xu et al., 2000; Laplante
et al., 1998; Ohashi et al., 2000, Hu et al., 1998; Mor et al.,
1992). HSP60 is also involved as an autoantigen in type 1 diabetes
and arthritis, and appears to down-regulate inflammation in models
of these autoimmune diseases (Elias et al., 1997, Quintana et al.,
2002, van Eden et al., 1988).
[0005] Numerous disclosures claim uses of heat shock proteins or
fragments thereof as immune modulators in diagnosis, treatment or
prevention of autoimmune diseases. Many of these disclosures relate
to the human HSP60 or to its bacterial equivalent HSP65, or
fragments thereof.
[0006] For example, the particular protein produced by the human
body during development of Insulin Dependent Diabetes Mellitus
(IDDM, type 1 diabetes), which serves as a diagnostic marker for
the incipient outbreak of IDDM, is the human heat shock protein
having a size of about 62 kD (human HSP60) or an antigen cross
reactive therewith as disclosed in EP 0417271, and in U.S. Pat.
Nos. 5,114,844, 5,578,303, 5,671,848, and 5,780,034. The p277
peptide, being the epitope of the human HSP60 involved in IDDM and
corresponding to positions 437-460 of the human HSP60 sequence (SEQ
ID NO:1), was first described in Israeli Patent No. 94241 of the
present applicant. It has been disclosed that fragments of this
HSP60 protein, including p277 and derivatives thereof, may serve as
therapeutically useful entities in preventing or alleviating IDDM
(U.S. Pat. No. 6,180,103, WO 96/19236, and WO 97/01959).
[0007] WO 89/12455 and WO 94/29459 disclose the use of stress
proteins and analogs for producing or enhancing an immune response
or for inducing immune tolerance, for prophylaxis or therapy of
autoimmune diseases and for treating or preventing infections or
cancers. A fusion protein is claimed comprising a stress protein
fused to a protein against which an immune response is desired.
[0008] WO 95/25744 discloses microbial stress protein fragments
containing epitopes homologous to related mammalian epitopes--used
to treat and prevent inflammatory autoimmune diseases and to
prevent transplant rejection. The protective epitopes are located
in short peptides comprising sequences of 5-15 amino acids of
stress proteins that are highly conserved between microorganisms
and animals.
[0009] WO 97/11966 and WO 96/10039 disclose polypeptides of up to
21 amino acids derived from microbial heat shock protein, which are
useful for prophylaxis or treatment of autoimmune diseases
especially arthritis.
[0010] WO 96/16083 discloses a peptide 25 amino acids long, derived
from the 10 kD heat shock protein (HSP10) of Mycobacterium
tuberculosis, which is useful in pharmaceutical products for the
treatment of inflammatory pathologies, especially rheumatoid
arthritis.
[0011] WO 91/02542 discloses the use of antigenic and/or
immuno-regulatory material derived from Mycobacterium vaccae and
specifically HSP60, for treating chronic inflammatory disorders
caused or accompanied by an abnormally high release of IL-6 and/or
TNF-.alpha..
[0012] WO 96/18646 discloses peptides of 9-20 amino acids derived
from Mycobacterial HSP60 used for treatment or prevention of
autoimmune CNS diseases, e.g. multiple sclerosis, chronic
inflammatory CNS disease and primary brain tumors.
[0013] WO 94/02509 discloses peptides of 7-30 amino acids derived
from DR3-restricted epitope of Mycobacterial HSP60 used for
treatment of HLA-DR3 related autoimmune diseases.
[0014] U.S. Pat. No. 5,958,416 describes heat shock protein
peptides and methods for modulating autoimmune central nervous
system diseases.
[0015] EP 262710 of Cohen et al. discloses the use of HSP65, or
fragments thereof for the preparation of compositions for the
alleviation, treatment and diagnosis of autoimmune diseases,
especially arthritic conditions. EP 322990 of Cohen et al.
discloses that a polypeptide having amino acid sequence 172-192 of
HSP65 is capable of inducing resistance to autoimmune arthritis and
similar autoimmune diseases. WO 92/04049 of Boog et al. discloses
peptides derived from Mycobacterium tuberculosis protein HSP65
containing at least 7 amino acid residues that inhibit antigen
recognition by T lymphocytes in treatment of arthritis and organ
rejection. The use of p277 in the treatment of arthritic conditions
has not been disclosed previously.
[0016] WO 02/16549 of Cohen et al. relates to DNA vaccines useful
for the prevention and treatment of ongoing autoimmune diseases.
The compositions and methods of the invention feature the CpG
oligonucleotide, preferably in a motif flanked by two 5' purines
and two 3' pyrimidines. The vaccines optionally further comprise
DNA encoding a peptide or a polypeptide selected from the group
consisting of HSP60, p277 or p277 variants. That disclosure is
directed to methods and compositions for the ameliorative treatment
of ongoing autoimmune disease in general and Insulin Dependent
Diabetes Mellitus (IDDM) in particular.
[0017] WO 03/096967 of Cohen et al. discloses DNA vaccines encoding
HSP60, HSP70 or HSP90 and active fragments thereof for the
treatment of autoimmune diseases such as arthritis.
[0018] U.S. Pat. No. 5,993,803 discloses that when HSP60, p277, or
other peptides and analogs thereof, are administered in a recipient
subject before transplantation of an organ or tissue, autoimmunity
to HSP60 is down-regulated, resulting in the prevention or
suppression of graft rejection of the transplanted organ or
tissue.
[0019] WO 00/27870 of Naparstek and colleagues discloses a series
of related peptides derived from heat shock proteins HSP65 and
HSP60, their sequences, antibodies, and use as vaccines for
conferring immunity against autoimmune and/or inflammatory
disorders such as arthritis. These peptides are intended according
to that disclosure to represent the shortest sequence or epitope
that is involved in protection of susceptible rat strains against
adjuvant induced arthritis. These sequences further disclose what
the inventors identify as the common "protective motif".
[0020] WO 01/43691 provides peptides and peptide analogs capable of
acting as antagonists of HSP60 characterized in that they have the
ability to reduce or prevent the induction of a pro-inflammatory
response of cells of the innate immune system by HSP60.
[0021] Apart from the disclosures utilizing the role of HSP60 as an
autoantigen involved in the progression of various autoimmune
diseases for modulating the development of such diseases, other
recent disclosures indicate that HSP60, via Toll-like receptor 2
(TLR-2), may directly inhibit T-cell migration in response to
CXCL12 (SDF-1.alpha.), and the expression of its receptor, CXCR4
(Zanin-Zhorov et al., 2003). WO 03/070761 of Cohen et al provides
novel conjugates comprising HSP60 peptides and their uses in
treating immune conditions, particularly inflammatory conditions
and autoimmune diseases. These conjugates comprise as a first part
an HSP60 epitope that is capable of reacting via TLR2 on T cells
and as a second segment a specific peptide capable of eliciting a
reaction via a T cell receptor (TcR). Conjugates comprising p277
and a suitable TcR epitope were suggested for the therapy of
various diseases, depending on the identity of the TcR epitope.
[0022] WO 03/063759 discloses peptides and peptide analogs of heat
shock proteins capable of interacting directly with dendritic
cells, and pharmaceutical compositions comprising dendritic cells
exposed to such peptides and analogs, exemplified by a p277 analog,
useful for prevention or treatment of inflammatory disorders and
autoimmune diseases or malignancies, viral infections and
allergy.
[0023] WO 04/098489 discloses HSP60 epitopes capable of binding to
LPS or to macrophages and pharmaceutical compositions comprising
these novel compounds, useful for prevention or treatment of
inflammatory and autoimmune diseases and disorders.
[0024] WO 2005/048914, published after the priority dates of the
present invention, discloses recombinant constructs encoding active
fragments of HSP60, which are effective in treating T cell-mediated
inflammatory autoimmune diseases by DNA vaccines. The HSP60
fragments of the disclosed invention are identified by their
ability to react with T cells isolated from an animal vaccinated
with DNA constructs encoding HSP70 to induce Th2/3 T-cell
responses.
[0025] None of the background art demonstrates, however, that p277
and analogs thereof may be used to down-regulate T cell mediated
inflammation irrespective of the involvement of HSP60 as an
autoantigen contributing to the development of the pathology, and
without being conjugated to or administered with a second TcR
antigen.
[0026] Hepatitis
[0027] Hepatitis is an inflammatory disease that predominantly
affects the liver. The disease is characterized by the initial
onset of symptoms such as anorexia, nausea, vomiting, fatigue,
malaise, arthralgias, myalgias, and headaches, followed by the
onset of jaundice. The disease may also be characterized by
increased serum levels of liver aminotransferases aspartate
aminotransferase (AST) and alanine aminotransferase (ALT);
Quantification of these enzymes in serum indicates the extent of
liver damage.
[0028] Infectious, autoimmune, as well as non-infectious processes
such as chemicals, are among the causes of hepatitis. Examples of
infectious diseases affecting the liver include, but are not
limited to: (i) viral hepatitis, e.g., hepatitis A, B, C, D, E, and
G and (ii) parasitic hepatitis, e.g., Schistosoma mansoni,
Schistosoma hematobium, and Schistosoma japonicum. Examples of
noninfectious diseases affecting the liver include, but are not
limited to, autoimmune diseases, such as autoimmune hepatitis and
primary biliary cirrhosis. Other forms of noninfectious hepatitis
are caused by hepatotoxic agents such as alcohol, drugs and toxins.
Regardless of whether the attack on the liver is infectious,
autoimmune or noninfectious, the liver responds to injury by
recruiting inflammatory cells into the site of attack. T cells, as
the primary regulators of the immune system, play an important role
in the pathogenesis of a variety of human liver disorders (Heneghan
et al., 2002), including autoimmune liver disease, viral hepatitis
(Rosen et al., 2002;), and alcoholic liver disease (Chedid et al.,
1993). Injection of the T cell mitogenic plant lectin concanavalin
A (ConA) is a well-established model to study T cell-mediated
hepatitis (Tiegs et al., 1992).
[0029] Unfortunately, there are few effective treatments for
hepatitis. For example, treatment of autoimmune chronic hepatitis
is generally limited to immunosuppressive treatment with
corticosteroids. While corticosteroid therapy has been shown to
extend life, improve biochemical abnormalities and enhance quality
of life in many patients, the beneficial effects of corticosteroids
are compensated by the often serious complications and side effects
associated with the prolonged treatment therewith. For the
treatment of hepatitis B and C, the FDA has approved administration
of recombinant interferon alpha. However, for adult patients with
hepatitis B infections only about 35% responded to such treatment,
and in perinatal infectees only about 10% responded to treatment.
For hepatitis C infections, despite apparent short-term success
utilizing such therapy, the rate of relapse after termination of
treatment is high. In addition, a further difficulty with alpha
interferon therapy is that the composition frequently has toxic
side effects such as thrombocytopenia, leukopenia, bacterial
infections, and influenza-like symptoms, which require reduced
dosages for sensitive patients. Other agents used to treat chronic
hepatitis B or C include the nucleoside analog ribovirin and
ursodeoxycholic acid; however, neither has been shown to be very
effective. Consequently, the need remains for finding new and
effective drugs and methods for treating hepatitis.
[0030] None of the background art demonstrates that HSP60 or a
fragment thereof may be an effective agent particularly useful for
preventing or treating hepatitis.
[0031] There exists a long-felt need for an effective means of
curing or ameliorating T cell mediated pathologies, including
hepatic disorders. Such a treatment should ideally control the
inappropriate T cell response, rather than merely reducing the
symptoms. The development of new agents capable of selectively
inhibiting the deleterious T cell mediated response with minimal
side effects is therefore desirable.
SUMMARY OF THE INVENTION
[0032] The present invention provides compositions and methods
utilizing heat shock protein 60 (HSP60) and peptides derived from
HSP60 including the known peptide designated p277 (positions
437-460 of human HSP60), nucleic acids encoding same, and analogs,
derivatives and salts thereof, for the treatment and prevention of
T cell mediated pathologies.
[0033] The application is based, in part, on the unexpected finding
that p277 and analogs thereof may be used for inhibiting T-cell
mediated inflammation, regardless of the specific antigen to which
these T cells are directed. p277 and analogs thereof were
surprisingly found to inhibit T cell mediated inflammation via
induction of an anti-inflammatory response characterized by at
least one of p277-specific anti-ergotypic response, stimulation of
CD4.sup.+CD25.sup.+ regulatory T cells, and SOCS3-mediated
inhibition of chemotaxis.
[0034] Thus, the invention provides novel uses for p277 and its
analogs and derivatives and nucleic acid sequences encoding them in
treating T cell mediated pathologies, beyond the known uses for
IDDM and graft rejection. Therapeutic and prophylactic T-cell
vaccination compositions and methods thereof are further
disclosed.
[0035] According to one embodiment, the invention specifically
demonstrates, for the first time, that HSP60, as well as a p277
analog designated p277(Val.sup.6Val.sup.11) (also known as
DiaPep277), is an effective agent for treating or preventing T
cell-mediated hepatitis. Thus, the invention provides novel
therapeutic methods utilizing HSP60 and p277, fragments, analogs,
homologs and derivatives thereof, and nucleic acids encoding same,
for the treatment and prevention of hepatic disorders by
suppression of inflammation.
[0036] Peptide p277(Val.sup.6Val.sup.11), also known as DiaPep277,
disclosed in U.S. Pat. No. 6,180,103, is a synthetic analog of a
native 24-amino acid fragment p277 of the 60 kDa human HSP60. The
terms p277 and Peptide p277 are used interchangeably throughout the
specification and in the claims and these terms are intended to
denote both the native sequence (SEQ ID NO:1) as well as synthetic
variants thereof, such as DiaPep277 (SEQ ID NO:2).
[0037] In various embodiments of the present invention, there are
provided T cell vaccine compositions and methods utilizing HSP60,
active fragments thereof, p277, and analogs, variants, derivatives
and salts thereof for the treatment of T cell mediated
pathologies.
[0038] In one aspect, the invention provides a pharmaceutical
composition comprising attenuated activated T cells exposed ex vivo
to a compound selected from the group consisting of: p277 (SEQ ID
NO:1), analogs, variants, derivatives and salts thereof.
[0039] In certain embodiments, the compound is a p277 analog
wherein at least one of the cysteine residues in positions 6 and 11
of p277 has been substituted. In another embodiment, the p277
analog is p277(Val.sup.6Val.sup.11) (SEQ ID NO:2). In another
embodiment, the p277 analog is p277(Ser.sup.6Ser.sup.11) (SEQ ID
NO:3).
[0040] In another aspect, the invention provides a pharmaceutical
composition comprising a first population of T cells cultured ex
vivo in the presence of a second population of T cells, the second
population of T cells being histocompatible attenuated activated T
cells exposed ex vivo to a compound selected from a group
consisting of: p277 (SEQ ID NO:1), analogs, variants, derivatives
and salts thereof.
[0041] In certain embodiments, the compound is a p277 analog
wherein at least one of the cysteine residues in positions 6 and 11
of p277 has been substituted. In another embodiment, the p277
analog is p277(Val.sup.6Val.sup.11) (SEQ ID NO:2). In another
embodiment, the p277 analog is p277(Ser.sup.6Ser.sup.11) (SEQ ID
NO:3).
[0042] In another aspect, the invention provides methods of
treating or preventing a T cell mediated pathology in a subject in
need thereof, comprising: (a) isolating T cells from the subject or
from a donor histocompatible with said subject; (b) activating the
T cells ex vivo to induce Major Histocompatibility Complex (MHC) II
expression; (c) exposing said activated cells to a compound
selected from a group consisting of: p277 (SEQ ID NO:1), analogs,
variants, derivatives and salts thereof; (d) attenuating said T
cells; and (e) introducing said T cells into the subject in an
amount sufficient to induce an anti-ergotypic response in said
subject.
[0043] In another embodiment, the attenuation step is performed
after activating the cells and prior to exposing them to said
compound. In another embodiment, the p277 analog is
p277(Val.sup.6Val.sup.11) (SEQ ID NO:2). In another embodiment, the
p277 analog is p277(Ser.sup.6Ser.sup.11) (SEQ ID NO:3). In various
embodiments, the T cell mediated pathology is selected from the
group consisting of: inflammatory diseases, autoimmune diseases,
allergic diseases and Th1 mediated diseases.
[0044] In another aspect, the invention provides methods of
treating or preventing a T cell mediated pathology in a subject in
need thereof, comprising: (a) isolating a first population of T
cells from the subject or from a donor histocompatible with said
subject; (b) culturing the first population of T cells in the
presence of a second population of histocompatible attenuated
activated T cells and a compound selected from a group consisting
of: p277 (SEQ ID NO:1), analogs, variants, derivatives and salts
thereof; and (c) introducing said first population of T cells into
the subject in an amount sufficient to induce an anti-ergotypic
response in said subject.
[0045] Advantageously, culturing includes repeated exposure of the
first population of T cells to fresh attenuated activated T cells
and said compound under conditions suitable for proliferation of
said first population of T cells.
[0046] In another embodiment, step (b) may alternatively comprise
culturing the first population of T cells in the presence of a
second population of histocompatible attenuated activated T cells
that were previously exposed to a compound selected from a group
consisting of: p277 (SEQ ID NO:1), analogs, variants, derivatives
and salts thereof. In another embodiment, the p277 analog is
p277(Val.sup.6Val.sup.11) (SEQ ID NO:2). In another embodiment, the
p277 analog is p277(Ser.sup.6Ser.sup.11) (SEQ ID NO:3). In various
embodiments, the T cell mediated pathology is selected from the
group consisting of: inflammatory diseases, autoimmune diseases,
allergic diseases and Th1 mediated diseases.
[0047] In another aspect, the invention provides methods of
treating hepatitis or liver damage associated therewith in a
subject in need thereof, comprising: (a) isolating T cells from the
subject or from a donor histocompatible with said subject; (b)
activating the T cells ex vivo to induce Major Histocompatibility
Complex (MHC) II expression; (c) exposing said activated cells to a
compound selected from a group consisting of: HSP60, an active
fragment thereof, p277 (SEQ ID NO:1), analogs, variants,
derivatives and salts thereof; (d) attenuating said T cells; and
(e) introducing said T cells into the subject in an amount
sufficient to induce an anti-ergotypic response in said
subject.
[0048] In another embodiment, the attenuation step is performed
after activating the cells and prior to exposing them to said
compound.
[0049] In another aspect, the invention provides methods of
treating hepatitis or liver damage associated therewith in a
subject in need thereof, comprising: (a) isolating a first
population of T cells from the subject or from a donor
histocompatible with said subject; (b) culturing the first
population of T cells in the presence of a second population of
histocompatible attenuated activated T cells and a compound
selected from a group consisting of: HSP60, an active fragment
thereof, p277 (SEQ ID NO:1), analogs, variants, derivatives and
salts thereof; and (C) introducing said first population of T cells
into the subject in an amount sufficient to induce an
anti-ergotypic response in said subject.
[0050] In another embodiment, step (b) may alternatively comprise
culturing the first population of T cells in the presence of a
second population of histocompatible attenuated activated T cells
that were previously exposed to a compound selected from a group
consisting of: p277 (SEQ ID NO:1), analogs, variants, derivatives
and salts thereof.
[0051] In another aspect, the invention provides methods of
treating or preventing a T cell mediated pathology other than
insulin-dependent diabetes mellitus and graft rejection in a
subject in need thereof, comprising administering to the subject a
therapeutically-effective amount of a compound selected from a
group consisting of: p277 (SEQ ID NO:1), analogs, variants,
derivatives and salts thereof.
[0052] In one embodiment, the compound is a p277 analog in which at
least one of the cysteine residues in positions 6 and 11 of p277
has been substituted. In another embodiment, the p277 analog is
p277(Val.sup.6Val.sup.11) (SEQ ID NO:2). In another embodiment, the
p277 analog is p277(Ser.sup.6Ser.sup.11) (SEQ ID NO:3).
[0053] In another embodiment, the compound is administered in the
form of a pharmaceutical composition further comprising a
pharmaceutically acceptable carrier, and optionally an adjuvant. In
another embodiment, the composition is administered in a manner
selected from: enteral (e.g. oral, rectal), buccal, transmucosal,
intranasal, bronchial and intrapulmonary administration. In another
embodiment, the composition comprises a sustained release
formulation of said compound.
[0054] In other embodiments, the T cell mediated pathology is
selected from the group consisting of: inflammatory diseases,
autoimmune diseases, allergic diseases and Th1 mediated
diseases.
[0055] Another aspect of the present invention is a method of
treating hepatitis comprising administering to a subject in need
thereof a therapeutically effective amount of a compound selected
from a group consisting of: HSP60 protein, an active fragment
thereof, and analogs, variants, derivatives and salts thereof.
[0056] In one preferred embodiment, the HSP60 protein is human
HSP60. In another embodiment, the HSP60 protein has an amino acid
sequence as set forth in any one of SEQ ID NOS:4 and 11. In another
embodiment, other mammalian HSP60 are used. In yet another
embodiment, the HSP60 polypeptide is bacterial HSP60. In another
embodiment, the HSP60 polypeptide is E. coli GroEL (Zanin-Zhorov et
al., 2003; SEQ ID NO:9). In another preferred embodiment, the
active fragment of HSP60 is the p277 peptide (SEQ ID NO:1). In
another embodiment, the HSP60 fragment analog has an amino acid
sequence as set forth in SEQ ID NO:2. In yet another embodiment,
the HSP60 fragment analog has an amino acid sequence as set forth
in SEQ ID NO:3. In another embodiment, the compound is administered
in the form of a pharmaceutical composition further comprising a
pharmaceutically acceptable carrier or diluent. In another
embodiment, the composition comprises a sustained release
formulation of said compound.
[0057] In another aspect, there is provided a method of treating or
preventing liver damage comprising administering to a subject in
need thereof a therapeutically effective amount of a compound
selected from a group consisting of: HSP60 protein, an active
fragment thereof, and analogs, variants, derivatives and salts
thereof.
[0058] In one embodiment, the HSP60 protein is selected from the
group consisting of: human HSP60, mammalian HSP60 and bacterial
HSP60. In a preferred embodiment, the HSP60 protein is human HSP60.
In another embodiment, the HSP60 protein has an amino acid sequence
as set forth in any one of SEQ ID NOS:4 and 11. In another
embodiment, the HSP60 protein has an amino acid sequence as set
forth in SEQ ID NO:9. In another particular embodiment, the HSP60
fragment is the p277 peptide, having an amino acid sequence as set
forth in SEQ ID NO:1. In another embodiment, the HSP60 fragment
analog has an amino acid sequence as set forth in SEQ ID NO:2. In
yet another embodiment, the HSP60 fragment analog has an amino acid
sequence as set forth in SEQ ID NO:3. In another embodiment, the
compound is administered in the form of a pharmaceutical
composition further comprising a pharmaceutically acceptable
carrier or diluent. In another embodiment, the composition
comprises a sustained release formulation of said compound.
According to various embodiments, the subject in need thereof is
afflicted with a disease selected from: (i) viral hepatitis (ii)
parasitic hepatitis (iii) autoimmune hepatitis (iv) primary biliary
cirrhosis (v) alcoholic liver disease.
[0059] In another aspect, the present invention provides methods of
treating or preventing a T cell mediated pathology other than
insulin-dependent diabetes mellitus and graft rejection in a
subject in need thereof, comprising administering to the subject a
therapeutically-effective amount of a pharmaceutical composition
comprising a recombinant construct, the recombinant construct
comprising a nucleic acid sequence encoding a peptide selected
from: p277 (SEQ ID NO:1), analogs and variants thereof, the nucleic
acid sequence being operably linked to one or more transcription
control sequences; and a pharmaceutically acceptable carrier.
[0060] In one embodiment, the encoded peptide is a p277 analog in
which at least one of the cysteine residues in positions 6 and 11
of p277 has been substituted. In another embodiment, the encoded
p277 analog is p277(Val.sup.6Val.sup.11) (SEQ ID NO:2). In another
embodiment, the encoded p277 analog is p277(Ser.sup.6Ser.sup.11)
(SEQ ID NO:3). In another embodiment, said construct comprises a
nucleic acid sequence as set forth in any one of SEQ ID NOS:5-7. In
another embodiment, the nucleic acid composition is administered as
naked DNA. In other embodiments, the T cell mediated pathology is
selected from the group consisting of: inflammatory diseases,
autoimmune diseases, allergic diseases and Th1 mediated
diseases.
[0061] In another aspect, the invention provides a method of
treating hepatitis, comprising administering to a subject in need
thereof a therapeutically effective amount of a pharmaceutical
composition comprising as an active ingredient a recombinant
construct, the recombinant construct comprising a nucleic acid
sequence encoding a polypeptide selected from a group consisting
of: HSP60 protein, an active fragment thereof, and analogs and
variants thereof, wherein the nucleic acid sequence is operably
linked to one or more transcription control sequences, thereby
treating hepatitis.
[0062] In one embodiment, the HSP60 protein is selected from the
group consisting of: human HSP60, mammalian HSP60 and bacterial
HSP60. In a preferred embodiment, the HSP60 protein is human HSP60.
In another embodiment, the HSP60 protein has an amino acid sequence
as set forth in any one of SEQ ID NOS:4 and 11. In another
embodiment, the HSP60 protein has an amino acid sequence as set
forth in SEQ ID NO:9. In another particular embodiment, the HSP60
fragment is the p277 peptide, having an amino acid sequence as set
forth in SEQ ID NO:1. In another embodiment, the HSP60 fragment
analog has an amino acid sequence as set forth in SEQ ID NO:2. In
yet another embodiment, the HSP60 fragment analog has an amino acid
sequence as set forth in SEQ ID NO:3. In another embodiment, said
construct comprises a nucleic acid sequence as set forth in any one
of SEQ ID NOS:5-8.
[0063] The invention provides, in another aspect, a method of
treating or preventing liver damage, comprising administering to a
subject in need thereof a therapeutically effective amount of a
pharmaceutical composition comprising as an active ingredient a
recombinant construct, the recombinant construct comprising an
isolated nucleic acid sequence encoding a polypeptide selected from
a group consisting of: HSP60 protein, an active fragment thereof,
and analogs and variants thereof, wherein the nucleic acid sequence
is operably linked to one or more transcription control sequences,
thereby treating or preventing liver damage.
[0064] In one embodiment, the HSP60 protein is selected from the
group consisting of: human HSP60, mammalian HSP60 and bacterial
HSP60. In a preferred embodiment, the HSP60 protein is human HSP60.
In another embodiment, the HSP60 protein has an amino acid sequence
as set forth in any one of SEQ ID NOS:4 and 11. In another
embodiment, the HSP60 protein has an amino acid sequence as set
forth in SEQ ID NO:9. In another particular embodiment, the HSP60
fragment is the p277 peptide, having an amino acid sequence as set
forth in SEQ ID NO:1. In another embodiment, the HSP60 fragment
analog has an amino acid sequence as set forth in SEQ ID NO:2. In
yet another embodiment, the HSP60 fragment analog has an amino acid
sequence as set forth in SEQ ID NO:3. In another embodiment, said
construct comprises a nucleic acid sequence as set forth in any one
of SEQ ID NOS:5-8. In another embodiment, the subject in need
thereof is afflicted with a disease selected from: (i) viral
hepatitis (ii) parasitic hepatitis (iii) autoimmune hepatitis (iv)
primary biliary cirrhosis (v) alcoholic liver disease.
[0065] In another aspect, there is provided a method of treating
hepatitis or liver damage associated therewith comprising the steps
of (a) obtaining cells from a subject; (b) introducing a
recombinant construct into the cells ex vivo, the construct
comprising an isolated nucleic acid sequence encoding a polypeptide
selected from a group consisting of: HSP60 protein, an active
fragment thereof, and analogs and variants thereof, wherein the
nucleic acid sequence is operably linked to one or more
transcription control sequences; and (c) reintroducing said treated
cells to the subject; wherein the HSP60 or fragment, analog or
variant thereof is expressed in vivo in said treated cells in an
amount sufficient to treat hepatitis or liver damage associated
therewith.
[0066] In one embodiment, the HSP60 protein is selected from the
group consisting of: human HSP60, mammalian HSP60 and bacterial
HSP60. In a preferred embodiment, the HSP60 protein is human HSP60.
In another embodiment, the HSP60 protein has an amino acid sequence
as set forth in any one of SEQ ID NOS:4 and 11. In another
embodiment, the HSP60 protein has an amino acid sequence as set
forth in SEQ ID NO:9. In another particular embodiment, the HSP60
fragment is the p277 peptide, having an amino acid sequence as set
forth in SEQ ID NO:1. In another embodiment, the HSP60 fragment
analog has an amino acid sequence as set forth in SEQ ID NO:2. In
yet another embodiment, the HSP60 fragment analog has an amino acid
sequence as set forth in SEQ ID NO:3. In another embodiment, said
construct comprises a nucleic acid sequence as set forth in any one
of SEQ ID NOS:5-8. In another embodiment, the subject in need
thereof is afflicted with a disease selected from: (i) viral
hepatitis (ii) parasitic hepatitis (iii) autoimmune hepatitis (iv)
primary biliary cirrhosis (v) alcoholic liver disease.
[0067] These and other embodiments of the present invention will
become apparent in conjunction with the figures, description and
claims that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1. DNA vaccination with HSP60 induces anti-ergotypic T
cells. A and B. Anti-ergotypic proliferative response of LNC from
rats vaccinated with pcDNA3, pHSP65 or pHSP60 (A) or pcDNA3, pI or
pII (B), taken 26 days after the induction of AA. Proliferative
responses are presented as the .DELTA.CPM.+-.SEM of quadruplicate
cultures. * p<0.05 compared to the pHSP65 (A) or the pcDNA3 (B)
groups. C. Monoclonal antibodies to MHC-II/RT1.B, MHC-II/RT1.D or
MHC-I were assayed for their ability to block the anti-ergotypic
proliferative response. Results are presented as the percent of
inhibition of proliferation.+-.SEM of quadriplicate cultures. D.
Anti-ergotypic cytokine response of LNC taken from rats vaccinated
with pcDNA3, pHSP65, pHSP60, pI or pII 26 days after the induction
of AA. IFN.gamma., TGF.beta.1, IL-10 and IL-4 were quantified in
the culture supernatants after 72 hr of stimulation with 10.sup.5
activated or resting, irradiated, A2b cells per well. The results
are presented as pg/ml.+-.SEM of triplicate cultures. Three
independent experiments produced similar results.
[0069] FIG. 2. Vaccination with HSP60 peptide Hu3 induces
anti-ergotypic T cells. A. Anti-ergotypic proliferative response of
LNC from rats vaccinated with PBS, Mt3 or Hu3 in IFA, taken 26 days
after AA induction. Proliferative responses are presented as the
.DELTA.CPM.+-.SEM of quadruplicate cultures. * p<0.05 compared
to the Mt3 group. B. Monoclonal antibodies to MHC-II/RT1.B,
MHC-II/RT1.D or MHC-I were assayed for their ability to block the
anti-ergotypic proliferative response. Results are presented as the
percent of inhibition of proliferation.+-.SEM of quadruplicate
cultures. C. Anti-ergotypic cytokine response of LNC taken from
rats vaccinated with PBS, Mt3 or Hu3 in IFA, 26 days after AA
induction. IFN.gamma., TGF.beta.1, IL-10 and IL-4 were quantified
in the culture supernatants after 72 hr of stimulation with
10.sup.5 activated or resting, irradiated, A2b cells per well. The
results are presented as pg/ml.+-.SEM of triplicate cultures. Three
independent experiments produced similar results.
[0070] FIG. 3. T-cell activation up-regulates cellular levels of
HSP60. A. LNC were stimulated with Con A for 24, 48 or 72 hr,
subjected to a 30 minutes 42.degree. C. heat shock (HS) or kept at
37.degree. C. (None). Cell lysates were prepared and HSP60
expression was analyzed by western blot with specific antibodies,
and quantified (in arbitrary units). B. A2b T-cells were stimulated
with various concentrations of the target peptide Mt176-90, a
control peptide (Mt3) for 72 hr, or with medium alone (None). Cell
lysates were prepared and HSP60 expression was analyzed by western
blot with specific antibodies, and quantified (in arbitrary units).
Two independent experiments produced similar results.
[0071] FIG. 4. MHC class II-restricted recognition of activated T
cells by HSP60-specific T-cells. A. Anti-ergotypic proliferative
response of Anti-HSP60 or Anti-MBP T cell lines. Proliferative
responses are presented as the .DELTA.CPM.+-.SEM of quadruplicate
cultures. B. Anti-ergotypic proliferative response of Anti-p277 or
Anti-MBP T cell lines. Proliferative responses are presented as the
.DELTA.CPM.+-.SEM of quadruplicate cultures. C. Monoclonal
antibodies to MHC-II/RT1.B, MHC-II/RT1.D or MHC-I were assayed for
their ability to block the anti-ergotypic proliferative response of
the Anti-HSP60 and the Anti-p277 T cell lines. Results are
presented as the percent of inhibition of proliferation.+-.SEM of
quadruplicate cultures. D. IFN.gamma., TGF.beta.1, IL-10 and IL-4
were quantified in the culture supernatants after 72 hr of
stimulation of the Anti-MBP, Anti-p277 or Anti-HSP60 T cell lines
with 10.sup.5 activated or resting, irradiated, A2b cells per well.
The results are presented as pg/ml.+-.SEM of triplicate cultures.
Three to five independent experiments produced similar results.
[0072] FIG. 5. The activation HSP60-specific anti-ergotypic T cells
requires co-stimulation. Monoclonal antibodies to CD28, CD80 or
CD86, or a control IgG (Control), were assayed for their ability to
block the anti-ergotypic proliferative response of Anti-HSP60
T-cells (Line) or of LNC prepared from pHSP60-vaccinated rats
(LNC). Results are presented as the percent of inhibition of
proliferation.+-.SEM of quadruplicate cultures. Three independent
experiments produced similar results.
[0073] FIG. 6. p277-specific anti-ergotypic T-cells control
arthritogenic T-cells in vivo. A and B. Anti-MBP or Anti-p277 T
cells were injected ip into naive Lewis rats and three days later
AA was induced. Twenty-six days after AA induction, at the peak of
AA, the AA clinical score (A) and the hind paw diameter (B) were
determined. The bars represent the mean values.+-.SEM for each
group of 8 rats. C. LNC were collected on day 26 after AA induction
and the secretion of IFN.gamma. upon stimulation with Mt176-90 was
studied. The results are presented as pg/ml.+-.SEM of triplicate
cultures. Three independent experiments produced similar results. *
p<0.05 and ** p<0.005 compared to the Anti-MBP group.
[0074] FIG. 7. HSP60-specific Anti-ergotypic T-cells control
arthritogenic T-cells in vitro. A. LNC from Mt immunized rats
(2.5.times.10.sup.5 per well) were activated with Mt176-90 for 72
hr in the presence of Anti-p277 or Anti-MBP T-cells
(5.times.10.sup.4 per well). The secretion of IFN.gamma. was
determined by ELISA, the results are presented as pg/ml.+-.SEM of
triplicate cultures. The differences between the groups were
significant (p<0.05) for antigen concentrations higher than 0.1
.mu.g/ml. Three independent experiments produced similar results.
B-D. anti MBP or anti-p277 lines were injected ip into naive Lewis
rats three days before the induction of AA. Twenty-six days later
LNC were collected and the proliferative responses to PPD, HSP65,
Mt176-90 (B); HSP60, p277 or Hu3 (C) or antiergotypic (D) were
studied.
[0075] FIG. 8. Anti-ergotypic HSP60-specific T cells become anergic
after interacting with activated T cells. Anti-ergotypic Anti-p277
T cells were stimulated for 3 days with irradiated, activated A2b
cells (A2b) or with irradiated APC fed with the p277 peptide (APC).
The Anti-p277 T cells were maintained for 4 additional days in
culture, and stimulated with APC and p277 peptide, Con A or
immobilized anti-TCR (.alpha.TCR) antibodies. T-cell proliferation
(A) and IFN.gamma. (B) release were measured after 3 days. The
proliferative responses are presented as the .DELTA.CPM (.+-.SEM)
(A), and the IFN.gamma. as pg/ml.+-.SEM (B) of triplicate cultures.
Three to five independent experiments produced similar results.
[0076] FIG. 9. HSP60 inhibits SDF-1.alpha.-induced chemotaxis
through fibronectin (A) and endothelial cells (B) of human T cells,
and homing of such cells into the bone marrows of NOD/SCID mice in
a TLR2-dependent manner (C and D). A. and B. Human T cells were
incubated with indicated concentrations of HSP60 for 1 hr (A), or
with 1 ng/ml of HSP60 for 1 hr (B), radioactively labeled, and
T-cell migration assays through FN-coated (A), or through
TNF-.alpha.-activated HUVEC monolayer (B) were performed (3 hr,
37.degree. C.) in Transwell apparati in the presence or absence of
SDF-1.alpha. (100 ng/ml). Results are expressed as the percent of
T-cells migrating towards SDF-1.alpha.. The average values.+-.SD of
five experiments are depicted. C and D. Human T cells, some of
which were pretreated with mouse anti-human CXCR4, TLR2, or
anti-TLR4 mAb, were treated with HSP60 (1 .mu.g/ml, 1 hr) and
injected into naive NOD/SCID mice. T-cell homing to the bone
marrows of mice was evaluated 16 hr later by flow cytometry. The
results are expressed as the number (see right hand corner of the
panels) of homing human cells per 10.sup.6 acquired cells (C), and
percent of control (D). Combined data from five experiments is
depicted. *P<0.05.
[0077] FIG. 10. HSP60 inhibits SDF-1.alpha.-induced mouse T-cell
chemotaxis in vitro and DTH in vivo. A. Lymph node cells of BALB/c
mice pre-sensitized to oxazolone were incubated in vitro with HSP60
(1 hr), washed, and their SDF-1.alpha.-mediated chemotaxis was
determined. B. HSP60-treated mouse lymph node cells from
oxazolone-sensitized mice were injected i.v. into naive recipients
whose earlobes were then painted with oxazolone. DTH reactivity
(ear swelling, 10.sup.-2 mm.+-.SD) was measured 24 hr later. DTH
index: 1-[(treated cells mice-control cells)].times.100. *
P<0.05. One experiment representative of 3 is depicted.
[0078] FIG. 11. HSP60 inhibits SDF-1.alpha.-induced ERK (A-C), Pyk2
(D, E), and AKT phosphorylation (F).
[0079] FIG. 12. HSP60 induces phosphorylation of MLC (A), inhibits
SDF-1.alpha.-induced phosphorylation of MLC (B) and polarized
morphology (C) in T cells.
[0080] FIG. 13. Activation by HSP60 of SOCS3 expression in T cells
via TLR2 signaling and STATS activation mediates the
down-regulation of SDF-1.alpha.-induced chemotaxis. Human T cells
(A, D, F, G, H), mouse lymph node lymphocytes (B) or mouse purified
T cells (E) were incubated with HSP60 at 0.01-1000 ng/ml for 1 hr,
or at 1 ng/ml for 0-120 min (C). Some cells were pre-treated with
anti-TLR2 or anti-TLR4 mAb (D), the JAK/STAT inhibitor AG490 or
control inhibitor AG9 (18 hr; F). E. Purified T cells from wild
type C57BL/6J and TLR2-knockout mice incubated with HSP60 1 ng/ml
for 1 hr. H and I, T cells were transfected with siRNA targeting
SOCS3, or with control siRNA, and exposed to HSP60 (2 hr). Cell
lysates were immunoblotted with anti-SOCS3 and anti-total ERK
[tERK; evaluation of total tERK served as a control (A-F, H)], or
with anti-pSTAT3 and anti-STATS (G). The levels of SOCS3, tERK,
pSTAT3, and tSTAT3 were estimated by densitometry and the average
percentage (.+-.SD) of the three experiments was calculated by OD
of SOCS3/tERK (pSTAT3/tSTAT3).times.100. H, cells were
.sup.51[Cr]-labeled, washed, and their ability to migrate in
response to SDF-1.alpha. was examined. Averages.+-.SD of three
different experiments are shown.
[0081] FIG. 14. Effects of HSP60 on SOCS3 expression are not due to
contamination with LPS. Human T cells were treated (1 hr;
37.degree. C.) with LPS (different concentrations in A, 100 ng/ml
in C), HSP60 (1 ng/ml, B), LPS or HSP60 (D), or p30 or p277 (E).
SOCS3 activation in the T-cell lysates was measured by
immunoblotting. B, T cells were pretreated (30 min) with anti-HSP60
mAb (20 .mu.g/ml), isotype matched mAb (IgM), or PMB (1 .mu.g/ml).
Alternatively, HSP60 or LPS were boiled (100.degree. C., 30 min)
before their addition to the T-cell cultures. D. LPS and HSP60 were
pre-incubated with PMB-conjugated agarose beads, the unbound
material was collected, checked for protein amount, and used to
pre-treat the cells. One experiment representative of five (A-C),
or three (D, E) is shown.
[0082] FIG. 15. HSP60 inhibits anti-CD3-induced IFN-.gamma. (a) and
TNF-.alpha. (b) secretion, and up-regulates IL-10 (c) secretion in
CD3.sup.+ T cells and CD4.sup.+, but not in CD8.sup.+ T cells.
Purified CD3.sup.+, CD4.sup.+, and CD8.sup.+ T cells were
pre-incubated with the indicated concentrations of HSP60 for 2
hours, washed and transferred to 24-well plates coated with mAb
anti-CD3 (OKT; 0.5 .mu.g/ml) in serum-free medium. The supernatants
were collected after 24 hr and analyzed for IFN-.gamma. (A),
TNF-.alpha. (B) and IL-10 (C) secretion. The means.+-.SD of five
different donors are shown. *P<0.05.
[0083] FIG. 16. HSP60 does not affect IFN-.gamma. and TNF-.alpha.
secretion by activated CD4.sup.+CD25.sup.- T cells, but
up-regulates IL-10 and TGF-.beta. secretion by activated
CD4.sup.+CD25.sup.+ T cells. A. and B. Unseparated CD4.sup.+,
CD4.sup.+CD25.sup.+, and CD4.sup.+CD25.sup.- T cells were stained
with 20 .mu.g/ml PE-conjugated mAb anti-CD25, FITC-conjugated mAb
anti-CD4, anti-CTLA4, or anti-CD45RO, followed by a secondary
FITC-conjugated Ab. Marker expression was determined by FACScan
analysis. Foxp3 levels were measured in cell lysates. One
representative experiment of five is shown. C. Unseparated
CD4.sup.+, CD4.sup.+CD25.sup.+, and CD4.sup.+CD25.sup.- T cells
were incubated with HSP60 (1 ng/ml) for 2 hours, washed and
transferred to 24-well plates coated with mAb anti-CD3 (OKT; 0.5
.mu.g/ml) in serum-free medium. The supernatants were analyzed for
IFN-.gamma., TNF-.alpha., IL-10, and TGF-.beta. secretion. The
means.+-.SD of five different donors are shown. *P<0.05.
[0084] FIG. 17. Treatment of CD4.sup.+CD25.sup.+ T cells with HSP60
affects cytokine secretion and proliferation in co-culture with
CD4.sup.+CD25.sup.- T cells. Purified CD4.sup.+CD25.sup.+ T cells
were incubated with HSP60 (1 ng/ml) for 2 hours, washed, mixed in
the indicated proportions with CD4.sup.+CD25.sup.- T cells, and
transferred to 24-well plates coated with mAb anti-CD3 (OKT; 0.5
.mu.g/ml) in serum-free medium. The supernatants were collected
after 24 hr and analyzed for IFN-.gamma. (A), TNF-.alpha. (B),
IL-10 (C) and TGF-.beta. (D). Cell proliferation was determined
after 24 and 96 hr (E). The means.+-.SD of six different donors are
shown.
[0085] FIG. 18. Treatment of CD4.sup.+CD25.sup.+ T cells with HSP60
affects cytokine secretion in co-culture with CD8.sup.+ T cells.
Purified CD4.sup.+CD25.sup.+ T cells were incubated with HSP60 (1
ng/ml) for 2 hours, washed, mixed in indicated proportions with
CD8.sup.+ T cells, and transferred to 24-well plates coated with
mAb anti-CD3 (OKT; 0.5 .mu.g/ml) in serum-free medium. The
supernatants were collected after 24 hr and analyzed for
IFN-.gamma. (A), TNF-.alpha. (B), IL-10 (C) and TGF-.beta. (D). The
means.+-.SD of five different donors are shown.
[0086] FIG. 19. The effects of HSP60 on CD4.sup.+CD25.sup.+ T cells
depend on TLR2 signaling, and are not due to contaminating LPS.
Purified CD4.sup.+CD25.sup.+ T cells were pretreated with
monoclonal anti-TLR2 or anti-TLR4 (20 .mu.g/ml, 30 min). Then, the
cells were incubated with HSP60 (1 ng/ml, 2 hr), washed, and
co-cultured with CD4.sup.+CD25.sup.- T cells (ratio 1:9) on
anti-CD3 in serum-free medium (A-C). D. Unseparated CD4.sup.+,
CD4.sup.+CD25.sup.+, and CD4.sup.+CD25.sup.- T cells were fixed,
permeabilizied and stained with anti-TLR2, or anti-TLR4 (20
.mu.g/ml). Receptor expression was determined by FACScan analysis;
E-G. CD4.sup.+CD25.sup.+ T cells were incubated (2 hr) with
untreated, PMB-treated, or boiled (100.degree. C., 30 min) HSP60 (1
ng/ml) and LPS (100 ng/ml). After washing, the CD4.sup.+CD25.sup.+
T cells were co-cultured with CD4.sup.+CD25.sup.- T cells (ratio
1:9) on anti-CD3 in serum-free medium. The supernatants were
collected after 24 hr and analyzed for IFN-.gamma. (A, E),
TNF-.alpha. (B, F) and IL-10 (C, G). The means.+-.SD of four
different donors are shown. *P<0.05.
[0087] FIG. 20. Treatment of CD4.sup.+CD25.sup.+ T cells with HSP60
peptide p277 inhibits cytokine secretion and proliferation in
co-culture with CD4.sup.+CD25.sup.- T cells. Purified
CD4.sup.+CD25.sup.+ T cells were incubated with HSP60, p277, or
MT-p277 (1 ng/ml) for 2 hours, washed, co-cultured with
CD4.sup.+CD25.sup.- T cells (ratio 1:9) on anti-CD3 in serum-free
medium. The supernatants were collected after 24 hr and analyzed
for IFN-.gamma. (A), TNF-.alpha. (B). Proliferation was determined
after 96 hr (C). The means.+-.SD of three different donors are
shown.
[0088] FIG. 21. HSP60-induced enhancement of CD4.sup.+CD25.sup.+
Treg function involves both contact-dependent and cytokine
dependent mechanisms. A. Purified CD4.sup.+CD25.sup.+ T cells were
incubated with HSP60 (1 ng/ml, 2 hr), washed, and co-cultured with
CD4.sup.+CD25.sup.- T cells (ratio 1:9) on anti-CD3 in the same
lower well or separately in the upper chamber of the Transwell. As
indicated, some CD4.sup.+CD25.sup.+ T cells were pretreated with
control or monoclonal anti-CTLA4 (20 .mu.g/ml, 30 min), and washed
(B), or blocking anti-IL-10, and anti-TGF-.beta. (10 .mu.g/ml) mAbs
(D) were added to the co-culture. The supernatants were collected
after 24 hr and analyzed for IFN-.gamma. and TNF-.alpha.. The
means.+-.SD of five different donors are shown. C. Purified
CD4.sup.+CD25.sup.+ T cells were incubated with HSP60 (1 ng/ml, 2
hr), washed, and plated on anti-CD3 for 24 hr in serum-free medium.
Then, the supernatants were collected, and added to
CD4.sup.+CD25.sup.- T cells in presence of anti-CD3 mAbs. The
supernatants from CD4.sup.+CD25.sup.- T cells were collected after
additional 24 hr and analyzed for IFN-.gamma. and TNF-.alpha.. The
means.+-.SD of four different donors are shown.
[0089] FIG. 22. HSP60 induces phosphorylation of AKT, Pyk-2, p38,
and down-regulates anti-CD3-induced ERK-phosphorylation in
activated CD4.sup.+CD25.sup.+ T cells. A. cytokine secretion; B.
Akt phosphorylation; C. Pyk-2 phosphorylation; D. p38
phosphorylation; E. Erk phosphorylation.
[0090] FIG. 23. Co-culture of CD4.sup.+CD25.sup.- T cells with
HSP60-treated CD4.sup.+CD25.sup.+ T cells down-regulates ERR
phosphorylation (A), inhibits, nuclear translocation of NF-.kappa.B
(B), and T-bet (C) expression in the CD4.sup.+CD25.sup.- T
cells.
[0091] FIG. 24 shows that HSP60 inhibits anti-CD3-induced
IFN-.gamma. (a) and TNF-.alpha. (b) secretion, and up-regulates
IL-10 (c) secretion by T cells. Purified human T cells were
pre-incubated with the indicated concentrations of HSP60 for 1
hour, washed and transferred to 24-well plates coated with mAb
anti-CD3 (OKT; 0.5 .mu.g/ml) in serum-free medium. The supernatants
were collected after 24 hr and analyzed for IFN-.gamma. (A),
TNF-.alpha. (B) and IL-10 (C) secretion. The means.+-.SD of five
experiments are shown. *P<0.05.
[0092] FIG. 25 demonstrates that the effects of HSP60 on T-cell
cytokine secretion are TLR2-dependent. Purified human T cells were
pretreated with monoclonal anti-TLR2, anti-TLR4 or anti-HSP60 (20
.mu.g/ml, 30 min), and washed. Then, the cells were incubated with
HSP60 (1 ng/ml, 1 hr), washed, and exposed to immobilized
monoclonal anti-CD3 in serum-free medium. The supernatants were
collected after 24 hr and analyzed for IFN-.gamma. (A), TNF-.alpha.
(B) and IL-10 (C) secretion. The means.+-.SD of three experiments
are shown. *P<0.05.
[0093] FIG. 26 shows that the effects of HSP60 on cytokine
secretion are not due to contaminating LPS. Purified human T cells
were treated (1 hr) with HSP60 (0.1 ng/ml) or LPS (100 ng/ml) after
pretreatment (30 min) with polymyxin B (PMB; 1 .mu.g/ml). As
indicated, the HSP60 and LPS were boiled (100.degree. C., 30 min)
in some samples before addition to the to the cell cultures. After
washing, the T cells were exposed to immobilized anti-CD3 in
serum-free medium. The supernatants were collected after 24 hr and
analyzed for IFN-.gamma. (A), TNF-.alpha. (B) and IL-10 (C)
secretion. The means.+-.SD of three experiments are shown.
*P<0.05.
[0094] FIG. 27 demonstrates that HSP60 inhibits anti-CD3-induced
nuclear translocation of NF-.kappa.B in T cells. Purified human T
cells were incubated with HSP60 at 0.01-1000 ng/ml for 1 hr (A, B)
or with 1 ng/ml for 0-240 min (C). Then, the T cells were washed
and exposed to immobilized mAb anti-CD3 (A-C) for 24 hr. Nuclear
(A, C) or cytoplasmic (B) lysates were immunoblotted with
anti-NF-.kappa.B (A-C), anti-Lamin B (A, C), or anti-total ERK
(tERK) (B). Abs against Lamin B and tERK served as a control. One
experiment representative of three is presented in each case. The
levels of NF-.kappa.B, Lamin B and tERK were estimated by
densitometry and the average percentage of three different
experiments was calculated by OD of NF-kB/Lamin B (total
ERK).times.100%. P<0.01.
[0095] FIG. 28 demonstrates that HSP60 inhibits anti-CD3-induced
NFATp activation in T cells. Purified human T cells were incubated
with HSP60 at 0.01-100 ng/ml for 1 hr (A) or at 1 ng/ml for 0-240
min (B). In (C), the cells were pre-treated with cycloheximide
(CHX; 50 .mu.M, 30 min), and exposed to HSP60 (1 ng/ml, 2 hr).
Then, the T cells were washed and exposed to immobilized anti-CD3
for 24 hr. Nuclear (A-C) and cytoplasmic (A) lysates were
immunoblotted with anti-NFATp. The upper NFATp band served as a
control. One experiment representative of three is presented in
each case. The levels of dephospho-NFATp were estimated by
densitometry and the average percentage of three different
experiments is shown. *P<0.05.
[0096] FIG. 29 demonstrates that HSP60 inhibits T-bet and
up-regulates GATA-3 expression in TLR-2-dependent signaling.
Purified human T cells (A, B, C) or activated mouse lymph node
cells (D) were incubated with HSP60 at 0.01-1000 ng/ml for 1 hr (A,
B, C,). Some cells were pre-treated with anti-TLR2 or anti-TLR4 (20
.mu.g/ml, 30 min), washed and exposed to HSP60 (0.1 ng/ml, 2 hr)
(B). Then, the cells were washed and incubated in full medium (A,
B, D), or in the presence of immobilized mAb anti-CD3 (C) for 24
hr. Nuclear lysates were immunoblotted with anti-T-bet Ab,
stripped, and the same blot was incubated with anti-GATA-3 Ab. One
experiment representative of three is presented in each case. The
levels of T-bet and GATA-3 were estimated by densitometry and the
average percentage derived from three different experiments was
shown. *P<0.05.
[0097] FIG. 30 demonstrates that HSP60 inhibits ConA-induced
hepatitis. The levels of hepatic enzymes, AST and ALT (A), and of
TNF-.alpha. (B) were examined 24 and 2 hr following ConA
administration, respectively, in sera obtained from untreated,
ConA-treated and HSP60 and ConA-treated BALB/c mice (7 mice per
group), (C). Histopathological analysis, using hematoxylin and
eosin staining, of liver sections of untreated and treated mice. T
cell were purified from the spleens of untreated or treated mice,
lysed, and immunoblotted with anti-SOCS3 (D) anti-T-bet, and
anti-GATA-3 (E). Each band in the gels is composed of a pool of
T-cell lysate from 2 mice. The columns show the levels of SOCS3,
T-bet, and GATA-3 expression were estimated by densitometry and the
average percentage (.+-.SD) of the various pools was calculated.
This experiment was repeated three times, and a representative
experiment is shown. *P<0.05.
[0098] FIG. 31 is a graph showing that p277 inhibits ConA-induced
hepatitis. The levels of hepatic enzymes, AST and ALT were examined
24 hr following ConA administration, in sera obtained from
untreated, ConA-treated, p277 and ConA-treated, or MTp277 and
ConA-treated BALB/c mice.
[0099] FIG. 32 illustrates that HSP60 and p277 modulate cytokine
secretion in ConA-induced hepatitis. The levels of TNF-.alpha. (A),
IL-6 (B), and IL-10 (C) were examined 2 hr following ConA
administration in sera obtained from PBS-, HSP60, p277, and
MT-p277-treated BALB/c mice (4 mice per group). *P<0.05.
DETAILED DESCRIPTION OF THE INVENTION
[0100] The invention provides novel compositions and methods
utilizing HSP60 and peptides thereof, including the p277 peptide
(SEQ ID NO:1, positions 437-460 of human HSP60) and analogs thereof
for the treatment of T cell mediated disorders.
[0101] It is herein demonstrated for the first time that p277 may
be used for inhibiting T-cell mediated inflammation, regardless of
the specific antigen to which these T cells are directed. Thus, the
invention provides novel uses for p277 and its analogs and
derivatives in treating T cell mediated pathologies beyond the
reported uses for IDDM and graft rejection.
[0102] The invention is based, in part, on the surprising discovery
that HSP60 and p277 are presented by activated T cells to
regulatory (anti-ergotypic) T cells. It is now disclosed for the
first time that HSP60 and p277-specific T cell lines demonstrating
anti-ergotypic activity inhibit IFN.gamma. production by activated
T-cells and ameliorate adjuvant arthritis (AA) when adoptively
transferred to rats.
[0103] The invention is further based, in part, on the unexpected
discovery that HSP60 and p277 act as co-stimulators of human
CD4.sup.+CD25.sup.+ T regulatory cells (Tregs), added before
mitogenic anti-CD3 activation. Treatment of Tregs with HSP60 or
p277 suppressed IFN-.gamma. and TNF-.alpha. secretion, and
proliferation of CD4.sup.+CD25.sup.- or CD8.sup.+ T cells in a
TLR-2-dependent manner.
[0104] The present invention is also based, in part, on the
unexpected discovery that HSP60 and p277 up-regulate suppressors of
cytokine signaling (SOCS)3 expression, thereby inhibiting the
down-stream effects of stromal cell-derived (SDF)-1.alpha.
(CXCL12)-CXCR4 interactions in vitro and in vivo.
[0105] In one aspect, the present invention provides methods for
treating or preventing a T cell mediated pathology. The term
"T-cell mediated pathology" as used herein indicates any condition
in which an inappropriate T cell response is a component of the
pathology. The term is intended to include both diseases directly
mediated by T cells, and also diseases in which an inappropriate T
cell response contributes to the production of abnormal
antibodies.
[0106] According to various embodiments, the T cell mediated
pathology includes, but is not limited to, autoimmune diseases,
allergic diseases, Th1 mediated diseases and other inflammatory
diseases. In one embodiment of the invention, the compositions and
methods of the invention are useful for treating a T cell-mediated
autoimmune disease, including but not limited to: multiple
sclerosis, rheumatoid arthritis, autoimmune neuritis, systemic
lupus erythematosus (SLE), psoriasis, Sjogren's disease, thyroid
disease, myasthenia gravis, sarcoidosis, autoimmune uveitis, and
inflammatory bowel disease (Crohn's and ulcerative colitis). In
other particular embodiments the compositions and methods of the
invention are useful for treating a Th1-associated inflammatory
disease, e.g. Th1 mediated allergic responses which result in skin
sensitivity and inflammation, such as contact dermatitis. In other
embodiments, the compositions and methods of the invention are
useful in treating a wide range of inflammatory diseases and
conditions. According to certain other particular embodiments, the
compositions and methods of the invention are useful in treating
inflammatory conditions in which chemotaxis of T cells in response
to a chemoattractant (particularly SDF-1.alpha.) results in
migration of the cells to a site of inflammation, e.g.
glomerulonephritis, post-viral myocarditis and atherosclerosis. It
should be emphasized, however, that the present invention is not
intended to include known therapeutic uses of p277, such as for
inhibiting IDDM and graft rejection.
[0107] The present invention is also based, in part, on the
unexpected discovery that HSP60, as well as a peptide thereof, i.e.
p277, is an effective agent for treating or preventing the symptoms
of T cell-mediated hepatitis. It is now disclosed for the first
time that HSP60 and p277 inhibit the clinical, histological, and
serological manifestations of concanavalin A (conA)-induced
hepatitis, an animal disease model of acute inflammatory hepatitis.
Without wishing to be bound by any theory or mechanism of action,
this phenomenon may be associated with a Toll-like receptor 2
(TLR2)-dependent modulation of the expression of Th1/Th2
transcription factors. HSP60 is herein demonstrated to
differentially modulate the expression of Th1/Th2 transcription
factors: down-regulating T-bet, NF-.kappa.B, and NFATp, and
up-regulating GATA-3, leading to decreased secretion of TNF.alpha.
and IFN.gamma. and enhanced secretion of IL-10. HSP60 and p277 also
modulate the secretion of IL-6, a well-known regulator of T cell
mediated hepatitis.
[0108] The term "hepatitis" is used herein to refer to a disease of
patients characterized in part by inflammation of the liver.
Causative agents of hepatitis include, for example, viral
infections, such as infections of hepatitis A, B, C, D, E, and G
viruses; parasitic infections, including, but not limited to
infections of Schistosoma mansoni, Schistosoma hematobium, and
Schistosoma japonicum; autoimmune diseases, including, but not
limited to, autoimmune hepatitis and primary biliary cirrhosis; and
non-infectious hepatotoxic agents, including, but not limited to,
alcohol, drugs and toxins. Liver damage due to inflammation is
associated with these hepatic disorders. The compositions and
methods of the invention are thus useful for treating or preventing
liver damage associated, for example, with inflammation secondary
to viral or parasitic infection.
[0109] Protein- and Peptide-Based Compositions and Methods
[0110] According to one aspect, the present invention is directed
to the use of pharmaceutical compositions comprising as an active
ingredient a compound selected from a group consisting of: p277
(VLGGGCALLRCIPALDSLTPANED, SEQ ID NO:1), analogs, variants,
derivatives and salts thereof, for the treatment and prevention of
T cell mediated pathologies. In one embodiment, the compositions
further comprise pharmaceutically acceptable carriers, excipients
or diluents.
[0111] In one embodiment, the active ingredient is a p277 analog,
in which at least one of the cysteine residues in positions 6 and
11 of p277 has been substituted. In one preferred embodiment, the
p277 analog is p277(Val.sup.6Val.sup.11) (VLGGGVALLRVIPALDSLTPANED,
SEQ ID NO:2).
[0112] In another preferred embodiment, the active ingredient is
p277(Ser.sup.6Ser.sup.11) (VLGGGSALLRSIPALDSLTPANED SEQ ID NO:3).
Surprisingly, it is now disclosed that certain p277 analogs that
may be inactive as specific antigens modulating IDDM, may be used
to regulate T cell function according to the present invention (see
Example 36). Thus, for example, p277(Ser.sup.6Ser.sup.11) that did
not reduce the incidence of diabetes upon immunization by a single
subcutaneous injection in mineral oil (see U.S. Pat. No.
6,180,103), may be useful for modulating T cell immunity (e.g. in
hepatitis) upon prolonged administration without an adjuvant, e.g.
as a sustained-release formulation.
[0113] Another aspect of the present invention relates to the use
of a compound selected from a group consisting of: p277 (SEQ ID
NO:1), analogs, variants, derivatives and salts thereof, for the
preparation of a pharmaceutical compositions for the treatment and
prevention of T cell mediated pathologies or symptoms associated
therewith.
[0114] In another aspect, there is provided a method for treating a
T cell mediated pathology other than insulin-dependent diabetes
mellitus and graft rejection in a subject in need thereof,
comprising administering to the subject a therapeutically-effective
amount of a compound selected from a group consisting of: p277 (SEQ
ID NO:1), analogs, variants, derivatives and salts thereof. In one
embodiment, the subject is human.
[0115] In another aspect, there is provided a method for preventing
a T cell mediated pathology other than insulin-dependent diabetes
mellitus and graft rejection in a subject in need thereof,
comprising administering to the subject a therapeutically-effective
amount of a compound selected from a group consisting of: p277 (SEQ
ID NO:1), analogs, variants, derivatives and salts thereof.
[0116] In another aspect, the invention is directed to a
composition comprising heat shock protein 60 (HSP60), or an active
fragment thereof, for treating or preventing the symptoms of
hepatitis. In one preferred embodiment, the HSP60 polypeptide is
human HSP60. In one embodiment, the human HSP60 has an amino acid
sequence as set forth in SEQ ID NO:4 (corresponding to accession
number: P10809). In another embodiment, the human HSP60 has an
amino acid sequence as set forth in SEQ ID NO:11 (corresponding to
accession number: gi:306890). In another embodiment, other
mammalian HSP60 are used. In yet another embodiment, the HSP60
polypeptide is bacterial HSP60. In another embodiment, the HSP60
polypeptide is E. coli GroEL (which have been previously implicated
in TLR-2 mediated T cell adhesion, see Zanin-Zhorov et al., 2003).
In one embodiment, the E. coli GroEL has an amino acid sequence as
set forth in SEQ ID NO:9 (accession number: AAS75782). In another
preferred embodiment, the active fragment of HSP60 is the p277
peptide (SEQ ID NO:1). According to other embodiments, the
composition may comprise homologs, analogs, derivatives and salts
thereof. In one preferred embodiment, the analog has an amino acid
sequence as set forth in SEQ ID NO:2. In another preferred
embodiment, the analog has an amino acid sequence as set forth in
SEQ ID NO:3.
[0117] Another aspect of the present invention relates to the use
of a compound selected from a group consisting of: HSP60 protein,
an active fragment thereof, and analogs, variants, derivatives and
salts thereof, for the preparation of a pharmaceutical compositions
for the treatment and prevention of the symptoms of hepatitis.
[0118] Another aspect of the present invention is a method of
treating hepatitis comprising administering to a subject in need
thereof a therapeutically effective amount of a compound selected
from a group consisting of: HSP60 protein, an active fragment
thereof, and analogs, variants, derivatives and salts thereof.
[0119] In another aspect, there is provided a method of treating or
preventing liver damage comprising administering to a subject in
need thereof a therapeutically effective amount of a compound
selected from a group consisting of: HSP60 protein, an active
fragment thereof, and analogs, variants, derivatives and salts
thereof.
[0120] The polypeptides or peptides of the invention may be
synthesized using any recombinant or synthetic method known in the
art, including, but not limited to, solid phase and solution phase
synthesis methods. A non-limitative example of peptide synthesis is
presented in the Examples; however, other methods known in the art
may be used. For solid phase peptide synthesis, a summary of the
many techniques may be found in: Stewart and Young, 1963; and
Meienhofer, 1973. For a review of classical solution synthesis, see
Schroder and Lupke, 1965.
[0121] The amino acid residues described herein are preferred to be
in the "L" isomeric form. However, residues in the "D" isomeric
form can be substituted for any L-amino acid residue, as long as
the peptide retains the desired functional property.
[0122] It should be understood that a polypeptide or a peptide of
the invention need not be identical to the amino acid sequence of
SEQ ID NOS:1, 2 or 3, so long as it retains their biological
activity with respect to T cell mediated pathologies, as described
herein. Several non-limitative examples of methods suitable for
determining p277 activity are described in the Examples below.
Similarly, a polypeptide or a peptide of the invention need not be
identical to the amino acid sequence of SEQ ID NOS:1-4, 9 or 11, so
long as it retains their biological activity with respect to
hepatitis, as described herein.
[0123] As used herein, the terms "peptide", "polypeptide" and
"protein" all refer to a primary sequence of amino acids that are
joined by covalent "peptide linkages". In general, a peptide
consists of a few amino acids, typically from 2-50 amino acids, and
is shorter than a protein. The term "polypeptide" encompasses
peptides and proteins. In some embodiments, the peptide,
polypeptide or protein is synthetic, while in other embodiments,
the peptide, polypeptide or protein is recombinant or naturally
occurring. A synthetic peptide is a peptide which is produced by
artificial means in vitro (i.e., was not produced in vivo). The
HSP60 fragments and peptides according to the present invention are
preferably 7-30 amino acids in length.
[0124] Whenever the terms "p277", "peptide p277", "fragment of
HSP60" or "peptide of HSP60" are mentioned in the invention, also
salts and functional derivatives thereof are contemplated, as long
as the biological activity of the peptide with respect to T cell
mediated pathologies and/or hepatitis is maintained. The present
invention encompasses any analog, derivative, and conjugate
containing a polypeptide or a peptide of the invention, so long as
the polypeptide or peptide is capable of inhibiting or preventing
hepatitis and/or other T cell mediated pathologies. Thus, the
present invention encompasses peptides containing non-natural amino
acid derivatives or non-protein side chains.
[0125] The term "analog" indicates molecule which has the amino
acid sequence according to the invention except for one or more
amino acid changes or one or more modification/replacement of an
amide bond. In one embodiment, the term relates to peptides in
which one or more residues have been conservatively substituted
with a functionally similar residue and which displays the
abilities as described herein. Examples of conservative
substitutions include the substitution of one non-polar
(hydrophobic) residue such as isoleucine, valine, leucine or
methionine for another, the substitution of one polar (hydrophilic)
residue for another such as between arginine and lysine, between
glutamine and asparagine, between glycine and serine, the
substitution of one basic residue such as lysine, arginine or
histidine for another, or the substitution of one acidic residue,
such as aspartic acid or glutamic acid for another. In other
embodiments, the term further includes non-conservative
substitutions in an amino acid that does not contribute to the
biological activity of the peptide. For example, without
limitation, at least one of the cysteine residues in positions 6
and 11 of p277 may be substituted.
[0126] The phrase "conservative substitution" also includes the use
of a chemically derivatized residue in place of a non-derivatized
residue provided that such polypeptide or peptide displays the
requisite inhibitory function on hepatic disorders and/or other T
cell mediated pathologies as specified herein.
[0127] The term "derivative" includes any chemical derivative of
the polypeptides or peptides of the invention having one or more
residues chemically derivatized by reaction of side chains or
functional groups. Such derivatized molecules include, for example,
those molecules in which free amino groups have been derivatized to
form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl
groups. Free carboxyl groups may be derivatized to form salts,
methyl and ethyl esters or other types of esters or hydrazides.
Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl
derivatives. The imidazole nitrogen of histidine may be derivatized
to form N-im-benzylhistidine. Also included as chemical derivatives
are those peptides, which contain one or more naturally occurring
amino acid derivatives of the twenty standard amino acid residues.
For example: 4-hydroxyproline may be substituted for proline;
5-hydroxylysine may be substituted for lysine; 3-methylhistidine
may be substituted for histidine; homoserine may be substituted or
serine; and ornithine may be substituted for lysine.
[0128] In addition, a peptide derivative can differ from the
natural sequence of the peptide of the invention by chemical
modifications including, but not limited to, amino terminal
acylation, acetylation, or thioglycolic acid amidation, and by
terminal-carboxlyamidation, e.g., with ammonia, methylamine, and
the like. Peptides can be either linear, cyclic or branched and the
like, which conformations can be achieved using methods well known
in the art.
[0129] Peptides of the present invention also include any peptide
having one or more additions and/or deletions of residues relative
to the sequence of p277, so long as they are able to inhibit T cell
mediated pathologies. Polypeptides or peptides of the present
invention also include any polypeptide or peptide having one or
more additions and/or deletions of residues relative to the
sequence of the HSP60 or p277, so long as they are able to inhibit
hepatitis and/or liver damage.
[0130] Addition of amino acid residues may be performed at either
terminus of the peptides of the invention for the purpose of
providing a "linker" by which the peptides of this invention can be
conveniently bound to a carrier. Such linkers are usually of at
least one amino acid residue and can be of 40 or more residues,
more often of 1 to 10 residues. Typical amino acid residues used
for linking are tyrosine, cysteine, lysine, glutamic and aspartic
acid, or the like.
[0131] A polypeptide or peptide of the present invention may be
coupled to or conjugated with another protein or polypeptide to
produce a conjugate. Such a conjugate may have advantages over the
polypeptide or peptide used alone.
[0132] A pharmaceutical composition useful in the practice of the
present invention typically contains a polypeptide or a peptide of
the invention formulated into the pharmaceutical composition as a
neutralized pharmaceutically acceptable salt form. Pharmaceutically
acceptable salts include the acid addition salts (formed with the
free amino groups of the polypeptide), which are formed with
inorganic acids, such as for example, hydrochloric or phosphoric
acid, or with organic acids such as acetic, oxalic, tartaric, and
the like.
[0133] Suitable bases capable of forming salts with the
polypeptides or peptides of the present invention include, but are
not limited to, inorganic bases such as sodium hydroxide, ammonium
hydroxide, potassium hydroxide and the like; and organic bases such
as mono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,
diisopropyl amine, methyl amine, dimethyl amine and the like) and
optionally substituted ethanolamines (e.g. ethanolamine,
diethanolamine and the like).
[0134] The preparation of pharmaceutical compositions, which
contain peptides or polypeptides as active ingredients is well
known in the art. Typically, such compositions are prepared as
indictable, either as liquid solutions or suspensions, however,
solid forms, which can be suspended or solubilized prior to
injection, can also be prepared. The preparation can also be
emulsified. The active therapeutic ingredient is mixed with
inorganic and/or organic carriers, which are pharmaceutically
acceptable and compatible with the active ingredient. Carriers are
pharmaceutically acceptable excipients (vehicles) comprising more
or less inert substances when added to a pharmaceutical composition
to confer suitable consistency or form to the composition. Suitable
carriers are, for example, water, saline, dextrose, glycerol,
ethanol, or the like and combinations thereof. In addition, if
desired, the composition can contain minor amounts of auxiliary
substances such as wetting or emulsifying agents and pH buffering
agents, which enhance the effectiveness of the active
ingredient.
[0135] In one particular embodiment, the composition is a vaccine
composition comprising an immunogenic adjuvant suitable for
enhancing a p277-specific anti-ergotypic reaction in said
subject.
[0136] The term "anti-ergotypic T cell response" refers to the
activation of regulatory anti-ergotypic T cells. In various
embodiments, the anti-ergotypic T cell response may be measured as
increased T cell proliferation response to activated syngeneic T
cells. Alternatively, the activation of regulatory anti-ergotypic T
cells may be determined by measuring the secretion level of
cytokines by said T cells.
[0137] The vaccine composition may optionally comprise adjuvants
such as vegetable oils or emulsions thereof, surface active
substances, e.g., hexadecylamin, octadecyl amino acid esters,
octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide,
N,N-dicoctadecyl-N'--N'bis(2-hydroxyethyl-propane diamine),
methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g.,
pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl
dipeptide, dimethylglycine, tuftsin; immune stimulating complexes;
oil emulsions (including, but not limited to, oil-in-water
emulsions having oil droplets in the submicron range, such as those
disclosed by U.S. Pat. Nos. 5,961,970, 4,073,943 and 4,168,308),
liposaccharides such as MPL.RTM. and mineral gels. The peptide
antigens of the present invention can be coupled to albumin or to
other carrier molecule in order to modulate or enhance the immune
response, all as are well known to those of ordinary skill in the
vaccine art. Metabolizable lipid emulsions, such as Intralipid or
Lipofundin, may also be used as vehicles for the p277 vaccination
in the manner disclosed in WO 97/02016, the entire contents of
which being hereby incorporated herein by reference. These lipid
emulsions may be formulated as oil-in-water submicron emulsion, as
disclosed in U.S. Pat. No. 5,961,970.
[0138] The vaccines can be administered to a human or animal by a
variety of routes, including but not limited to parenteral,
intradermal, transdermal (such as by the use of slow release
polymers), intramuscular, intraperitoneal, intravenous,
subcutaneous, oral and intranasal routes of administration,
according to protocols well known in the art. The vaccine
compositions of the invention are administered in a dose which is
suitable to elicit an immune response in said subject. The
particular dosage of the p277 peptide will depend upon the age,
weight and medical condition of the subject to be treated, as well
as on the identity of the antigen and the method of administration.
Suitable doses will be readily determined by the skilled artisan. A
preferred dose for human intramuscular, subcutaneous and oral
vaccination is between about 12.5 .mu.g to about 20 mg per kg body
weight, preferably between about 25 .mu.g to about 10 mg per kg
body weight, and more preferably between about 125 pg to about 2 mg
per kg body weight. Adjustment and manipulation of established
dosage ranges used with traditional carrier antigens for adaptation
to the present vaccine is well within the ability of those skilled
in the art.
[0139] In other particular embodiments, the compound is formulated
for oral, buccal, rectal, transmucosal, transnasal, enteral,
bronchial, or intrapulmonary administration. In these embodiments,
the compound is formulated without an adjuvant. The preparation of
such formulations is well within the ability of one skilled in the
art.
[0140] Sustained-release oral delivery systems are also
contemplated and are preferred method of mucosal administration.
Non-limiting examples of sustained-release oral dosage forms
include those described in: U.S. Pat. Nos. 4,704,295; 4,556,552;
4,309,404; 4,309,406; 5,405,619; 5,371,109; 5,356,635 and
5,151,272. In one preferable embodiment, the p277, analogs
derivatives and salts thereof of the invention are derivatized by
reversible pegylation as described for example by WO/2004/089280,
hereby fully incorporated herein by reference. In this embodiment,
the active ingredient is a peptide-PEG conjugate having an
increased half-life in the circulation, from which PEG can be
released by hydrolysis under physiological conditions.
[0141] Sustained-release oral dosage forms coated with bioadhesives
may also be used. Examples of such compositions are disclosed by:
WO 85/02092, EP 516141 and EP 205282; U.S. Pat. Nos. 4,226,848;
4,713,243; and 4,940,587; WO 85/02092
[0142] Commercially available sustained-release oral delivery
formulations and devices include those marketed by ALZA
Corporation, Palo Alto, Calif., USA, under the trade names
OROS.RTM., ALZET.RTM., INFUSET.TM., IVOS.TM., and OSMET.RTM., and
those described by: U.S. Pat. Nos. 5,284,660; 5,141,750; 5,110,597;
4,917,895; 4,837,027; 3,993,073; 3,948,262; 3,944,064; and
3,699,963; PCT/US93/10077 and PCT/US93/11660; and EP 259013 and EP
354742.
[0143] For by-inhalation administration (i.e., delivery to the
bronchopulmonary mucosa), suitable sprays and aerosols can be used,
for example, with a nebulizer, such as those described in U.S. Pat.
Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. The aerosol
material is inhaled by the subject to be treated.
[0144] Other systems of aerosol delivery, such as the pressurized
metered-dose inhaler (MDI) and the dry powder inhaler, as disclosed
by Newman, S. P. (1984) (Aerosols and the Lung, Clarke, S. W.
Davis, D., eds., pp. 197-224, Butterworths, London, England) can be
used when practicing the present invention.
[0145] Aerosol delivery systems of the type disclosed herein are
available from numerous commercial sources, including Fisons
Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, N.J.),
and American Phannoseal Co. (Valencia, Calif.).
[0146] Formulations for nasal administration can be administered,
for example, as a dry powder or in an aqueous solution. Preferred
aerosol-based pharmaceutical formulations may comprise, for
example, a physiologically acceptable buffered saline solution
containing p277, analogs derivatives and salts thereof of the
present invention.
[0147] The nasally administered formulation of the present
invention may comprise a thermosetting gel, which increases in
viscosity at body temperature upon contact with the nasal
mucosa.
[0148] Formulations for buccal administration may comprise a
mucoadhesive mixed with effective amounts of p277, analogs
derivatives and salts thereof. Effective amounts are anticipated to
vary according to the formulation employed and other factors, as is
known to one skilled in the art.
[0149] The amount of the p277, analogs derivatives and salts
thereof in a pharmaceutical composition for enteral or mucosal
administration without an adjuvant, which will be effective in the
treatment of a particular disorder or condition, will depend on the
nature of the disorder or condition, and can be determined by
standard clinical techniques. In addition, in vitro assays may
optionally be employed to help identify optimal dosage ranges.
Suitable dosage ranges are between 5 ng and 1000 mg per day. The
precise dose to be employed in the formulation will also depend on
the route of administration, and the seriousness of the disease or
disorder, and should be decided according to the judgment of the
practitioner and each patient's circumstances. For example, for
formulation administered by inhalation, the effective amount is
likely to be less than that of the oral dose. This amount can be
further refined by well-known methods, such as establishing a
matrix of dosages and frequencies of administration. For sustained
release formulations, the dose is determined such that the
effective dose in the serum will be between about 1 and 100 ng/ml.
Such determination is well within the abilities of the skilled
artisan.
[0150] In various embodiments, compositions according to the
invention can be delivered by a variety of means including
intravenous, intramuscularly, infusion, oral, intranasal,
intraperitoneal, subcutaneous, rectal, topical, buccal,
transmucosal, transnasal, enteral, bronchial, or intrapulmonary or
into other regions, such as into synovial fluids. Delivery of the
composition transdermally is also contemplated, such by diffusion
via a transdermal patch. For oral ingestion it is possible to
prepare peptide analogs or specific peptide formulations having
improved oral bioavailability and enhanced resistance to
degradation as are known in the art.
[0151] The composition is administered in a manner compatible with
the dosage formulation, and in a therapeutically effective amount.
The quantity to be administered depends on the subject to be
treated, capacity of the subject's blood hemostatic system to
utilize the active ingredient. Precise amounts of active ingredient
required to be administered depend on the judgment of the
practitioner and are peculiar to each individual.
[0152] In order to treat a subject with a disease, a pharmaceutical
composition of the present invention is administered to the subject
in an effective manner such that the composition is capable of
treating that subject from disease. According to the present
invention, treatment of a disease refers to alleviating a disease
and/or preventing the development of a secondary disease resulting
from the occurrence of a primary disease. An effective
administration protocol (i.e., administering a pharmaceutical
composition in an effective manner) comprises suitable dose
parameters and modes of administration that result in treatment of
a disease. Effective dose parameters and modes of administration
can be determined using methods standard in the art for a
particular disease. Such methods include, for example,
determination of survival rates, side effects (i.e., toxicity) and
progression or regression of disease.
[0153] In accordance with the present invention, a suitable single
dose size is a dose that is capable of treating a subject with
disease when administered one or more times over a suitable time
period. Doses can vary depending upon the disease being treated.
Doses of a pharmaceutical composition of the present invention
suitable for use with direct injection techniques can be used by
one of skill in the art to determine appropriate single dose sizes
for systemic administration based on the size of a subject.
[0154] A suitable single dose of a composition according to the
invention is a sufficient amount of the active ingredient to
reduce, and preferably eliminate, the T-cell mediated disorder, or
the symptoms thereof. For example, a suitable single dose of a
pharmaceutical composition comprising HSP60 or a HSP60 fragment is
a sufficient amount of the HSP60 or HSP60 fragment to reduce, and
preferably eliminate, a T-cell mediated hepatic disorder. A
preferred single dose of HSP60 or HSP60 fragment for the treatment
of hepatitis or preventing liver damage is between 5 ng and 50
.mu.g, and preferably between 50 ng and 5 .mu.g.
[0155] The pharmaceutical compositions of the invention may be used
alone or in combination with one ore more therapeutic agents. For
example, a pharmaceutical compositions for the treatment of
hepatitis according to the invention may be administered in
combination with a drug including, but not limited to: interferon,
ribavirin, or one or more other agents known in the art for the
treatment or prevention of hepatitis, all of which administered
together or separately, e.g., prior to, concurrently with or
following the administration of the pharmaceutical compositions the
invention.
[0156] Nucleic Acid-Based Compositions and Methods
[0157] According to another aspect, the present invention is
directed to the use of a pharmaceutical composition comprising a
recombinant construct comprising a nucleic acid sequence encoding a
peptide selected from: p277 (SEQ ID NO:1), analogs and variants
thereof, the nucleic acid sequence being operably linked to one or
more transcription control sequences, and a pharmaceutically
acceptable carrier, for the treatment and prevention of T cell
mediated pathologies.
[0158] In one embodiment, the nucleic acid molecule encodes a p277
analog, in which at least one of the cysteine residues in positions
6 and 11 of p277 has been substituted. In one preferred embodiment,
the encoded peptide is the p277 analog p277(Val.sup.6Val.sup.11)
(SEQ ID NO:2). In another preferred embodiment, the encoded peptide
is the p277 analog p277(Ser.sup.6Ser.sup.11) (SEQ ID NO:3). In
another embodiment, said construct comprises a nucleic acid
sequence as set forth in any one of:
TABLE-US-00001 (encoding p277, SEQ ID NO: 5) gtt ttg gga ggg ggt
tgt gcc ctc ctt cga tgc att cca gcc ttg gac tca ttg act cca gct aat
gaa gat; (encoding p277(Val.sup.6Val.sup.11), SEQ ID NO: 6) gtt ttg
gga ggg ggt gtt gcc ctc ctt cga gtc att cca gcc ttg gac tca ttg act
cca gct aat gaa gat; and (encoding p277(Ser.sup.6Ser.sup.11), SEQ
ID NO: 7) gtt ttg gga ggg ggt tct gcc ctc ctt cga tcc att cca gcc
ttg gac tca ttg act cca gct aat gaa gat.
[0159] In another aspect, the invention is directed to the use of a
recombinant construct comprising a nucleic acid sequence encoding a
peptide selected from: p277 (SEQ ID NO:1), analogs and variants
thereof, the nucleic acid sequence being operably linked to one or
more transcription control sequences for the preparation of a
pharmaceutical composition for the treatment and prevention of T
cell mediated pathologies or symptoms associated therewith.
[0160] In another aspect, the present invention provides a method
of treating a T cell mediated pathology other than
insulin-dependent diabetes mellitus and graft rejection in a
subject in need thereof, comprising administering to the subject a
therapeutically-effective amount of a pharmaceutical composition
comprising a recombinant construct comprising a nucleic acid
sequence encoding a peptide selected from: p277 (SEQ ID NO:1),
analogs and variants thereof, the nucleic acid sequence being
operably linked to one or more transcription control sequences; and
a pharmaceutically acceptable carrier.
[0161] In another aspect, the present invention provides methods of
preventing a T cell mediated pathology other than insulin-dependent
diabetes mellitus and graft rejection in a subject in need thereof,
comprising administering to the subject a therapeutically-effective
amount of a pharmaceutical composition comprising a recombinant
construct comprising a nucleic acid sequence encoding a peptide
selected from: p277 (SEQ ID NO:1), analogs and variants thereof,
the nucleic acid sequence being operably linked to one or more
transcription control sequences; and a pharmaceutically acceptable
carrier.
[0162] In another aspect, the present invention provides methods of
treating or preventing a T cell mediated pathology other than
insulin-dependent diabetes mellitus and graft rejection in a
subject in need thereof, comprising administering to the subject a
therapeutically-effective amount of a pharmaceutical composition
comprising a recombinant construct comprising a nucleic acid
sequence encoding a peptide selected from: p277 (SEQ ID NO:1),
analogs and variants thereof, the nucleic acid sequence being
operably linked to one or more transcription control sequences; and
a pharmaceutically acceptable carrier.
[0163] According to another aspect, the present invention is
directed to the use of a pharmaceutical composition comprising a
recombinant construct, the recombinant construct comprising a
nucleic acid sequence encoding a polypeptide or peptide selected
from a group consisting of: HSP60 protein, an active fragment
thereof, and analogs and variants thereof, the nucleic acid
sequence being operably linked to one or more transcription control
sequences, and a pharmaceutically acceptable carrier, for the
treatment of hepatitis.
[0164] In one embodiment, the HSP60 protein is selected from the
group consisting of: human HSP60, mammalian HSP60 and bacterial
HSP60. In another embodiment, the HSP60 protein has an amino acid
sequence as set forth in any one of SEQ ID NOS:4 and 11. In another
embodiment, the HSP60 protein has an amino acid sequence as set
forth in SEQ ID NO:9. In another particular embodiment, the HSP60
fragment is the p277 peptide, having an amino acid sequence as set
forth in SEQ ID NO:1. In another embodiment, the HSP60 fragment
analog has an amino acid sequence as set forth in SEQ ID NO:2. In
yet another embodiment, the HSP60 fragment analog has an amino acid
sequence as set forth in SEQ ID NO:3. In another embodiment, said
construct comprises a nucleic acid sequence as set forth in any one
of SEQ ID NOS:5-7. In another embodiment, the HSP60 protein is
encoded by a nucleic acid sequence as set forth in SEQ ID NO:8
(encoding human HSP60, accession number M34664).
[0165] According to another aspect, the present invention is
directed to the use of a pharmaceutical composition comprising a
recombinant construct, the recombinant construct comprising a
nucleic acid sequence encoding a polypeptide or peptide selected
from a group consisting of: HSP60 protein, an active fragment
thereof, and analogs and variants thereof, the nucleic acid
sequence being operably linked to one or more transcription control
sequences, and a pharmaceutically acceptable carrier, for
preventing liver damage.
[0166] According to another aspect, the present invention is
directed to the use of a recombinant construct comprising a nucleic
acid sequence encoding a polypeptide selected from a group
consisting of: HSP60 protein, an active fragment thereof, and
analogs and variants thereof, the nucleic acid sequence being
operably linked to one or more transcription control sequences for
the preparation of a pharmaceutical composition useful for the
treatment of hepatitis and/or prevention of liver damage.
[0167] In another aspect, the invention provides a method of
treating hepatitis, comprising administering to a subject in need
thereof a therapeutically effective amount of a pharmaceutical
composition comprising as an active ingredient a recombinant
construct, the recombinant construct comprising a nucleic acid
sequence encoding a polypeptide selected from a group consisting
of: HSP60 protein, an active fragment thereof, and analogs and
variants thereof, wherein the nucleic acid sequence is operably
linked to one or more transcription control sequences, thereby
treating hepatitis.
[0168] The invention provides, in another aspect, a method of
treating or preventing liver damage, comprising administering to a
subject in need thereof a therapeutically effective amount of a
pharmaceutical composition comprising as an active ingredient a
recombinant construct, the recombinant construct comprising an
isolated nucleic acid sequence encoding a polypeptide selected from
a group consisting of: HSP60 protein, an active fragment thereof,
and analogs and variants thereof, wherein the nucleic acid sequence
is operably linked to one or more transcription control sequences,
thereby treating or preventing liver damage.
[0169] In another aspect, there are provided methods of treating
hepatitis and/or treating or preventing liver damage comprising the
steps of (a) obtaining cells from a subject; (b) introducing a
recombinant construct into the cells ex vivo, the construct
comprising an isolated nucleic acid sequence encoding a polypeptide
selected from a group consisting of: HSP60 protein, an active
fragment thereof, and analogs and variants thereof, wherein the
nucleic acid sequence is operably linked to one or more
transcription control sequences; and (c) reintroducing said treated
cells to the subject; wherein the HSP60 or fragment, analog or
variant thereof is expressed in vivo in said treated cells in an
amount sufficient to treat hepatitis.
[0170] The nucleic acid sequence corresponding to mammalian heat
shock proteins may include DNA, RNA, or derivatives of either DNA
or RNA. An isolated nucleic acid sequence encoding heat shock
proteins can be obtained from its natural source, either as an
entire (i.e., complete) gene or a portion thereof. A nucleic acid
molecule can also be produced using recombinant DNA technology
(e.g., polymerase chain reaction (PCR) amplification, cloning) or
chemical synthesis. Nucleic acid sequences include natural nucleic
acid sequences and homologues thereof, including, but not limited
to, natural allelic variants and modified nucleic acid sequences in
which nucleotides have been inserted, deleted, substituted, and/or
inverted in such a manner that such modifications do not
substantially interfere with the nucleic acid molecule's ability to
encode a functional heat shock protein or an active fragment or
peptide of the invention.
[0171] A nucleic acid molecule homolog can be produced using a
number of methods known to those skilled in the art (see, for
example, Sambrook et al., 1989). For example, nucleic acid
molecules can be modified using a variety of techniques including,
but not limited to, classic mutagenesis techniques and recombinant
DNA techniques, such as site-directed mutagenesis, chemical
treatment of a nucleic acid molecule to induce mutations,
restriction enzyme cleavage of a nucleic acid fragment, ligation of
nucleic acid fragments, polymerase chain reaction (PCR)
amplification and/or mutagenesis of selected regions of a nucleic
acid sequence, synthesis of oligonucleotide mixtures and ligation
of mixture groups to "build" a mixture of nucleic acid molecules
and combinations thereof. Nucleic acid molecule homologs can be
selected from a mixture of modified nucleic acids by screening for
the function of the peptide encoded by the nucleic acid. Techniques
to screen for HSP60 and p277 activity are known to those of skill
in the art (several non-limitative examples of these methods are
described in the Examples below).
[0172] A polynucleotide or oligonucleotide sequence can be deduced
from the genetic code of a protein, however, the degeneracy of the
code must be taken into account. Nucleic acid sequences of the
invention include sequences, which are degenerate as a result of
the genetic code, which sequences may be readily determined by
those of ordinary skill in the art.
[0173] The oligonucleotides or polynucleotides of the invention may
contain a modified internucleoside phosphate backbone to improve
the bioavailability and hybridization properties of the
oligonucleotide or polynucleotide. Linkages are selected from the
group consisting of phosphodiester, phosphotriester,
methylphosphonate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoroanilidate, phosphoramidate,
phosphorothioate, phosphorodithioate or combinations thereof.
[0174] Additional nuclease linkages include alkylphosphotriester
such as methyl- and ethylphosphotriester, carbonate such as
carboxymethyl ester, carbamate, morpholino carbamate,
3'-thioformacetal, silyl such as dialkyl (C1-C6)-- or
diphenylsilyl, sulfamate ester, and the like. Such linkages and
methods for introducing them into oligonucleotides are described in
many references, e.g. reviewed generally by Peyman and Ulmann,
Chemical Reviews, 90:1543-584 (1990).
[0175] The present invention includes a nucleic acid sequence of
the present invention operably linked to one or more transcription
control sequences to form a recombinant molecule. The phrase
"operably linked" refers to linking a nucleic acid sequence to a
transcription control sequence in a manner such that the molecule
is able to be expressed when transfected (i.e., transformed,
transduced or transfected) into a host cell. Transcription control
sequences are sequences which control the initiation, elongation,
and termination of transcription. Particularly important
transcription control sequences are those which control
transcription initiation, such as promoter, enhancer, operator and
repressor sequences. Suitable transcription control sequences
include any transcription control sequence that can function in at
least one of the recombinant cells of the present invention. A
variety of such transcription control sequences are known to those
skilled in the art. Preferred transcription control sequences
include those which function in animal, bacteria, helminth, insect
cells, and preferably in animal cells. More preferred transcription
control sequences include, but are not limited to RSV control
sequences, CMV control sequences, retroviral LTR sequences, SV-40
control sequences and .beta.-actin control sequences as well as
other sequences capable of controlling gene expression in
eukaryotic cells. Additional suitable transcription control
sequences include tissue-specific promoters and enhancers (e.g., T
cell-specific and liver specific enhancers and promoters).
Transcription control sequences of the present invention can also
include naturally occurring transcription control sequences
naturally associated with a gene encoding HSP60.
[0176] According to still further features in the described
preferred embodiments the recombinant construct is a eukaryotic
expression vector.
[0177] According to still further features in the described
preferred embodiments the expression vector is selected from the
group consisting of pcDNA3, pcDNA3.1(+/-), pZeoSV2(+/-), pSecTag2,
pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pCI, pBK-RSV,
pBK-CMV, pTRES and their derivatives.
[0178] According to the present invention, a host cell can be
transfected in vivo (i.e., in an animal) or in vitro (i.e., outside
of an animal, such as in tissue culture). Transfection of a nucleic
acid molecule into a host cell can be accomplished by any method by
which a nucleic acid molecule can be inserted into the cell.
Transfection techniques include, but are not limited to,
transfection, electroporation, microinjection, lipofection,
adsorption, and protoplast fusion. Preferred methods to transfect
host cells in vivo include lipofection and adsorption.
[0179] A recombinant cell of the present invention comprises a cell
transfected with a nucleic acid molecule that encodes HSP60, p277
or an analog, fragment or variant thereof.
[0180] It may be appreciated by one skilled in the art that use of
recombinant DNA technologies can improve expression of transfected
nucleic acid molecules by manipulating, for example, the number of
copies of the nucleic acid molecules within a host cell, the
efficiency with which those nucleic acid molecules are transcribed,
the efficiency with which the resultant transcripts are translated,
and the efficiency of post-translational modifications. Recombinant
techniques useful for increasing the expression of nucleic acid
molecules of the present invention include, but are not limited to,
operably linking nucleic acid molecules to high-copy number
plasmids, integration of the nucleic acid molecules into one or
more host cell chromosomes, addition of vector stability sequences
to plasmids, substitutions or modifications of transcription
control signals (e.g., promoters, operators, enhancers),
substitutions or modifications of translational control signals
(e.g., ribosome binding sites, Shine-Dalgarno sequences),
modification of nucleic acid molecules of the present invention to
correspond to the codon usage of the host cell, and deletion of
sequences that destabilize transcripts. The activity of an
expressed recombinant peptide of the present invention may be
improved by fragmenting, modifying, or derivatizing nucleic acid
molecules encoding such a peptide.
[0181] The pharmaceutical composition of the invention is
administered to a subject in need of said treatment in a
therapeutically effective amount. According to the present
invention, a "therapeutically effective amount" is an amount that
when administered to a patient is sufficient to inhibit, preferably
to eradicate, a T cell mediated pathology, or in other embodiments,
the T-cell mediated hepatic disorder or symptoms thereof. In a
preferred embodiment, the subject is human.
[0182] In another embodiment of the present invention, a
therapeutic composition further comprises a pharmaceutically
acceptable carrier. As used herein, a "carrier" refers to any
substance suitable as a vehicle for delivering a nucleic acid
molecule of the present invention to a suitable in vivo or in vitro
site. As such, carriers can act as a pharmaceutically acceptable
excipient of a therapeutic composition containing a nucleic acid
molecule of the present invention. Preferred carriers are capable
of maintaining a nucleic acid molecule of the present invention in
a form that, upon arrival of the nucleic acid molecule to a cell,
the nucleic acid molecule is capable of entering the cell and being
expressed by the cell. Carriers of the present invention include:
(1) excipients or formularies that transport, but do not
specifically target a nucleic acid molecule to a cell (referred to
herein as non-targeting carriers); and (2) excipients or
formularies that deliver a nucleic acid molecule to a specific site
in a subject or a specific cell (i.e., targeting carriers).
Examples of non-targeting carriers include, but are not limited to
water, phosphate buffered saline, Ringer's solution, dextrose
solution, serum-containing solutions, Hank's solution, other
aqueous physiologically balanced solutions, oils, esters and
glycols. Aqueous carriers can contain suitable auxiliary substances
required to approximate the physiological conditions of the
recipient, for example, by enhancing chemical stability and
isotonicity.
[0183] Suitable auxiliary substances include, for example, sodium
acetate, sodium chloride, sodium lactate, potassium chloride,
calcium chloride, and other substances used to produce phosphate
buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances
can also include preservatives, such as thimerosal, m- and
o-cresol, formalin and benzol alcohol. Preferred auxiliary
substances for aerosol delivery include surfactant substances
non-toxic to a subject, for example, esters or partial esters of
fatty acids containing from about six to about twenty-two carbon
atoms. Examples of esters include, caproic, octanoic, lauric,
palmitic, stearic, linoleic, linolenic, olesteric, and oleic acids.
Other carriers can include metal particles (e.g., gold particles)
for use with, for example, a biolistic gun through the skin.
Therapeutic compositions of the present invention can be sterilized
by conventional methods.
[0184] Targeting carriers are herein referred to as "delivery
vehicles". Delivery vehicles of the present invention are capable
of delivering a therapeutic composition of the present invention to
a target site in a subject. A "target site" refers to a site in a
subject to which one desires to deliver a therapeutic composition.
Examples of delivery vehicles include, but are not limited to,
artificial and natural lipid-containing delivery vehicles. Natural
lipid-containing delivery vehicles include cells and cellular
membranes. Artificial lipid-containing delivery vehicles include
liposomes and micelles. A delivery vehicle of the present invention
can be modified to target to a particular site in a subject,
thereby targeting and making use of a nucleic acid molecule of the
present invention at that site. Suitable modifications include
manipulating the chemical formula of the lipid portion of the
delivery vehicle and/or introducing into the vehicle a compound
capable of specifically targeting a delivery vehicle to a preferred
site, for example, a preferred cell type. Specifically targeting
refers to causing a delivery vehicle to bind to a particular cell
by the interaction of the compound in the vehicle to a molecule on
the surface of the cell. Suitable targeting compounds include
ligands capable of selectively (i.e., specifically) binding another
molecule at a particular site. Examples of such ligands include
antibodies, antigens, receptors and receptor ligands. For example,
an antibody specific for an antigen found on the surface of a
target cell can be introduced to the outer surface of a liposome
delivery vehicle so as to target the delivery vehicle to the target
cell. Manipulating the chemical formula of the lipid portion of the
delivery vehicle can modulate the extracellular or intracellular
targeting of the delivery vehicle. For example, a chemical can be
added to the lipid formula of a liposome that alters the charge of
the lipid bilayer of the liposome so that the liposome fuses with
particular cells having particular charge characteristics.
[0185] A preferred delivery vehicle of the present invention is a
liposome. A liposome is capable of remaining stable in a subject
for a sufficient amount of time to deliver a nucleic acid molecule
of the present invention to a preferred site in the subject. A
liposome of the present invention is preferably stable in the
subject into which it has been administered for at least about 30
minutes, more preferably for at least about 1 hour and even more
preferably for at least about 24 hours.
[0186] A liposome of the present invention comprises a lipid
composition that is capable of targeting a nucleic acid molecule of
the present invention to a particular, or selected, site in a
subject. Preferably, the lipid composition of the liposome is
capable of targeting to any organ of a subject, more preferably to
the lung, liver, spleen, heart brain, lymph nodes and skin of a
subject.
[0187] A liposome of the present invention comprises a lipid
composition that is capable of fusing with the plasma membrane of
the targeted cell to deliver a nucleic acid molecule into a cell.
Preferably, the transfection efficiency of a liposome of the
present invention is about 0.5 microgram (.mu.g) of DNA per 16
nanomole (nmol) of liposome delivered to about 10.sup.6 cells, more
preferably about 1.0 .mu.g of DNA per 16 nmol of liposome delivered
to about 10.sup.6 cells, and even more preferably about 2.0 .mu.g
of DNA per 16 nmol of liposome delivered to about 10.sup.6
cells.
[0188] A preferred liposome of the present invention is between
about 100 and 500 nanometers (nm), more preferably between about
150 and 450 nm and even more preferably between about 200 and 400
nm in diameter.
[0189] Suitable liposomes for use with the present invention
include any liposome. Preferred liposomes of the present invention
include those liposomes standardly used in, for example, gene
delivery methods known to those of skill in the art. More preferred
liposomes comprise liposomes having a polycationic lipid
composition and/or liposomes having a cholesterol backbone
conjugated to polyethylene glycol.
[0190] Complexing a liposome with a nucleic acid molecule of the
present invention can be achieved using methods standard in the
art. A suitable concentration of a nucleic acid molecule of the
present invention to add to a liposome includes a concentration
effective for delivering a sufficient amount of nucleic acid
molecule to a cell such that the cell can produce sufficient
therapeutic protein or peptide to regulate effector cell immunity
in a desired manner. Preferably, from about 0.1 .mu.g to about 10
.mu.g of nucleic acid molecule of the present invention is combined
with about 8 nmol liposomes, more preferably from about 0.5 .mu.g
to about 5 .mu.g of nucleic acid molecule is combined with about 8
nmol liposomes, and even more preferably about 1.0 .mu.g of nucleic
acid molecule is combined with about 8 nmol liposomes.
[0191] Another preferred delivery vehicle comprises a recombinant
virus particle vaccine. A recombinant virus particle vaccine of the
present invention includes a therapeutic composition of the present
invention, in which the recombinant molecules contained in the
composition are packaged in a viral coat that allows entrance of
DNA into a cell so that the DNA is expressed in the cell. A number
of recombinant virus particles can be used, including, but not
limited to, those based on alphaviruses, poxviruses, adenoviruses,
herpesviruses, arena virus and retroviruses.
[0192] In order to treat a subject with disease, a therapeutic
composition of the present invention is administered to the subject
in an effective manner such that the composition is capable of
treating that subject from disease. For example, a recombinant
molecule, when administered to a subject in an effective manner, is
able to stimulate effector cell immunity in a manner that is
sufficient to alleviate the disease afflicting the subject.
According to the present invention, treatment of a disease refers
to alleviating a disease and/or preventing the development of a
secondary disease resulting from the occurrence of a primary
disease. An effective administration protocol (i.e., administering
a therapeutic composition in an effective manner) comprises
suitable dose parameters and modes of administration that result in
treatment of a disease. Effective dose parameters and modes of
administration can be determined using methods standard in the art
for a particular disease. Such methods include, for example,
determination of survival rates, side effects (i.e., toxicity) and
progression or regression of disease.
[0193] In accordance with the present invention, a suitable single
dose size is a dose that is capable of treating a subject with
disease when administered one or more times over a suitable time
period. Doses can vary depending upon the disease being treated.
Doses of a therapeutic composition of the present invention
suitable for use with direct injection techniques can be used by
one of skill in the art to determine appropriate single dose sizes
for systemic administration based on the size of a subject. A
suitable single dose of a therapeutic composition to treat a T-cell
mediated pathology is a sufficient amount of recombinant sequence
to reduce, and preferably eliminate, the T-cell mediated pathology
following transfection of the recombinant molecules into cells. A
preferred single dose of HSP60, p277 or fragments and analogs
thereof encoding recombinant molecule is an amount that, when
transfected into a target cell population leads to the production
of from about 250 femtograms (fg) to about 1 .mu.g, preferably from
about 500 fg to about 500 picogram (pg), and more preferably from
about 1 pg to about 100 pg of p277 per transfected cell.
[0194] A preferred single dose of HSP60, p277 or fragments and
analogs thereof-encoding recombinant molecule complexed with
liposomes, is from about 100 .mu.g of total DNA per 800 nmol of
liposome to about 2 mg of total recombinant molecules per 16
micromole (.mu.mol) of liposome, more preferably from about 150
.mu.g per 1.2 .mu.mol of liposome to about 1 mg of total
recombinant molecules per 8 .mu.mol of liposome, and even more
preferably from about 200 .mu.g per 2 .mu.mol of liposome to about
400 .mu.g of total recombinant molecules per 3.2 .mu.mol of
liposome.
[0195] A preferred single dose of HSP60, p277 or fragments and
analogs thereof-encoding recombinant molecule in a non-targeting
carrier to administer to a subject, is from about 12.5 .mu.g to
about 30 mg of total recombinant molecules per kg body weight, more
preferably from about 25 .mu.g to about 10 mg of total recombinant
molecules per kg body weight, and even more preferably from about
125 .mu.g to about 3 mg of total recombinant molecules per kg body
weight.
[0196] It will be obvious to one of skill in the art that the
number of doses administered to a subject is dependent upon the
extent of the disease and the response of an individual patient to
the treatment. Thus, it is within the scope of the present
invention that a suitable number of doses includes any number
required to cause regression of a disease. A preferred protocol is
monthly administrations of single doses (as described above) for up
to about 1 year. A preferred number of doses of a therapeutic
composition comprising a recombinant molecule of the invention in a
non-targeting carrier or complexed with liposomes is from about 1
to about 10 administrations per patient, preferably from about 2 to
about 8 administrations per patient, and even more preferably from
about 3 to about 5 administrations per person. Preferably, such
administrations are given once every 2 weeks until signs of
remission appear, then once a month until the disease is gone.
[0197] A therapeutic composition is administered to a subject in a
fashion to enable expression of the administered recombinant
molecule of the present invention into a curative protein in the
subject to be treated for disease. A therapeutic composition can be
administered to a subject in a variety of methods including, but
not limited to, local administration of the composition into a site
in a subject, and systemic administration.
[0198] Therapeutic compositions to be delivered by local
administration include: (a) recombinant molecules of the present
invention in a non-targeting carrier (e.g., as "naked" DNA
molecules, such as is taught, for example in Wolff et al., 1990);
and (b) recombinant molecules of the present invention complexed to
a delivery vehicle of the present invention. Suitable delivery
vehicles for local administration comprise liposomes. Delivery
vehicles for local administration can further comprise ligands for
targeting the vehicle to a particular site.
[0199] Therapeutic compositions useful in systemic administration,
include recombinant molecules of the present invention complexed to
a targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
liposomes comprising ligands for targeting the vehicle to a
particular site. Systemic administration is particularly
advantageous when organs, in particular difficult to reach organs
(e.g., heart, spleen, lung or liver) are the targeted sites of
treatment.
[0200] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art. Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
1992, which is incorporated herein by reference in its entirety).
Oral delivery can be performed by complexing a therapeutic
composition of the present invention to a carrier capable of
withstanding degradation by digestive enzymes in the gut of a
subject. Examples of such carriers include plastic capsules or
tablets, such as those known in the art. Topical delivery can be
performed by mixing a therapeutic composition of the present
invention with a lipophilic reagent (e.g., DMSO) that is capable of
passing into the skin.
[0201] T Cell Vaccination
[0202] In another aspect, the invention provides a pharmaceutical
composition comprising attenuated activated T cells exposed ex vivo
to a compound selected from a group consisting of: p277 (SEQ ID
NO:1), analogs, variants, derivatives and salts thereof.
[0203] In certain embodiments, at least one of the cysteine
residues in positions 6 and 11 of p277 has been substituted. In
another embodiment, the p277 analog is p277(Val.sup.6Val.sup.11)
(SEQ ID NO:2). In another embodiment, the p277 analog is
p277(Ser.sup.6Ser.sup.11) (SEQ ID NO:3).
[0204] According to this aspect, such T cell vaccines (TCV)
preferably include cell vaccines in which allogeneic (i.e., cells
derived from a source other than a patient, but that are
histocompatible with the patient) or autologous (i.e., cells
isolated from a patient) cells are activated in vitro to induce
Major Histocompatibility Complex (MHC) II expression, exposed to
p277 or analogs, variants, derivatives and salts thereof contained
in a therapeutic composition, attenuated and administered to a
patient by, for example, intradermal, intravenous or subcutaneous
injection. In one embodiment, the patient is human.
[0205] Suitable antigen-nonspecific agents capable of activating T
cells are known in the art and include, but are not limited to,
mitogens such as concanavalin A, phytohemagglutinin, and pokeweed
mitogen. Additional activating agents are antibodies to T
cell-surface structures, including but not limited to, antibodies
to the CD3 cell-surface molecule, antibodies to the CD2
cell-surface molecule, antibodies to the CD28 cell-surface
molecule, and the natural ligands of CD2 or CD28. Other activating
agents include phorbol esters, such as phorbol myristate acetate,
or a combination of a phorbol ester and a calcium ionophore, such
as ionomycin. Also intended as T cell activating agents are
antibodies to the T cell receptor chains. Upon activation by such
agents, T cells up regulate various surface markers, including, but
not limited to major histocompatibility complex (MHC) II, and may
express p277 epitopes in the context of MHC II, as disclosed
herein.
[0206] The T lymphocyte activation step of the present invention
may or may not include the addition of T cell growth factors or
stimulatory factors, such as, for example, IL-1, IL-2 or IL-4, to
the culture medium for part or all of the activation interval.
[0207] Treatment to attenuate the T lymphocytes, may include, but
is not limited to, gamma- or X-irradiation, or treatment with
mitomycin C, by methods well known in the art, may also be used
according to the invention (Ben-Nun, et al., 1987, Holoshitz et
al., 1983). In one particular embodiment, the cells are attenuated
by exposure to gamma irradiation (2000-10000 rads).
[0208] In another aspect, the invention provides methods of
treating or preventing a T cell mediated pathology in a subject in
need thereof, comprising: (a) isolating T cells from the subject or
from a donor histocompatible with said subject; (b) activating the
T cells ex vivo to induce Major Histocompatibility Complex (MHC) II
expression; (c) exposing said activated cells to a compound
selected from a group consisting of: p277 (SEQ ID NO:1), analogs,
variants, derivatives and salts thereof; (d) attenuating said T
cells; and (e) introducing said cells into the subject in an amount
sufficient to induce an anti-ergotypic response in said
subject.
[0209] In certain embodiments, at least one of the cysteine
residues in positions 6 and 11 of p277 has been substituted. In
another embodiment, the p277 analog is p277(Val.sup.6Val.sup.11)
(SEQ ID NO:2). In another embodiment, the p277 analog is
p277(Ser.sup.6Ser.sup.11) (SEQ ID NO:3).
[0210] Effective amounts of cells to be introduced into the subject
may be extrapolated from animal model test bioassays or systems.
Suitable amounts of attenuated p277-loaded T cells are preferably
between 10.sup.6-10.sup.8 cells per administration.
[0211] In other aspects, the invention provides T cell vaccine
compositions and methods thereof using adoptive transfer of
p277-specific anti-ergotypic cells.
[0212] The generation of antigen-specific cell lines is within the
abilities of those of skill in the art, and is currently being
applied for the development of therapeutic TCV (see, for example,
Achiron et al., 2004). For the generation of p277-specific
anti-ergotypic cells suitable for adoptive transfer TCV, a first
population of T cells is activated by incubation in the presence of
a second population of p277-loaded attenuated activated T cells as
described above. Such attenuated T cells may be incubated with p277
or analogs, variants, derivatives and salts thereof prior to
incubation with the first T cell population, or alternatively be
incubated with the first T cell population in the presence of p277
or analogs, variants, derivatives and salts thereof. p277-specific
anti-ergotypic T cells present in the first population recognize
p277 epitopes presented on MHC II molecules of the second activated
T cell population. It is to be understood, therefore, that both T
cell populations used are histiotype compatible (histocompatible)
with each other as well as with the subject in need of said
treatment. Advantageously, this activation step is repeated at
least once (and is typically performed 2-3 times), in order to
enrich the resulting T cell population for the desired
p277-specific anti-ergotypic T cells. The method may optionally
further comprise one or more steps of expanding the resulting
p277-specific anti-ergotypic-enriched T cell population, e.g. by
culturing in the presence of IL-2. The resulting T cell population
is then administered to said subject in an amount sufficient to
induce an anti-ergotypic response in said subject. Suitable amounts
of p277 specific anti-ergotypic-enriched T cells are preferably
between 10.sup.7-3.times.10.sup.7 cells per administration.
[0213] In one embodiment, T cells are the majority of the cells
used to produce the T cell vaccines according to the invention. In
some embodiments, the T cells are substantially pure of other
antigen presenting cells. In various embodiments, the T cell
populations used in accordance with the present invention contain
at least 90%, preferably at least 95% and more preferably at least
97% T cells of the total cell population.
[0214] The use of HSP60, active fragments thereof, p277, analogs,
variants, derivatives and salts thereof for the preparation of a T
cell vaccine for the treatment of hepatitis or liver damage
associated therewith is further disclosed by the present
invention.
[0215] In another aspect, the invention provides methods of
treating hepatitis or liver damage associated therewith in a
subject in need thereof, comprising: (a) isolating T cells from the
subject or from a donor histocompatible with said subject; (b)
activating the T cells ex vivo to induce Major Histocompatibility
Complex (MHC) II expression; (c) exposing said activated cells to a
compound selected from a group consisting of: HSP60, an active
fragment thereof, p277 (SEQ ID NO:1), analogs, variants,
derivatives and salts thereof; (d) attenuating said T cells; and
(e) introducing said T cells into the subject in an amount
sufficient to induce an anti-ergotypic response in said
subject.
[0216] In another aspect, the invention provides methods of
treating hepatitis or liver damage associated therewith in a
subject in need thereof, comprising: (a) isolating a first
population of T cells from the subject or from a donor
histocompatible with said subject; (b) culturing the first
population of T cells in the presence of a second population of
histocompatible attenuated activated T cells and a compound
selected from a group consisting of: HSP60, an active fragment
thereof, p277 (SEQ ID NO:1), analogs, variants, derivatives and
salts thereof; and (C) introducing said first population of T cells
into the subject in an amount sufficient to induce an
anti-ergotypic response in said subject.
[0217] It is to be noted that the compositions and methods of the
present invention do not include the obligatory presence of a
second peptide capable of eliciting a reaction via a T cell
receptor conjugated to p277 to form a "dual-effect ligand", as
disclosed in WO 03/070761.
[0218] The following examples are presented in order to more fully
illustrate some embodiments of the invention. They should, in no
way be construed, however, as limiting the broad scope of the
invention.
EXAMPLES
A. P277 is Presented by Activated T Cells to Anti-Ergotypic
Regulatory T Cells
[0219] Rats.
[0220] Female Lewis rats were raised and maintained under
pathogen-free conditions in the Animal Breeding Center of this
institute. Experiments were carried out under the supervision and
guidelines of the Animal Welfare Committee. The rats were 1-2
months old at the start of the experiments.
[0221] Antigens, Peptides, Antibodies and Adjuvants.
[0222] M. tuberculosis (Mt) strain H37Ra was obtained from Difco
(Detroit, Mich., USA). Mt purified protein derivative (PPD) was
provided by the Statens Seruminstitut (Copenhagen, Denmark).
Recombinant mycobaterial 65 kDa HSP (HSP65) was kindly provided by
Dr. Ruurd van der Zee (Institute of Infectious Diseases and
Immunology, Faculty of Veterinary Medicine, Utrecht, The
Netherlands). Recombinant HSP60 was prepared as described (Quintana
et al., 2000). Guinea pig myelin basic protein (MBP) was purchased
from Sigma (Rehovot, Israel). Two HSP65 peptides were used:
1\4076-190 (aa 176-190) EESNTFGLQLELTEG (Anderton et al., 1994, SEQ
ID NO:14) and Mt3 (aa 5-24) AYDEEARRGLERGLNALADA (Quintana et al.,
2003, SEQ ID NO:15). The Mt176-90 peptide used in this work
includes the 180-188 epitope (van Eden et al., 1988). Two peptides
derived from HSP60 were used in Examples 1-8: p277 (aa 437-460)
VLGGGCALLRCIPALDSLTPANED (SEQ ID NO:1) and Hu3 (aa 31-50)
KFGADARALMLQGVDLLADA (SEQ ID NO:16). Peptides were synthesized by a
standard Fmoc procedure, purified by reverse-phase HPLC and their
compositions confirmed by aa analysis. Concanavalin A (Con A) was
purchased from Sigma. Incomplete Freund's Adjuvant (IFA) was
purchased from Difco.
[0223] A monoclonal antibody reactive to rat TCR (clone R73) was
purified by us from the hybridoma. Monoclonal antibodies to MI-IC
class-I (MHC-I), MHC class-II RT1.B (MHC-II/RT1.B), MHC class-II
RT1.D (MHC-II/RT1.D), CD28, CD80 and CD86 were purchased from
Serotec (Oxford, UK). Purified rabbit anti-human HSP60 polyclonal
IgG antibodies were provided by Dr Gabriel Nussbaum (Department of
Immunology, The Weizmann Institute of Science, Israel).
[0224] T-Cell Lines and Clones
[0225] T-cell lines were raised and expanded using antigen
presenting cells (APC) and antigens or peptides as described (Mor
et al., 1992). Three Lewis rat T-cell lines were used in our
experiments: Anti-HSP60, raised against recombinant human HSP60
(human HSP60 is 97% identical to rat HSP60 at the aa level);
Anti-p277, raised against the p277 peptide of human HSP60 (96%
identical its rat counterpart at the aa level) and Anti-MBP, raised
against guinea pig MBP. For ergotypic stimulation, the A2b T-cell
clone, specific for the 180-188 epitope of HSP65 (van Eden et al.,
1988) was used; similar results were obtained when other rat T-cell
clones were used as targets. A2b expresses MHC-1 molecules
constitutively and CD80, CD86 and MHC-II molecules upon activation,
and can present peptide epitopes to T cells. Activated A2b cells
were used on day 3 of their stimulation, and resting A2b cells were
used on day 14-16 of their rest cycle, unless stated otherwise.
[0226] DNA and peptide vaccination. The vectors containing the
full-length cDNA of the human hsp60 gene (pHSP60) or the cDNA
corresponding to aa 1-140 (pI) or aa 130-260 (pII) have been
previously described (Quintana et al.,. 2002, Quintana et al.,
2002b, Quintana et al.,. 2003, Quintana et al.,. 2000). The vector
coding for mycobacterial HSP65 (pHSP65) was kindly provided by Dr.
Douglas Lowrie (Medical Research Council, London, UK) (Ragno et
al., 1997). The empty vector pcDNA3 was used as a DNA vaccination
control.
[0227] Plasmid DNA was prepared in large scale and injected after
pretreatment with cardiotoxin (Sigma) as previously described
(Quintana et al.,. 2002b). Briefly, rats were vaccinated in the
quadriceps three times (on days -40, -26 -12 relative to AA
induction) with 150 .mu.g of pcDNA3, pHSP65 or pHSP60. Endotoxin
levels were checked by the Limulus amoebocyte lysate assay and
found always to be under acceptable levels for in vivo use (less
than 0.02 EU/.mu.g DNA).
[0228] Female Lewis rats were immunized intraperitoneally (ip) with
a single dose of 100 .mu.g of peptide emulsified in IFA. AA was
induced 12 days after the completion of vaccination with DNA or
peptide.
[0229] AA Induction and Assessment.
[0230] AA was induced using heat-killed Mt strain H37Ra (Difco)
suspended in IFA, as described (Quintana et al.,. 2002b). The day
of AA induction was designated as day 0. Disease severity was
assessed by direct observation of all 4 limbs in each animal. A
relative score between 0 and 4 was assigned to each limb, based on
the degree of joint inflammation, redness and deformity; thus the
maximum possible score for an individual animal was 16. Arthritis
was also quantified by measuring hind limb diameter with a caliper.
Measurements were taken on the day of the induction of AA and 26
days later, at the peak of AA (Quintana et al., 2002b); the results
are presented as the mean.+-.SEM of the difference between the
values for hind limb diameter taken on days 0 and 26.
[0231] Anti-Ergotypic T-Cell Proliferation Assay.
[0232] T-cell lines or lymph node cells (LNC, prepared from
inguinal and popliteal lymph nodes) were cultured in
quadruplicates, 2.5.times.10.sup.5 per well, in round-bottom
microtiter wells (Nunc, Roskilde, Denmark). Activated or resting
A2b stimulator cells were irradiated (5000 R) and added to the test
cultures in 2-fold dilutions, starting from 10.sup.5 cells per
well, with no other APC. Con A (1.25 .mu.g/ml) was used as a
positive control for T-cell proliferation, and in some experiments
the cells were activated with immobilized anti-TCR antibodies as
described (Wang et al., 2002). Monoclonal antibodies, 10 .mu.g/ml,
were added where indicated to test for MHC restrictions or
co-stimulation requirements of the anti-ergotypic T cells. Cultures
were incubated for 72 hr at 37.degree. C. in 7% CO.sub.2, and
pulsed for the last 16 hr with 1 .mu.Ci/well of [methyl
.sup.3H]-thymidine (Amersham, Buckinghamshire, UK). The cultures
were harvested and cpm were determined using a beta counter. The
.DELTA.CPM was computed as the difference between the mean cpm of
wells containing activated or resting A2b stimulator cells to
control wells cultured with medium alone.
[0233] Cytokine Assays.
[0234] Supernatants were collected after 72 hr of stimulation with
test antigens or stimulator cells. Pharmingen's OPTEIA IL-10, IL-4
and IFN.gamma. kits (Pharmingen, San Diego, USA) and the TGF.beta.1
E.sub.max.RTM. ImmunoAssay System (Promega, Madison, USA) were used
to quantify cytokine release to culture supernatants, as previously
described (Quintana et al.,. 2002b). The lower limits of detection
for the experiments described in this paper were 15 pg/ml for
TGF.beta.1, IL-10, IL-4 and IFN.gamma..
[0235] Western Blotting.
[0236] Cell lysates of resting or activated T cells were prepared
by treatment for 15 minutes in the following lysis buffer: NP40 1%,
NaCl 0.9%, Tris 50 mM, EDTA 1 mM, PMSF 0.4 mM, pepstatin A 4
.mu.g/ml, leupeptin 4 .mu.g/ml and aprotinin 4 .mu.g/ml. The
lysates were centrifuged for 15 min at 14000 rpm and the protein
concentration in the supernatant was determined using a BCA protein
assay kit (Pierce, Rockford, Ill., USA). The lysates were subjected
to PAGE-SDS using a mini-gel apparatus (Bio-Rad Laboratories,
Hercules, Calif.); 100 .mu.g of each sample were loaded per well.
Two identical gels were run each time in parallel: one gel was
stained with Coomassie Brilliant Blue R-250 according to the
manufacturer's protocol (Bio-Rad) and the other was
electro-transferred to nitrocellulose membranes (Schleicher and
Schuell, Dassel, Germany).
[0237] The nitrocellulose membranes were washed with PBS and then
blocked for 1 hr with 2% bovine serum albumin (Sigma), 2.5% milk
powder (Bio-Rad), Tris (Sigma) pH 7.5 10 mM, NaCl 150 mM and 0.02%
thimerosal (Sigma). After washing with PBS/Tween 20 (PBST; 0.02%,
Sigma), the membranes were incubated in blocking solution for 2 hr
with HSP60-specific polyclonal antibodies. The membranes were
washed with PBST and incubated with a peroxidase-conjugated goat
anti-rabbit IgG (Jackson Immuno-Research, West Grove, Pa.) at a
1/10000 dilution in blocking solution for 1 hr. Finally, the
membranes were developed using the Western Blotting Luminol Reagent
(Santa Cruz Biotechnology Inc., Santa Cruz, Calif., USA), exposed
to X-ray film and quantified using the NIH Image 1.63 program
(National Institutes of Health, USA). Size was determined using
pre-stained broad-range protein standard markers (Bio-Rad).
[0238] Adoptive Transfer of Anti-Ergotypic T Cells.
[0239] Anti-p277 or Anti-MBP T cells were activated for 3 days in
culture. Blast cells were isolated using a LymphoPrep gradient
(Nycomed, Oslo, Norway), washed, and 5.times.10.sup.6 cells per rat
were injected ip. Three days later, AA was induced.
[0240] Statistical Significance.
[0241] The InStat 2.01 program was used for statistical analysis.
Student's t-test and the Mann-Whitney test were carried out to
assay significant differences between the different experimental
groups.
Example 1
DNA Vaccination with pHSP60 Activates Anti-Ergotypic Responses
[0242] The inventors have reported that DNA vaccination with the
hsp60 gene (pHSP60) or with its N-terminal fragments--constructs pI
or pII--induced HSP60-specific T cells and inhibited the
development of AA (Quintana et al.,. 2002b, Quintana et al.,.
2003). DNA vaccination with mycobacterial HSP65 (pHSP65) also
protected rats against AA (Ragno et al., 1997), but this
vaccination was significantly less effective than was vaccination
with self-HSP60 (Quintana et al.,. 2002b). Does protective HSP60
vaccination activate anti-ergotypic reactivity? To approach this
question, the anti-ergotypic T-cell responses in rats vaccinated
with pHSP60, pI, pII, pHSP65 or pcDNA3, 26 days after the induction
of AA, were studied. Lymph node cells (LNC) of the vaccinated rats
were incubated with irradiated activated or resting A2b T cells,
and proliferative responses were measured to different numbers of
A2b stimulator cells. FIG. 1A shows that vaccination with pHSP60
induced a proliferative anti-ergotypic T-cell response, which was
significantly (p<0.05) higher than that induced by pHSP65;
control vaccination with pcDNA3 was least effective. Moreover,
vaccination with the pI or pII constructs of HSP60 also induced a
significant (p<0.05) anti-ergotypic response compared to pcDNA3
(FIG. 1B). Using neutralizing antibodies, the inventors found that
the anti-ergotypic response induced by DNA vaccination included
both MHC-II (RT1.B) and MHC-I restricted T cells (FIG. 1C).
[0243] Note that the pHSP60 DNA vaccine also increased the response
to resting A2b T cells, but to a lower extent than to activated A2b
T cells (FIG. 1A). However, only activated A2b T cells induced
cytokine secretion--characterized by secretion of IFN.gamma. and
TGF.beta.1, but not of IL-10 or IL-4 (FIG. 1D).
Example 2
Peptide Hu3 of HSP60 Activates an Anti-Ergotypic Response
[0244] Vaccination with the HSP60 peptide Hu3 (aa 31-50) can also
inhibit AA (Quintana et al.,. 2003). Does effective HSP60-peptide
vaccination also induce an anti-ergotypic response? FIG. 2A shows
that vaccination with peptide Hu3 was significantly (p<0.05)
more effective in inducing an anti-ergotypic proliferative response
than was vaccination with the homologous, immunogenic Mt3 peptide
from mycobacterial HSP65. The anti-ergotypic proliferative response
induced by peptide Hu3 was also more focused in its MHC-II
restriction (FIG. 2B); recall that HSP60 DNA vaccination led to
anti-ergotypic proliferative responses that included both MHC-I and
MHC-II restricted T cells (FIG. 1C). The anti-ergotypic T cells
induced by Hu3 peptide vaccination secreted IFN.gamma. and
TGF.beta.1, but not IL-10 or IL-4 in response to activated A2b T
cells (FIG. 2C).
Example 3
T-Cell Activation Up-Regulates HSP60 Expression
[0245] The above results (FIGS. 1 and 2) indicated that the
inhibition of AA by HSP60 DNA or peptide vaccination was associated
with the induction of anti-ergotypic proliferative and cytokine
responses to activated, syngeneic T cells; but do epitopes of HSP60
function as ergotopes? Is HSP60 up-regulated and presented on
activated T cells to anti-ergotypic T cells? To study this
question, the inventors compared the expression of HSP60 in
activated or resting T cells by western blot. LNC were incubated
for 1, 2 or 3 days with the T-cell mitogen Con A, or left
untreated. Cell lysates were prepared at the end of the incubation,
standardized by protein content, and analyzed by western blot for
the expression of HSP60. As a positive control for the induction of
HSP60, LNC were also heat shocked for 30 minutes at 42.degree. C.
and allowed to recover for 4 hr at 37.degree. C. FIG. 3A shows that
T-cell activation with Con A or heat shock triggered a similar
increase in the expression levels of HSP60. No differences in total
protein content were seen when the different samples were analyzed
by PAGE-SDS.
[0246] The inventors also detected up-regulation of HSP60 following
activation of the T-cell clone A2b by its target peptide epitope
Mt176-90 but not by the control peptide Mt3 (FIG. 3B); no
differences in total protein were seen when the samples were
analyzed by PAGE-SDS. The up-regulation of HSP60 protein is in
agreement with previous studies done at the level of mRNA
expression (Ferris et al., 1988), and demonstrates that T-cell
activation by specific antigen leads to the up-regulation of
cellular HSP60.
Example 4
Activated T Cells Stimulate HSP60-Specific and p277-Specific T
Cells
[0247] Are HSP60 epitopes actually presented by activated T cells?
The inventors studied this question using HSP60-specific T-cell
lines as probes for HSP60-epitope presentation, and a control
T-cell line specific for MBP. Activated or resting A2b T cells were
irradiated to inhibit their proliferation, and their presentation
of HSP60 epitopes was probed with the test T-cell lines. FIG. 4A
shows that the Anti-HSP60 T cells proliferated upon incubation with
activated A2b; the response to resting A2b T-cells was marginal.
The reaction to HSP60 was specific; the Anti-MBP T cells failed to
respond to the A2b T cells, irrespective of their state of
activation. Thus, only the activated A2b T cells presented HSP60
epitopes recognizable by the Anti-HSP60 line. The proliferation of
the Anti-HSP60 line was restricted through the MHC-II/RT1.B
molecule (FIG. 4C).
[0248] The 437-60 region of HSP60 (contained in the HSP60 peptide
designated p277) is an immunodominant T-cell epitope in the Lewis
rat (Reizis et al., 1996). The inventors could therefore use an
Anti-p277 T-cell line to investigate whether activated A2b T cells
presented the defined HSP60 peptide epitope p277. Although less
than the Anti-HSP60 T-cell line (compare FIGS. 4A and 4B), the
Anti-p277 T cells showed a significant proliferation upon
incubation with activated A2b T cells (FIG. 4B), but not with
resting A2b T cells. This anti-ergotypic proliferative response was
MHC-II/RT1.B restricted (FIG. 4C). Thus, activated T cells can
present a specific epitope of their up-regulated HSP60 molecules;
that this occurs in the absence of any other APC, suggests that
activated T cells can process and present epitopes of their own
HSP60. Thus, T-cell presentation of HSP60 can reveal the state of
activity of a T cell.
[0249] To investigate how the anti-ergotypic response to HSP60
might function, the inventors analyzed the cytokines produced by
Anti-HSP60 and Anti-p277 T cells in response to either activated or
resting A2b T cells. FIG. 4D shows that both the Anti-HSP60 and
Anti-p277 T-cells secreted relatively small amounts of IFN.gamma.
and relatively high amounts of TGF.beta.1 upon stimulation with
activated A2b T cells only. The T cells did not secrete IL-10 or
IL-4.
Example 5
The Activation of HSP60-Specific Anti-Ergotypic T Cells Requires
Co-Stimulation
[0250] Complete T-cell activation is achieved when TCR-mediated
signaling is reinforced by signals originating from co-stimulatory
molecules such as CD28. CD28 interacts with CD80 and CD86 molecules
displayed on the surface the APC. Activated T cells and activated
A2b express CD80 and CD86 molecules on their surface. The inventors
therefore studied the need for CD80, CD86 and CD28 in the
activation of HSP60-specific T cells by activated T cells. LNC
prepared from pHSP60 vaccinated rats, or Anti-HSP60 T cells, were
stimulated with irradiated A2b T cells in the presence of blocking
antibodies to CD80, CD86 or CD28. FIG. 5 shows that incubation with
each one of these antibodies produced a significant inhibition in
the anti-ergotypic response of HSP60-specific T cells. Hence,
co-stimulation by way of CD80, CD86 and CD28 appears to be required
for the activation of anti-ergotypic HSP60-specific T cells by
activated T cells.
Example 6
p277-Specific Anti-Ergotypic T Cells Ameliorate AA
[0251] If HSP60-specific anti-ergotypic T cells are indeed
regulatory, then it should be possible to inhibit inflammatory
disease by adoptively transferring them. The inventors tested the
effects of Anti-p277 T cells on AA by transferring 10.sup.7 cells
to rats 3 days before the active induction of AA. As a control, the
Anti-MBP T-cell line was used. The rats were scored for signs of
arthritis, and the hind paw diameter was measured with a caliper on
day 26, the peak of AA (Quintana et al.,. 2002b). FIG. 6 shows that
the recipients of the Anti-p277 cells showed a significant
reduction in the signs of AA, both in terms of arthritis score and
of limb swelling. The Anti-MBP T cells had no effect on the
progression of AA (FIG. 6).
[0252] The arthritogenic T cells that drive AA have a Th1
phenotype. Accordingly, lymph node T cells from rats suffering from
AA secrete high levels of IFN.gamma. in response to in vitro
stimulation with the Mt176-90 peptide (Quintana et al.,. 2002b),
containing the pathogenic 180-88 T-cell epitope of the
mycobacterial 65 kDa HSP (van Eden et al., 1988). The inhibition of
AA achieved by vaccination with HSP60 or its peptides is reported
to be associated with a reduction in INF.gamma. production induced
by Mt176-90 (Quintana et al.,. 2002b, Quintana et al.,. 2003). The
inventors therefore isolated LNC from rats adoptively transferred
with Anti-p277 or Anti-MBP T cells, and studied the secretion of
IFN.gamma. upon stimulation with Mt176-90. FIG. 6C shows that the
transfer of Anti-p277 T cells led to a significant reduction in the
secretion of IFN.gamma. in response to the AA target peptide
Mt176-190. Thus, HSP60-specific T cells, demonstrating
anti-ergotype activity, down-regulate IFN.gamma. secretion by the
candidate pathogenic T cells at the time they adoptively
down-regulate AA.
Example 7
p277-Specific Anti-Ergotypic T Cells Modulate Effector T-Cells In
Vitro
[0253] The results obtained in the AA model demonstrated that
anti-ergotypic HSP60-specific T-cells can control effector T cells
by adoptive transfer in vivo. To further investigate the effect of
HSP60-specific anti-ergotypic T cells, the inventors tested whether
these T cells might be able to directly regulate in vitro the
IFN.gamma. secretion of LNC taken from rats on day 26, at the peak
of AA. LNC of rats with actively induced AA were prepared and
activated with Mt176-90 in the presence of the Anti-p277
anti-ergotypic T-cell line, or in the presence of the control
Anti-MBP T-cell line. T cells reactive with Mt176-90 have been
shown to transfer AA to irradiated naive Lewis rats. Co-incubation
with the Anti-p277 line, but not with the Anti-MBP line, led to a
significant decrease in the secretion of IFN.gamma. (FIG. 7A). The
inventors did not detect a concomitant induction of IL-10 (not
shown). Thus, anti-ergotypic T cells can directly control in vitro
the arthritogenic T-cell IFN.gamma. cytokine response.
[0254] FIG. 7 further shows that the decrease in the clinical signs
of AA was associated with an increased reactivity against
Mt-derived antigens. 5.times.10.sup.6 T cells of anti MBP or
anti-p277 lines were injected ip into naive Lewis rats three days
before the induction of AA. Twenty-six days later LNC were
collected and the proliferative responses to PPD, HSP65, Mt176-90
(B); HSP60, p277 or Hu3 (C) or antiergotypic (D) were studied. The
results are expressed as the mean.+-.SEM stimulation index of
quadruplicate cultures. Three independent experiments produced
similar results.
[0255] An increase in the T-cell response to HSP60 (FIG. 7C) and to
activated A2b (anti-ergotypic response, FIG. 7D), could also
detected. Thus, the decrease in the clinical signs of AA induced by
the transfer of the anti-p277 specific T-cell line is associated
with changes in the T-cell responses to Mt-derived antigens and to
HSP60.
Example 8
HSP60-Specific Regulators Become Anergic Following their Regulation
of Activated T Cells
[0256] The inventors have shown in the previous Examples that
HSP60-specific anti ergotypic T cells can recognize and
down-regulate arthritogenic T cells, in vitro and in vivo. However,
any regulatory mechanism has to be regulated; uncontrolled
down-regulation of immunity would be as detrimental to the organism
as uncontrolled autoimmunity. The inventors therefore studied
whether the stimulation of HSP60-specific anti-ergotypic T cells by
activated T cells might itself affect the regulators. In other
words, might the anti-ergotypic HSP60-specific T-cell lines be
affected differently by seeing their HSP60 epitopes presented by
activated T cells compared to recognizing HSP60 presented by
classical APC? To study this possibility, the Anti-p277 line was
incubated for 3 days with either irradiated APC and p277 peptide or
with irradiated, activated A2b T cells. The Anti-p277 T cells were
then recovered from the cultures, maintained for 4 additional days
in culture without APC or A2b T cells, and then stimulated with APC
and p277 peptide, with mitogenic Con A or immobilized anti-TCR.
FIG. 8 presents the outcome. It can be seen that culturing the
Anti-p277 line with activated A2b T cells rendered the Anti-p277
line anergic; the line now failed to proliferate in response to APC
and p277 or to either of the two mitogens (FIG. 8A). FIG. 8B shows
that the Anti-p277 T cells could still secrete IFN.gamma. (but not
TGF.beta.1, IL-10 or IL-4; not shown), despite their failure to
proliferate. The Anti-p277 line cells, however, went on to die in
vitro after their exposure to the activated A2b T cells. Thus, it
appears that the interaction of anti-ergotypic T-cell lines with
their target activated effector T cells leads to anergy and
eventual loss of the anti-ergotypic T cells; activated effector T
cells and regulator T cells can down-regulate each other. In
contrast, as known in the art, anti-ergotypic T cells can be
readily maintained in culture by APC and specific peptide
antigen.
B. P277 Activates Cytokine-Associated Negative Regulator SOCS3 in T
Cells
[0257] Reagents.
[0258] The following reagents and chemicals were purchased as
indicated: recombinant HSP60 (StressGen Biotechnologies; Victoria,
BC, Canada); RPMI-1640 (Gibco BRL; Paisley, UK); FCS, antibiotics,
sodium pyruvate (Biological Industries; Kibbutz Beit-Haemek,
Israel); fibronectin (FN; Chemicon; Temecula, Calif.);
SDF-1.alpha., (R&D Systems; Minneapolis, Minn.), phosphatase
inhibitor cocktail, PMB and PMB-agarose beads (Sigma-Aldrich;
Rehovot, Israel); AG9 and AG490 (Calbiochem; San-Diego, Calif.),
and Na.sub.2.sup.51[Cr]O.sub.4 (Amersham Pharmacia Biotech; Little
Chalfont, UK). Monoclonal antibodies (mAb): anti-human CXCR4 (clone
12G5; R&D Systems; MN); anti-SOCS3 (H-103; Santa-Cruz Biotech);
anti-TLR2 and TLR4 (eBioscience; San-Diego, Calif.), and anti-human
recombinant HSP60 (designated clone P5, IgM fraction; kindly
provided by F. Quintana, The Weizmann Institute of Science).
Antibodies anti-phosphorylated Pyk2 (clone py881) and
anti-phosphorylated ERK1/2 (Biosource; Camarillo, Calif.);
anti-total Pyk2 (clone N-19), anti-phosphorylated MLC (pMLC) and Ab
anti-MLC (FL-172), anti-phosphorylated STAT3 (B7), and anti-total
STAT3 (H-190) (Santa-Cruz Biotech; Santa-Cruz, Calif.); and
anti-total ERK1/2 (Sigma); anti-phosphorylated AKT (pAKT) and
anti-AKT (Cell Signaling Technology, Beverly Mass.). The
recombinant HSP60 (StressGen Biotechnologies; Victoria, BC, Canada)
used in this study contained less than 0.001 EU/ml (0.1 pg/ml) of
bacterial endotoxin, as determined using a kinetic-turbidimetric
LAL test method (Biological Industries, Kibutz Beit-Haemek,
Israel). The peptides used in this study were prepared using
standard FMOC chemistry as previously described (Raz et al., 2001).
The sequence of p277 that was used in the following Examples 9-15
is VLGGGVALLRVIPALDSLTPANED (p277(Val.sup.6Val.sup.11), SEQ ID
NO:2). The sequence of p30 is FNEETVSFWLRVPKVSASHLE, residue
947-967 of Tetanus toxoid (SEQ ID NO:12).
[0259] Human Cells.
[0260] T cells were purified from the peripheral blood of healthy
human donors (Blood Bank; Tel-Hashomer Hospital, Israel) as
previously described (Zanin-Zhorov et al., 2003b). Whole blood was
incubated (20 min, 22.degree. C.) with RosetteSep.TM. human T-cell
enrichment cocktail (StemCell Technologies, Vencouver, BC, Canada).
After which, unsedimented cells were loaded onto Lymphocyte
Separation Medium (ICN Biomedicals; Belgium), T cells were isolated
by density centrifugation, and washed with PBS. Purified cells
(>97% CD3.sup.+ T cells) thus obtained were cultured in RPMI
containing antibiotics and 10% heat-inactivated FCS. Human
umbilical cord vein endothelial cells (HUVEC) were isolated from
umbilical cord veins and cultured as described (Ponomaryov et al.,
2000). Primary cultures were serially passaged, and passages 2 and
3 were taken for experiments.
[0261] Mice and Mouse T Cells.
[0262] Female C57BL/6J were obtained from Harlan Olac (Bicester,
U.K.). TLR2-knockout mice on the C57BL/6J background were kindly
provided by Dr. S. Akira (Osaka University, Osaka, Japan).
CD3.sup.+ T cells were isolated by negative selection with
anti-mouse antibody cocktail (Pan T cell kit; Miltenyi Biotec; CITY
Germany). The labeled cells were then passed through separation
columns (MidiMACS columns, Miltenyi Biotec). The purified cells
(>97% T cells) were untreated or treated with HSP60 as
described. Similarly, T cells were purified from the lymph nodes of
BALB/c mice (females, 1.5 months of age).
[0263] T-Cell Migration Assay.
[0264] Migration of .sup.51[Cr]-labeled human and mouse T cells
through FN or endothelial cells to recombinant human SDF-1.alpha.
was examined in 48-well Transwell chemotaxis apparatus (5-.mu.m
pore filters, 6.5-mm diameter; Corning, N.Y.), as previously
described (Zanin-Zhorov et al., 2003b). For transendothelial
migration assays, 2.times.10.sup.4 cells of HUVEC were layered on
the filters and grown in 10% FCS M199 for 2 days before the
performance of each assay. Then, HUVEC were stimulated with
TNF-.alpha. (50 ng/ml) for 24 hr and washed.
[0265] Western Blotting of T-Cell Lysates.
[0266] T cells were incubated (24 hr, 37.degree. C.) in starvation
medium (RPMI medium without serum). Aliquots
(5.times.10.sup.6/sample) of the starved cells were preincubated
(for various intervals) with different concentrations of HSP60
prior to exposure to SDF-1.alpha. (100 ng/ml, 10 min, 37.degree.
C., tissue culture conditions). These reactions were terminated by
freezing the plates (-70.degree. C., 10 min). The plates were
thawed, and the cells solubilized by incubating (60 min, 4.degree.
C.) in lysis buffer [EDTA (0.5 mM), NaCL (150 mM), NaF (10 nM),
Tris pH 7.5 (25 mM), Triton X-100 (1%), PMSF (200 .mu.g/ml), and
phosphatase inhibitor cocktail (1%)]. Lysates were cleared by
centrifugation (30 min, 14.times.10.sup.3 rpm, 4.degree. C.), and
the resulting supernatants analyzed for protein content. Sample
buffer was added, the samples were boiled, and equal amounts of
proteins were separated by 10% SDS-PAGE and transferred to
nitrocellulose membranes. The membranes were blocked with TBST
buffer [Tris pH 7.5 (20 mM), NaCl (135 mM) and Tween 20 (0.1%)]
containing low-fat milk (5%), and probed with the following mAb in
the same buffer: anti-phosphorylated (p) ERK (0.2 .mu.g/ml),
anti-total (t) ERK (diluted 1:20,000), anti-pPyk2 (1.5 .mu.g/ml),
anti-tPyk2 (0.2 .mu.g/ml), anti-pAKT (diluted 1:1000), anti-tAKT
(diluted 1:1000), anti-pMLC (diluted 1:250), and anti-tMLC (diluted
1:1000), anti-pSTAT3 (diluted 1:250), and anti-STATS (diluted
1:500). Immunoreactive protein bands were visualized using a
horseradish peroxidase-conjugated goat anti-mouse Ab and the
enhanced ECL system. Phosphorylation levels of the 3-5 independent
experiments were estimated by densitometry, and an average
percentage of phosphorylation.+-.SD was calculated as OD of
pERK/tERK, or pPyk2/tPyk2, or pAKT/tAKT, pMLC/tMLC, or
pSTAT3/tSTAT3.times.100%.
[0267] Cell Morphology.
[0268] T cells (3.times.10.sup.6 cells/ml) were seeded in
flat-bottom, 24-well plates in 500 .mu.l of RPMI on coverslips
coated with FN (25 .mu.g/ml). Some T cells were pretreated (1 hr,
37.degree. C., in a 7.5% CO.sub.2 humidified atmosphere) with
anti-TLRs mAb (20 .mu.g/ml), and then with HSP60 (1 hr). After
which, SDF-1.alpha. (200 ng/ml) was added, and the cells allowed to
adhere (30 min, 37.degree. C., in a 7.5% CO.sub.2 humidified
atmosphere). The cells were then fixed in 3.7% paraformaldehyde and
permeabilized (5 min, room temperature) with 0.5% TritonX-100. For
actin visualization, cells were stained with rhodamine-labeled
phalloidin (diluted 1/100), and examined with a laser-scanning
confocal microscope (LSM510, Zeiss).
[0269] In Vivo Homing Assay.
[0270] Purified human T cells were incubated (1 hr, tissue culture
conditions) with HSP60 (1 mg/ml) in tissue culture conditions. The
cells were then washed with PBS, and, where indicated, further
incubated (30 min, 4.degree. C.) with mouse anti-human CXCR4 mAb
(10 .mu.g/3.times.10.sup.6 cells), anti-human TLR2 or anti-TLR4 mAb
(each at 20 .mu.g/ml). The cells were then washed and injected
(5.times.10.sup.6 cells/0.5 ml per mouse) i.v. into the tails of
irradiated (375 cGY from a .sup.60[Co] source), 8-week-old NOD/SCID
mice, and the entrance of human T cells into the bone marrow of
recipient mice was evaluated as previously described (Rollet et
al., 2001). Each tested sample contained 1.5.times.10.sup.6
cells.
[0271] DTH Assays.
[0272] BALB/c mice (females; 1.5 months of age) were sensitized by
painting of their shaved abdominal walls with Oxazolone (2%)
emulsified in 100 .mu.l of acetone/olive oil (Sigma-Aldrich). On
day 5, mice were sacrificed, their draining (inguinal, mesenteric,
and cervical) lymph nodes were collected, and a single cell
suspension was prepared. The cells were treated with HSP60 (1
.mu.g/ml; 1 hr; tissue culture conditions), washed, and injected
(5.times.10.sup.7 cells per mouse) i.v. into the tails of naive
mice (at least 6 mice per group). Painting of the earlobes with
oxazolone (10 .mu.l of 0.5% in acetone/olive oil), which generates
a DTH response, was performed immediately after cell inoculation.
The area of the ears that was painted was measured before challenge
and 24 hr later with a micrometer. Changes in earlobe thickness are
indicative of DTH reactivity. All animal studies were performed in
compliance with and were approved by laws and animal welfare
guidelines of The Weizmaim Institute of Science (IACUC).
[0273] RNA Interference.
[0274] The inventors synthesized a silent RNA (siRNA) sequence
targeting SOCS3 position 80-101 relative to the start codon:
5'-AAGAGCGAGTACCAGCTGGTG-3' (SEQ ID NO:13); a double-stranded RNA
targeting luciferase (GL-2) was used as control (Dharmacon
Research). Transfections of freshly purified T cells were performed
using the Human T cell Nucleofector.TM. kit (Amaxa Biosystems,
Cologne, Germany). In brief, 5.times.10.sup.6 CD3.sup.+ T cell were
resuspended in 100 .mu.l of Human T Cell Nucleofector solution,
mixed with a total of 3 .mu.g of siRNA duplex, and pulsed using the
Nucleofector program U-14. Transfected cells were cultured in RPMI
containing 10% heat-inactivated FCS and 10 ng/ml of IL-2. Cell
migration was evaluated as described 48 h after transfection.
Transfection efficiency was controlled by evaluating SOCS3 levels
by Western Blotting.
Example 9
Exposure of T Cells to HSP60 for 1 Hr Inhibits SDF-1.alpha.-Induced
Chemotaxis Through Fibronectin and Endothelial Cells
[0275] Purified human T cells were incubated with various
concentrations of HSP60 for 1 hour and assayed the effect on
chemotaxis. The inventors observed a significant inhibition of
T-cell chemotaxis toward SDF-1.alpha. both through fibronectin
(FIG. 9A) and through TNF-.alpha.-activated endothelial cells (FIG.
9B). These findings suggested that HSP60 might decrease the
migration of T cells towards SDF-1.alpha. in vivo.
Example 10
HSP60 Decreases SDF-1.alpha.-Mediated Homing of Human T Cells to
the Bone Marrow of NOD/SCID Mice
[0276] Migration of T human cells in NOD/SCID mice is regulated
primarily by interactions of SDF-1.alpha., with CXCR4; following
exposure to sub-lethal doses of irradiation, the bone marrow of
these mice express high levels of SDF-1.alpha. (Kollet et al.,
2001). Therefore, the inventors examined the homing of
HSP60-treated human T cells into the bone marrow of irradiated
NOD/SCID mice. T cells were pretreated with HSP60 (1 .mu.g/ml for 1
hr) and/or anti-CXCR4, anti-TLR2, or anti-TLR4 mAb, and then the T
cells were injected into irradiated NOD/SCID mice. After 16 hr,
homing of the T cells to the bone marrow of the mice was determined
by assessing the expression of human CD45 and CXCR4 on bone marrow
cells.
[0277] T-cell homing into mouse bone marrow was significantly
abrogated by pre-treatment with anti-CXCR4 mAb (FIG. 9C); this
indicates that CXCR4--SDF-1.alpha. interactions are indeed involved
in the navigation of human T cells into this organ in vivo.
Exposure of T cells to HSP60 for 1 hr significantly (P<0.05)
decreased their homing to the bone marrow (FIG. 9C). Pre-treatment
of human T cells with neutralizing anti-TLR2 mAb, but not anti-TLR4
mAb (both are mouse IgG2a antibodies), abrogated the inhibitory
effect of HSP60 on T-cell homing into recipient bone marrow (FIG.
9D). Thus, the down-regulation of SDF-1.alpha.-induced human T-cell
chemotaxis and homing by HSP60 requires functional TLR2.
Example 11
HSP60 Inhibits SDF-1.alpha.-Induced Mouse T-Cell Chemotaxis In
Vitro and DTH In Vivo
[0278] Recently, SDF-1.alpha. was shown to be involved in the
recruitment of antigen-specific CD4.sup.+ T cells to sites of DTH
reactions in mice. Therefore, the inventors examined whether HSP60
can inhibit chemotaxis of mouse T cells in vitro and a T
cell-dependent DTH response in vivo. The effect of human HSP60 on
SDF-1.alpha.-mediated chemotaxis of BALB/c lymph node-cells in
vitro was similar to that on human T cells: HSP60 inhibited
migration of mouse lymph node cells towards SDF-1.alpha. (FIG.
10A). The inventors also studied the effect of human HSP60 on DTH
in mice. Exposure in vitro of oxazolone-reactive mouse lymph node
cells to HSP60 (1 .mu.g/ml for 1 hr) inhibited by 50% their ability
to adoptively transfer DTH in vivo (FIG. 10B). Thus, HSP60, which
inhibits SDF-1.alpha.-induced T-cell chemotaxis, appears capable of
down-regulating T-cell homing to and function at inflammatory sites
in vivo.
Example 12
HSP60 Effects on T Cell Signal Transduction
[0279] As can be seen in FIG. 11, HSP60 inhibits
SDF-1.alpha.-induced ERK (A-C), Pyk2 (D, E), and AKT
phosphorylation (F). Human T cells were exposed to HSP60 for 1 hr
(A, C--F) or to 1 ng of HSP60/ml for 0-120 min (B), washed and then
treated with SDF-1.alpha. (100 ng/ml, 10 min) (A-C, E, F) or not
(D). Lysates of these cells were immunoblotted with antibodies:
anti-phospho-ERK (pERK) and anti-total ERK (tERK) (A-C), anti-pPyk2
and anti-tPyk2 (D, E) or with anti-pAKT and anti-tAKT (F). C. T
cells pretreated with mAb anti-TLR2 or anti-TLR4 mAb (20 .mu.g/ml,
30 min) were then incubated with HSP60 (1 ng/ml, 1 hr), and washed
followed by SDF-1.alpha. (100 ng/ml, 10 min). The blot of 1
experiment representative of three (A, B) or five (C--F) is
presented. Phosphorylation levels of the experiments were estimated
by densitometry, and an average percentage of phosphorylation.+-.SD
was calculated as OD of pERK/tERK, or pPyk2/tPyk2, or
pAKT/tAKT.times.100%.
[0280] As can be seen in FIG. 12, HSP60 induces phosphorylation of
MLC (A), inhibits SDF-1 a-induced phosphorylation of MLC (B) and
polarized morphology (C) in T cells. Human T cells were treated
with HSP60 (1 hr; A) followed by SDF-1.alpha. (100 ng/ml, 10 min;
B). Cell lysates were immunoblotted with anti-pMLC and anti-tMLC
Ab. Phosphorylation levels of the five experiments were estimated
by densitometry, and an average percentage of phosphorylation was
calculated as the OD of pMLC/tMLC.times.100%. C. T cells were
plated onto FN-coated coverslips in the absence or presence of
HSP60 (1 .mu.g/ml). After 1 hr, some of the cells were exposed to
SDF-1.alpha. (100 ng/ml). Immunofluorescent staining for actin in
one experiment representative of 4 is shown.
[0281] Thus, inhibition by HSP60 of SDF-1.alpha.-induced
intracellular phosphorylation of signaling elements involved in
T-cell activation and chemotaxis is accompanied by an inhibition of
the morphological changes required for T-cell adhesion and
migration
Example 13
HSP60 Inhibits SDF-1.alpha.-Induced Activation and Migration of T
Cells by Up-Regulation of SOCS3
[0282] SOCS family proteins have been identified as negative
feedback regulators of cytokine-induced JAK/STAT activation,
through their binding to JAK kinases. Chemokine activation too, has
been shown to be regulated by SOCS proteins; up-regulation of SOCS3
inhibited CXCR4 signaling and blocked chemotaxis to SDF-1.alpha..
Since T-cell responses to SDF-1.alpha. are also attenuated by
HSP60, the inventors tested whether HSP60 too might induce SOCS3
activation. T cells were incubated (1 hr) with HSP60 (0-1000 ng/ml)
and intracellular activation of SOCS3 and tERK, which is
constitutively expressed, were determined by Western blotting. FIG.
13A shows that HSP60 activated the expression of SOCS3 (P<0.05).
The effects of human HSP60 are not restricted to human T cells:
HSP60 also induced the expression of SOCS3 in BALB/c lymph node
cells (FIG. 13B). These effects were also dose-dependent, like the
HSP60-induced expression of SOCS3 in human T cells (FIG. 13A). This
effect of HSP60 on SOCS3 expression in BALB/c lymphocytes also
explains the suppression of mouse T-cell chemotaxis to SDF-1.alpha.
noted above in vitro (FIG. 10A) and in vivo (induction of DTH; FIG.
10B).
[0283] The dose-response curve of the activation of SOCS3 in human
T cells was biphasic, displaying maximal activities at HSP60
concentrations of 1 ng/ml and 1 .mu.g/ml. With 1 ng/ml of HSP60,
activation was already apparent after 30 min and peaked at 1 to 2
hr (FIG. 13C).
[0284] To test whether TLR2 or TLR4 might be functionally involved
in the activation of SOCS3 by HSP60, the inventors assayed the
effect of pre-incubating T cells with blocking antibodies to the
TLR molecules. FIG. 13D shows that this activation of SOCS3 was
TLR2-dependent, since anti-TLR2 abrogated the activation by HSP60.
Moreover, the involvement of TLR2 was confirmed by using T cells
purified from TLR2-knockout and wild type C57BL/6J mice. FIG. 13E
shows that, as expected, treatment of wild type C57BL/6J-derived T
cells with HSP60 (1 ng/ml, 1 hr) induced SOCS3 expression at levels
similar to those induced by HSP60 in human T cells. In contrast,
SOCS3 expression remained at the low levels of untreated cells when
T cells from TLR2-knockout mice were treated with HSP60. Thus,
HSP60 induces SOCS3 expression in T cells via signaling through
TLR2.
[0285] Up-regulation of SOCS3 expression requires prior activation
of the JAK/STAT pathway. Therefore, the inventors tested the effect
of a specific blocker of JAK/STAT activation, AG490, on
HSP60-induced up-regulation of SOCS3 in human T cells. Pretreatment
(18 hr) of T cells with AG490 (10 nM), but not with its control
counterpart AG9, abrogated the effect of HSP60 on SOCS3 expression
(FIG. 13F), whereas pertussis toxin, a G.sub..alpha.i protein
inhibitor, had no effect (not shown). Furthermore, HSP60 induced
phosphorylation of STAT3 (FIG. 13G), which is essential for SOCS3
up-regulation (Alexander et al., 1999, Starr et al., 1997). The
dose-response curve of induction of STAT3 phosphorylation (FIG.
13G) was very similar to that of activation of SOCS3 by HSP60 (FIG.
13A). Thus, HSP60 induced SOCS3 expression through activation of
intracellular signaling involving up-regulation of STAT3
phosphorylation.
Example 14
Inhibition of SDF-1.alpha.-Induced T-Cell Chemotaxis by HSP60 is
Prevented By Silencing SOCS3 Gene Expression
[0286] To confirm the conclusion that the inhibitory effect of
HSP60 on SDF-1.alpha.-induced T-cell chemotaxis is mediated through
SOCS3 up-regulation, SOCS3 gene expression was specifically
silenced using RNA interference. This treatment abrogated the
induction of SOCS3 expression by HSP60; transfection with control
siRNA had no effect (FIG. 13H). Moreover, the inhibitory effect of
HSP60 on SDF-1.alpha.-induced T-cell chemotaxis was completely
prevented by specifically silencing the SOCS3 gene (FIG. 13I).
Example 15
Effects of HSP60 on SOCS3 Expression are not Due to Contamination
With LPS, and are Retained with p277
[0287] The LPS-TLR2 interaction results in intracellular signals,
and several batches of recombinant human HSP were shown to contain
minimal residual LPS and LPS-lipoproteins, which are biologically
active on macrophages. Using a kinetic-turbidimetric test method,
the inventors found that the recombinant human HSP60 used in this
study contained less than 0.001 EU/.mu.g protein (0.1 pg/.mu.g) of
bacterial endotoxin.
[0288] The following studies were performed to exclude the
possibility that such a minute amount of LPS could affect SOCS3
activation in T cells. First the inventors examined whether LPS
alone can affect SOCS3 expression by exposing T cells to increasing
concentrations of LPS. LPS up-regulates SOCS3 expression in human T
cells, but only at concentrations of 10-100 ng/ml (FIG. 14A), which
is 100-1000 fold higher than the amount of LPS in the HSP60
preparation used. Similarly, the inventors previously found that
1000 fold greater concentrations of LPS were needed to induce
T-cell adhesion to fibronectin, an effect which was still less than
that induced by human HSP60 (Zanin-Zhorov et al., 2003).
[0289] The exclusion of LPS in activation of SOCS3 by HSP60 was
further confirmed by using anti-HSP60 mAb, by boiling (which
denatures HSP60, but not LPS), and by using an LPS inhibitor,
polymyxin B (PMB). The results, shown in FIGS. 14, B and C,
demonstrate that activation of SOCS3 by HSP60 (0.1 ng/ml; upper
panel) was inhibited by boiling, but not by PMB (FIG. 14B), whereas
activation of SOCS3 by LPS (100 ng/ml; lower panel) was inhibited
by PMB, but not by boiling (FIG. 14C). Furthermore, anti-human
HSP60 mAb inhibited the activation of SOCS3 by HSP60, but not the
activation induced by LPS.
[0290] Recently, lipoproteins extracted from the LPS of Escherichia
coli were shown to activate macrophages via TLR-2. These
lipoproteins can be removed from LPS by passage through PMB-coupled
agarose beads. Pre-incubation with PMB-conjugated agarose beads did
not block the efficacy of our HSP60 preparation in inducing SOCS3
expression by T cells. In contrast, the efficacy of the LPS
preparation was abolished (FIG. 14D).
[0291] The inventors used p277 synthetic peptide to further rule
out the possibility that the effects of HSP60 on SOCS3 expression
could be due to contamination by LPS. Peptide p2.77 (1-1000 ng/ml),
but not the control p30 peptide of Tetanus toxoid, induced a marked
expression of activated SOCS3 in T cells (FIG. 14E). Thus, the
effect of our HSP60 preparation on SOCS3 expression was not due to
LPS.
C. D277 is a Co-Stimulator of CD4.sup.+CD25.sup.+ Regulatory T
Cells
[0292] Reagents.
[0293] The following reagents and chemicals were obtained as
indicated: RPMI-1640 (Gibco BRL; Paisley, UK), FCS, HEPES buffer,
antibiotics, sodium pyruvate (Biological Industries; Kibbutz
Beit-Haemek, Israel); phosphatase inhibitor cocktail, LPS
(Sigma-Aldrich, Rehovot, Israel). Monoclonal antibodies (mAb)
directed to CD4 and CD45RO were obtained from Serotec (Oxford, UK);
anti-CTLA4 (BNI3) was from BD Pharmingen (San Diego, Calif.),
PE-conjugated anti-CD25 was from Miltenyi Biotec (Bergisch
Gladbach, Germany). Monoclonal antibodies (mAb) anti-TLR2 and
anti-TLR4 were purchased from eBioscience (San-Diego, Calif.).
Antibodies anti-phosphorylated Pyk2 (clone py881),
anti-phosphorylated p38, and anti-phosphorylated ERK1/2 were
obtained from Biosource (Camarillo, Calif.); and anti-total ERK1/2
(Sigma); anti-phosphorylated AKT (pAKT) and anti-AKT (Cell
Signaling Technology, Beverly Mass.). Polyclonal Ab anti-total
ERK1/2 was obtained from Sigma (Rehovot, Israel); anti-NF-.kappa.B
p65 (A), anti-T-bet (39D), anti-total Pyk2 (clone N-19), anti-total
p38, and anti-Lamin B (C-20) were purchased from Santa-Cruz Biotech
(Santa-Cruz, Calif.). The anti-Foxp3 antibody (clone Poly6238) was
purchased from BioLegend (San-Diego, Calif.). The inhibitors of
intracellular protein kinases: GF109203X, were from LC Laboratories
(Woburn, Mass.); wortmanin, PD 98059, SB 203580, and Pertussis
Toxin were from Calbiochem (La Jolla, Calif.). Neutralizing
antibodies anti-IL-10 (clone 23738.11) and anti-TGF-.beta. (clone
9016) were purchased from R&D Systems (Minneapolis, Minn.). The
recombinant HSP60 (StressGen Biotechnologies; Victoria, BC, Canada)
used in this study contained less than 0.001 EU/ml (0.1 pg/ml) of
bacterial endotoxin, as determined using a kinetic-turbidimetric
LAL test method (Biological Industries, Kibutz Beit-Haemek,
Israel). The peptides used in this study were prepared using
standard FMOC chemistry as previously described (Raz et al., 2001).
The sequence of p277 that was used in the following Examples 16-25
is VLGGGVALLRVIPALDSLTPANED, (p277(Val.sup.6Val.sup.11), SEQ ID
NO:2). The sequence of MTp277 is VAGGGVTLLQAAPTLDELKLEG of HSP65 of
Mycobacterium tuberculosis (SEQ ID NO:10).
[0294] Purified T-Cell Populations.
[0295] T cells were purified from the peripheral blood of healthy
human donors (Blood Bank; Tel-Hashomer, Israel). The whole blood
was incubated (20 min, 22.degree. C.) with RosetteSep.TM. human
T-cell enrichment cocktail (CD3.sup.+ T cells), or with
RosetteSep.TM. human CD4.sup.+ and CD8.sup.+ T-cell enrichment
cocktail, (StemCell Technologies, Vencouver, BC, Canada). The
remaining un-sedimented cells were then loaded onto Lymphocyte
Separation Medium (ICN Biomedicals; Belgium), isolated by density
centrifugation, and washed with PBS. The purified cells (>97%
CD3.sup.+ T cells, >95% CD4.sup.+ T cells, >95% CD8.sup.+ T
cells) so obtained were cultured in RPMI containing antibiotics and
10% heat-inactivated FCS. In a second round of purification,
CD4.sup.+ T cells were separated to CD25.sup.- and CD25.sup.+
populations with magnetically coupled mAb against human CD25
(Miltenyi Biotec; Bergisch Gladbach, Germany). The purified cells
obtained (usually >90% CD4.sup.+CD25.sup.+ or >99%
CD4.sup.+CD25.sup.- T cells) were cultured in RPMI containing
antibiotics and 10% heat-inactivated FCS.
[0296] Cytokine Secretion.
[0297] Cytokine secretion was determined by ELISA as described in
section D of the Examples herein, using the appropriate mAb,
according to the manufacturer's instructions (Biosource; Camarillo,
Calif.).
[0298] Flow Cytometry.
[0299] Indicated populations of T cells (10.sup.6 cells per sample)
were stained (30 min, 4.degree. C.) with anti-CD25-PE,
anti-CD4-FITC, or anti-CD45RO-PE, washed with PBS (containing 0.05%
BSA and 0.05% sodium azide). For intracellular staining, cells were
fixed with 3% paraformaldehyde (20 min, at 25.degree. C.), and
washed. Cell-membranes were permeabilizied with 0.5% TritonX-100 in
PBS (10 min, at 25.degree. C.), washed, and stained (30 min,
4.degree. C.) with anti-CTLA4, anti-TLR2, or anti-TLR4 (20
.mu.g/ml), washed, and incubated (30 min, 4.degree. C.) with an
FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Lab.
Inc., West Grove, Pa.). After washing, T cell staining was analyzed
using a Becton Dickinson FACSort (Mountain View, Calif.) using the
Cell Quest software.
[0300] Co-Culture and Transwell Analysis.
[0301] In co-culture experiments, CD4.sup.+CD25.sup.+ T cells were
incubated with HSP60 (1 ng/ml, 2 hr), washed, and added at
different percentage (1%, 10%, and 25%) to CD4.sup.+CD25.sup.- T
cells. The cells were co-cultured on anti-CD3 mAb pre-coated
24-well plates for 24 hr (cytokine secretion), or 24 and 96 hr
(proliferation). In Transwell analysis, CD4.sup.+CD25.sup.+ and
CD4.sup.+CD25.sup.- T cells were cultured in Transwell plates
(Costar, Corning, N.Y.). Both chambers of each Transwell were
coated with anti-CD3 mAb (1 .mu.g/ml). The supernatants for
cytokine analysis were collected from both chambers after 24
hr.
[0302] CFSE Labeling of CD4.sup.+CD25.sup.- T Cells.
[0303] CD4.sup.+CD25.sup.- T cells were labeled with Vybrant.TM.
CFDA SE Cell Tracer Kit (Molecular Probes; Eugene, Oreg.) according
to the manufacturer's instructions. Labeled CD4.sup.+CD25.sup.- T
cells were co-cultured with CD4.sup.+CD25.sup.+ T cells on anti-CD3
mAb, as described above, for 6 hr. After co-culture,
CD4.sup.+CD25.sup.- T cells were isolated by sorting using a Becton
Dickinson FACSVantage (Mountain View, Calif.).
[0304] Proliferation Assay.
[0305] Proliferation was assessed by the 2,3
bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide
(XTT) assay (Mosmann, 1983).
[0306] Western Blot Analysis of T-Cell Lysates.
[0307] Indicated populations of T cells (5.times.10.sup.6) were
preincubated with 1 ng/ml of HSP60 for 10 min (37.degree. C. in a
7% CO.sub.2, humidified atmosphere). The cells were then washed and
replated in the same concentration on anti-CD3 mAb pre-coated
24-well plates for 4 hr (37.degree. C. in a 7% CO.sub.2, humidified
atmosphere). Total cell lysates, nuclear and cytoplasmic extracts
were prepared as previously described (Zanin-Zhorov et al., 2003,
section D of the Examples herein), analyzed for protein content.
Sample buffer was then added and, after boiling, the samples,
containing equal amounts of proteins, were separated on 10%
SDS-PAGE gel and transferred to nitrocellulose membranes. The
membranes were blocked and probed with the following mAb in the
same buffer: anti-phosphorylated (p) ERK (0.2 .mu.g/ml), anti-total
(t) ERK (diluted 1:20,000), anti-pPyk2 (1.5 .mu.g/ml), anti-tPyk2
(0.2 .mu.g/ml), anti-pAKT (diluted 1:1000), anti-tAKT (diluted
1:1000), anti-p38 (diluted 1:500), and anti-tp38 (diluted 1:1000),
anti-Foxp3 (diluted 1:250), anti-NF-.kappa.B (diluted 1:1000),
anti-T-bet (diluted 1:1000), and anti-Lamin B (diluted 1:1000).
Immunoreactive protein bands were visualized using a horseradish
peroxidase-conjugated goat anti-mouse Ab and the enhanced ECL
system. Phosphorylation levels of the 3-5 independent experiments
were estimated by densitometry.
[0308] Statistics.
[0309] Data were analyzed by Student's t-test. P<0.05 was
considered statistically significant.
Example 16
HSP60 Inhibits IFN.gamma. and TNF.alpha. Secretion and Up-Regulates
IL-10 Secretion in CD3.sup.+ and in CD4.sup.+ T Cells, but not in
CD8.sup.+ T Cells
[0310] The inventors analyzed the effects of HSP60 on the cytokine
secretion profile of freshly isolated and purified human CD3.sup.+,
CD4.sup.+, and CD8.sup.+ T cells that were activated by mitogenic
anti-CD3 in Ab. HSP60 alone (at a range of concentrations of
0.01-1000 ng/ml) did not induce cytokine secretion by the T cells.
However, when the CD3.sup.+ and CD4.sup.+ T cells were pre-treated
with different concentrations of HSP60, washed and then activated
by anti-CD3, the secretion of the Th1-related cytokines IFN-.gamma.
and TNF-.alpha. was inhibited by about 50% (FIGS. 15A and 15B),
while IL-10 secretion was enhanced 2-3 fold (FIG. 15C). In
contrast, CD8.sup.+ T cells were unresponsive to HSP60 at all
concentrations tested (FIG. 15). Recently, the inventors reported
that the biological effects of HSP60 on T cells manifested a
bi-phasic bell-shaped dose-response curve (Zanin-Zhorov et al.,
2003, Example 27 herein); here too the dose-response also
manifested a bi-phasic bell-shaped. Significant effects were
achieved with relatively low concentrations of HSP60 (0.1-1.0
ng/ml; P<0.05), while higher doses (in the order of 10 ng/ml)
did not affect cytokine secretion. However, cytokine secretion was
again affected significantly at higher concentrations of HSP60
(0.1-1 .mu.g/ml; P<0.05). Thus, HSP60 specifically affects
cytokine secretion in CD3.sup.+ and me, but not CD8.sup.+ T
cells.
Example 17
Depletion of CD4.sup.+CD25.sup.+ T Cells Prevents the Inhibitory
Effect of HSP60 on IFN-.gamma. and TNF-.alpha.Secretion in
CD4.sup.+CD25.sup.- T Cells
[0311] Cell surface staining of purified human CD4.sup.+ peripheral
blood T cells revealed that about 10% of these cells expressed the
CD25 marker (FIG. 16A). After purification, the CD4.sup.+CD25.sup.+
population expressed relatively high levels of intracellular CTLA4
and surface CD45RO markers, and transcription factor Foxp3 (FIG.
16B). To test whether CD4.sup.+CD25.sup.+ T cells are essential for
the inhibitory effect of HSP60 on T-cell cytokine secretion,
unseparated CD4.sup.+ T cells, purified CD4.sup.+CD25.sup.+ T
cells, or purified CD4'CD25.sup.- T cells were incubated with HSP60
(1 ng/ml, 2 hours), washed the cells, and then activated them using
mitogenic anti-CD3 mAbs for 24 hours. Depletion of the
CD4.sup.+CD25.sup.+ T cells completely prevented the inhibition of
IFN-.gamma. and TNF-.alpha. secretion by HSP60 treatment (FIG.
16C).
Example 18
HSP60 Induces IL-10 and TGF-.beta. Secretion in
CD4.sup.+CD25.sup.+, but not in CD4.sup.+CD25.sup.- T Cells
[0312] Although the relative contribution of the immunosuppressive
cytokines IL-10 and TGF-.beta. in the function of Tregs is
controversial, the supernatants described above were analyzed (FIG.
16C) for levels of IL-10 and TGF-.beta.. Treatment of un-separated
CD4.sup.+ or CD4.sup.+CD25.sup.+, but not of CD4.sup.+CD25.sup.- T
cells with HSP60 significantly up-regulated the secretion of IL-10
and TGF-.beta. induced by mitogenic anti-CD3 (FIG. 16C).
Example 19
Treatment of CD4.sup.+CD25.sup.+ T Cells with HSP60 Affects
Cytokine Secretion and Proliferation in Co-Culture with
CD4.sup.+CD25.sup.- T Cells
[0313] To confirm the involvement of CD4.sup.+CD25.sup.+ Tregs in
the effects of HSP60 on cytokine secretion, the inventors incubated
only this population with HSP60 and washed the cells. The
HSP60-treated CD4.sup.+CD25.sup.+ T cells were then co-cultured
with the CD4.sup.+CD25.sup.- T cells in different proportions on
mitogenic anti-CD3 for 24 hr, and the cytokine levels were
measured.
[0314] FIGS. 17A and 17B show that the co-culture of untreated
CD4.sup.+CD25.sup.+ T cells with the CD4.sup.+CD25.sup.- T cells
resulted in a relatively moderate effect on cytokine secretion.
However, HSP60 treatment of the CD4.sup.+CD25.sup.+ T cells
significantly enhanced the down-regulation of secretion of
IFN-.gamma. and TNF-.alpha. secretion and up-regulated the
secretion of IL-10 and TGF-.beta. in the co-cultures (FIG.
17A-17D).
Example 20
The Effects of HSP60 on T-Cell Proliferation were Measured in these
Co-Cultures after 24 and 96 Hours
[0315] HSP60 treatment of the CD4.sup.+CD25.sup.+ T cells did not
suppress anti-CD3-induced proliferation of the CD4.sup.+CD25.sup.-
T cells at 24 hours. However, at 96 hours there was a significant
inhibition of proliferation of the CD4.sup.+CD25.sup.- T cells
(FIG. 17E). Thus, HSP60 treatment of CD4.sup.+CD25.sup.+ T cells
enhanced their ability to inhibit both cytokine secretion and
proliferation in co-cultures with untreated CD4.sup.+CD25.sup.- T
cells activated by mitogenic anti-CD3.
Example 21
Treatment of CD4.sup.+CD25.sup.+ T Cells with HSP60 Affects
Cytokine Secretion in Co-Culture with CD8.sup.+ T Cells
[0316] The inventors found that HSP60 did not directly affect
cytokine secretion in cultures of CD8.sup.+ T cells free of
CD4.sup.+ T cells (FIG. 15). It has been reported, however, that
CD4.sup.+CD25.sup.+ T cells can suppress CD8.sup.+ T cells as well
as CD4.sup.+CD25.sup.- T cells. The inventors therefore co-cultured
purified CD8.sup.+ T cells with HSP60-treated or untreated
CD4.sup.+CD25.sup.+ T cells in different proportions on mitogenic
anti-CD3 for 24 hr, and measured the cytokine levels. FIGS. 18A and
18B show that, in contrast to CD4.sup.+CD25.sup.- T cells (FIGS.
17A and 17B), co-culture of CD8.sup.+ T cells with untreated
CD4.sup.+CD25.sup.+ T cells (in all proportions) did not affect
IFN-.gamma. or TNF-.alpha. secretion. However, the addition of 1%
or more HSP60-treated CD4.sup.+CD25.sup.+ T cells led to inhibition
of IFN-.gamma. or TNF-.alpha.cytokine secretion by 3-4 fold (FIGS.
18A and 18B). In addition, the secretion of IL-10 and TGF-.beta.
was up-regulated in the co-culture of HSP60-treated
CD4.sup.+CD25.sup.+ and CD8.sup.+ T cells. Thus, co-culture of
untreated CD8.sup.+ T cells with HSP60-treated CD4.sup.+CD25.sup.+
T cells significantly modulated the cytokine secretion profile;
HSP60 can affect CD8.sup.+ T cells indirectly through the
activation of CD4.sup.+CD25.sup.+ Tregs. However, in the studies
below of mechanisms and signal transduction events underling the
effects of HSP60 treatment on CD4.sup.+CD25.sup.+ T cells,
co-culture with the CD4.sup.+CD25.sup.- population was used as a
read-out.
Example 22
The Effects of HSP60 on CD4.sup.+CD25.sup.+ T Cells Depend on TLR2
Signaling
[0317] The inventors previously reported that HSP60 affects T-cell
behavior innately via TLR2 signaling (Zanin-Zhorov et al., 2003,
section D of the Examples herein). Here, whether TLR2 or TLR4 was
functionally involved in the effects of HSP60 on
CD4.sup.+CD25.sup.+ T cells was tested. CD4.sup.+CD25.sup.+ T cells
were pre-incubated with anti-TLR2 or TLR4 mAb (both are mouse IgG2a
antibodies) prior to activation with HSP60, and assayed the
cytokine secretion profile induced by anti-CD3 in the co-culture
(ratio 1:9) of the CD4.sup.+CD25.sup.+ T cells with untreated
CD4.sup.+CD25.sup.- T cells. The inventors found that the mAb to
TLR2, but not the mAb to TLR4, blocked the effects of HSP60 on the
down-regulation of IFN-.gamma. and TNF-.alpha. secretion (FIGS. 19A
and 19B) and the up-regulation of IL-10 secretion (FIG. 19C). Thus,
TLR2 appears to play a role in mediating the effects of HSP60 on
CD4.sup.+CD25.sup.+ Treg cells.
[0318] The inventors tested whether the
CD4.sup.+CD25.sup.+population expresses higher levels of TLR2 than
do CD4.sup.+CD25.sup.- T cells. In contrast to the results reported
in mouse cells, the inventors found by intracellular FACS staining
that CD4.sup.+CD25.sup.+ T cells were much richer in TLR2 than in
TLR4. However, no significant differences were found in TLR2 levels
between CD4.sup.+CD25.sup.- and CD4.sup.+CD25.sup.- T cells (FIG.
19D).
Example 23
The Effects of HSP60 on CD4.sup.+CD25.sup.+ T Cells are not Due to
Contaminating LPS
[0319] LPS-TLR2 interactions can transmit intracellular activation
signals in various types of leukocytes. The following studies were
performed to further exclude the possibility that even minute
amounts of LPS or other lipoprotein contaminants from the
Escherichia coli recombinant HSP60 might activate the
CD4.sup.+CD25.sup.+ T cells. The inventors incubated the HSP60 with
PMB-coupled agarose beads (Gao et al., 2003), collected the unbound
material, assayed the protein content, and tested the effect of the
PMB-treated HSP60 on CD4.sup.+CD25.sup.+ T cells. The inventors
found that the effects of HSP60 on CD4.sup.+CD25.sup.+ T cells were
completely inhibited by boiling (which denatures proteins, although
not LPS), but not by the PMB-beads (FIG. 19E-19G). Pre-incubation
with PMB-conjugated agarose beads completely abolished the efficacy
of LPS on TNF-.alpha. secretion in macrophages. Most importantly,
in contrast to its effects on mouse regulatory T cells (Caramalho
et al., 2003), the present inventors found that purified LPS did
not activate human CD4.sup.+CD25.sup.+ Treg cells (FIGS. 19E and
19F). Consequently, the effects of HSP60 on CD4.sup.+CD25.sup.+ T
cells could not be attributed to LPS.
Example 24
Treatment of CD4.sup.+CD25.sup.+ T Cells with HSP60-Derived Peptide
p277 Inhibits Cytokine Secretion and Proliferation in Co-Culture
with CD4.sup.+CD25.sup.- T Cells
[0320] A synthetic peptide derived from HSP60, amino acid sequence
437-460 called p277, was reported to arrest beta-cell destruction
in type 1 diabetes in human patients and in mice (Elias et al.,
1997, Raz et al., 2001). These observations suggested that peptide
p277 might also enhance the immuno-modulatory effects of Tregs.
Treatment of CD4.sup.+CD25.sup.+ T cells with peptide p277 (1
ng/ml, 2 hours), but not with a partially homologous, immunogenic
control peptide of Mycobacterium Tuberculosis, significantly
inhibited IFN-.gamma. and TNF-.alpha. secretion, and proliferation
in co-culture of the treated CD4.sup.+CD25.sup.+with untreated
CD4.sup.+CD25.sup.- T cells (FIG. 20). Thus, peptide p277 too can
function as a co-activator of Tregs. Moreover, these results using
a synthetic peptide cannot easily be attributed to LPS.
Example 25
HSP60-Induced Enhancement of CD4.sup.+CD25.sup.+ Treg Function
Involves Both Contact-Dependent and Cytokine-Dependent
Mechanisms
[0321] Transwell experiments were performed to investigate whether
cell-to-cell contact or soluble mediators were involved in the
enhanced effects of HSP60 on the function of CD4.sup.+CD25.sup.+
Tregs. FIG. 21A demonstrates that separation of HSP60-treated
CD4.sup.+CD25.sup.+ Treg cells from untreated CD4.sup.+CD25.sup.- T
cells by a semi-permeable membrane in transwell chambers reduced
the suppressive capacity of CD4.sup.+CD25.sup.+ T cells for
cytokine secretion by about 50%.
[0322] Recently it was reported that the CTLA4 molecule was
involved in the suppression mediated by mouse and human
CD4.sup.+CD25.sup.+regulatory T cells. To clarify the role of
CTLA4, CD4.sup.+CD25.sup.+ Treg cells were pre-incubated with
blocking anti-CTLA4 mAb before exposure to HSP60. Then, the treated
CD4.sup.+CD25.sup.+ T cells were co-cultured with
CD4.sup.+CD25.sup.- T cells on mitogenic anti-CD31n Ab. FIG. 21B
shows that the anti-CTLA4 rnAb blocked by about 50% the ability of
HSP60 treatment of the Tregs to inhibit cytokine secretion by the
CD4.sup.+CD25.sup.- T cells. Thus, the affect of HSP60-treated
CD4.sup.+CD25.sup.+ Tregs on CD4.sup.+CD25.sup.- T cells partially
depends on cell contact and the CTLA4 molecule.
[0323] To study whether cytokines had any role, CD4.sup.+CD25.sup.+
T cells were incubated with or without HSP60 (1 ng/ml, 2 hr),
washed, and activated on anti-CD3 mAbs for 24 hr. The supernatants
from the CD4.sup.+CD25.sup.+ Treg cells were collected and added
the supernatants to cultured CD4.sup.4CD25.sup.- T cells for an
additional 24 hr on anti-CD31n Abs. The inventors assayed
IFN-.gamma. and TNF-.alpha. secreted by the activated
CD4.sup.+CD25.sup.- T cells. The inventors found that the
supernatants from anti-CD3-activated CD4.sup.+CD25.sup.+ Tregs
untreated by HSP60 did not affect cytokine secretion by the
CD4.sup.+CD25.sup.- T cells (FIG. 21C). However, HSP60 treatment of
the CD4.sup.+CD25.sup.+ Treg cells induced them to secrete soluble
factors that inhibited IFN-.gamma. and TNF-.alpha.secretion by the
CD4.sup.+CD25.sup.- T cells by about 30% (FIG. 21C). Thus, soluble
factors account in part for the enhanced function of Tregs induced
by HSP60 treatment.
[0324] Because HSP60 induced IL-10 and TGF-.beta. secretion by
CD4.sup.+CD25.sup.+ T cells (see FIG. 16C), neutralizing anti-IL-10
and anti-TGF-.beta. mAb were used in the co-culture of
CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.- T cell populations.
Each antibody alone reversed HSP60-induced inhibition of
IFN-.gamma. and TNF-.alpha. secretion by about 40%; the
isotype-matched control mAb did not. Furthermore, presence of both
anti-IL-10 and anti-TGF-.beta. antibodies in the co-culture
markedly prevented the inhibitory effect on IFN-.gamma. and
TNF-.alpha. secretion. Thus, HSP60 treatment enhanced the
regulatory activity of CD4.sup.+CD25.sup.+ T cells involved both in
contact-dependent (CTLA4) and cytokine-dependent (IL-10,
TGF-.beta.) mechanisms.
Example 26
HSP60 Induces Phosphorylation of AKT, Pyk-2, p38, and
Down-Regulates Anti-CD3-Induced ERK-Phosphorylation in
CD4.sup.+CD25.sup.+ Treg Cells, and Co-Culture of
CD4.sup.+CD25.sup.- T Cells with HSP60-Treated CD4.sup.+CD25.sup.+
T Cells Down-Regulates ERK Phosphorylation, and Inhibits Nuclear
Translocation of NF-.kappa.B and T-Bet Expression in the
CD4.sup.+CD25.sup.- T Cells
[0325] As can be seen in FIG. 22, HSP60 induces phosphorylation of
AKT, Pyk-2, p38, and down-regulates anti-CD3-induced
ERK-phosphorylation in activated CD4.sup.+CD25.sup.+ T cells. A.
Purified CD4.sup.+CD25.sup.+ T cells were pretreated with
intracellular signal transduction inhibitors wortmanin (5 nM),
GF109203X (20 nM), SB203580 (10 .mu.m), pertussis toxin (2
.mu.g/ml), or PD98059 (10 .mu.m). Then, the cells were incubated
with HSP60 (1 ng/ml, 2 hrs), washed, co-cultured with
CD4.sup.+CD25.sup.- T cells (ratio 1:9) on anti-CD3 in serum-free
medium. The supernatants were collected after 24 hr and analyzed
for IFN-.gamma. and TNF-.alpha.; B-E. Unseparated CD4.sup.+,
CD4.sup.+CD25.sup.+, and CD4.sup.+CD25.sup.- T cells were incubated
with HSP60 (1 ng/ml) for 10 min, washed, and some cells (E) were
exposed to anti-CD3 mAbs (60 min). Lysates of these cells were
immunoblotted with antibodies: anti-pAKT and anti-tAKT (B),
anti-pPyk2 and anti-tPyk2 (C), anti-pp 38 and anti-tp38 (D), or
anti-phospho-ERK (pERK) and anti-total ERK (tERK) (E). The blot of
1 experiment representative of three different donors is presented.
Phosphorylation levels of the experiments were estimated by
densitometry, and an average percentage of phosphorylation.+-.SD
was calculated as OD of pAKT/tAKT, or pPyk2/tPyk2, or pp 38/tp38
pERK/tERK.times.100%.
[0326] Thus, HSP60 induced phosphorylation of AKT, Pyk-2, and p38,
whereas ERK phosphorylation induced by anti-CD3 was inhibited in
CD4.sup.+CD25.sup.+ T cells.
[0327] As can be seen in FIG. 23, Co-culture of CD4.sup.+CD25.sup.-
T cells with HSP60-treated
[0328] CD4.sup.+CD25.sup.+ T cells down-regulates ERK
phopsphorylation (A), inhibits, nuclear translocation of
NF-.kappa.B (B), and T-bet (C) expression in the
CD4.sup.+CD25.sup.- T cells. Purified CD4.sup.+CD25.sup.- T cells
were labeled with CFSE, washed, and co-cultured with HSP60-treated
(1 ng/ml, 2 hr) CD4.sup.+CD25.sup.+ T cells (ratio 1:9) on anti-CD3
in serum-free medium for 6 hr. The CD4.sup.+CD25.sup.- T cells were
re-isolated by FACS sorting and cell lysates of these cells were
immunoblotted with antibodies: anti-phospho-ERK (pERK) and
anti-total ERK (tERK) (A), anti-NF-.kappa.B and anti-Lamin B (B),
anti-T-bet and anti-total ERK (tERK) (C). The blot of 1 experiment
representative of four different donors is presented. The levels of
ERK phosphorylation, NF-.kappa.B, and T-bet were estimated by
densitometry and the average percentage derived from four different
donors was shown.
[0329] Thus, co-culture of CD4.sup.+CD25.sup.- T cells with
HSP60-treated CD4.sup.+CD25.sup.+ Treg cells resulted in
down-regulation of ERK phosphorylation, inhibition of nuclear
translocation of NF-KB and T-bet expression in the regulated
CD4.sup.+CD25.sup.- T cells.
D. HSP60 and p277 Inhibit Th1-Mediated Hepatitis Model Via Innate
Regulation of Th1/Th2 Transcription Factors and Cytokines
[0330] Reagents.
[0331] The following reagents and chemicals were obtained as
indicated: RPMI-1640 (Gibco BRL; Paisley, UK), FCS, HEPES buffer,
antibiotics, sodium pyruvate (Biological Industries; Kibbutz
Beit-Haemek, Israel); phosphatase inhibitor cocktail,
cycloheximide, PMA, LPS (Sigma-Aldrich, Rehovot, Israel).
Monoclonal antibodies (mAb) anti-TLR2 and anti-TLR4 (eBioscience;
San-Diego, Calif.); anti-human recombinant HSP60 (designated clone
P5, IgM fraction; kindly provided by F. Quintana of The Weizmann
Institute of Science). Polyclonal Ab anti-total ERK1/2 was obtained
from Sigma (Rehovot, Israel); anti-SOCS3 (H-103), anti-NF-.kappa.B
p65 (A), anti-T-bet (39D), anti-GATA-3 (HG3-31), and anti-Lamin B
(C-20) were purchased from Santa-Cruz Biotech (Santa-Cruz, Calif.).
Purified mouse anti-NFATp/NFATc2 (4G6-G5.1) was purchased from BD
Pharmingen (San-Diego, Calif.). The recombinant HSP60 (StressGen
Biotechnologies; Victoria, BC, Canada) used in this study contained
less than 0.001 EU/ml (0.1 pg/ml) of bacterial endotoxin, as
determined using a kinetic-turbidimetric LAL test method
(Biological Industries, Kibutz Beit-Haemek, Israel). The peptides
p277 (having the amino acid sequence of VLGGGVALLRVIPALDSLTPANED,
p277(Val.sup.6Val.sup.11), SEQ ID NO:2) and MTp277 (having the
amino acid sequence of VAGGGVTLLQAAPTLDELKLEG, SEQ ID NO:10) that
were used in Examples 27-34 were prepared using standard FMOC
chemistry as previously described (Raz et al., 2001), and contained
less than 0.001 EU/mg protein (0.1 pg/mg) of bacterial endotoxin as
determined using a kinetic-turbidimetric LAL test method.
[0332] Human T Cells.
[0333] T cells were purified from the peripheral blood of healthy
human donors (Blood Bank; Tel-Hashomer, Israel). The whole blood
was incubated (20 min, 22.degree. C.) with RosetteSep.TM. human
T-cell enrichment cocktail (StemCell Technologies, Vencouver, BC,
Canada). The remaining unsedimented cells were then loaded onto
Lymphocyte Separation Medium (ICN Biomedicals; Belgium), isolated
by density centrifugation, and washed with PBS. The purified cells
(>95% CD3.sup.+ T cells) so obtained were cultured in RPMI
containing antibiotics and 10% heat-inactivated FCS.
[0334] Cytokine Secretion.
[0335] T cells (2.times.10.sup.6 cells per ml) were activated (1
hr, 37.degree. C.) with the indicated concentrations of HSP60 in
24-well plates in RPMI containing 10% heat-inactivated FCS. The
cells were then washed and re-plated at the same concentration on
anti-CD3 mAb pre-coated 24-well plates (0.5 .mu.g/ml; non tissue
culture grade plates) in serum free medium containing 0.1% BSA at
4.degree. C. for 24 hr. The supernatants were collected, and the
cytokine content (TNF-.alpha., IFN-.gamma., and IL-10) was
determined by ELISA, using the appropriate mAb, according to the
manufacturer's instructions.
[0336] Western Blot Analysis of T-Cell Nuclear Extracts.
[0337] Purified T cells (5.times.10.sup.6) were preincubated with
different concentrations of HSP60 for the indicated periods of time
(37.degree. C. in a 7% CO.sub.2, humidified atmosphere). The cells
were then washed and replated in the same concentration of HSP60 on
anti-CD3 mAb pre-coated 24-well plates for 24 hr (37.degree. C. in
a 7% CO.sub.2, humidified atmosphere). T cells were lysed in 10 mM
HEPES, 1.5 mM MgCl.sub.2, 1 mM dithiothreitol (DTT), 1 mM PMSF,
0.5% Nonidet P-40. The lysates were incubated on ice for 10 min and
centrifuged at 2000 rpm for 10 min at 4.degree. C. The supernatants
(cytoplasmic extracts) were transferred and the pellet (nuclei) was
suspended in buffer containing 30 mM HEPES, 450 mM NaCl, 25%
Glycerol, 0.5 mM EDTA, 6 mM DTT, 12 mM MgCl.sub.2 1 mM PMSF, 10
.mu.g/ml leupeptin, 10 .mu.g/ml pepstatin, 1% phosphatase inhibitor
cocktail, and the suspension was incubated on ice for 30 mM. The
lysates were cleared by centrifugation (30 min, 14.times.10.sup.3
rpm, 4.degree. C.), and the resulting supernatants analyzed for
protein content. For NFATp analysis nuclear extracts were prepared
with a NE-PER Nuclear and Cytoplasmic Extraction reagent (Pierce,
Rockford, Ill.) according to the manufacturer's protocol. Sample
buffer was then added and, after boiling, the samples, containing
equal amounts of proteins, were separated on 10% SDS-PAGE gel and
transferred to nitrocellulose membranes. The membranes were blocked
with TBST buffer containing low-fat milk (5%), Tris pH 7.5 (20 mM),
NaCl (135 mM) and Tween 20 (0.1%), and probed with the following
mAb in the same buffer: anti-NF-.kappa.B (diluted 1:1000),
anti-total (t) ERIC (diluted 1:20,000), anti-NFATp (diluted 1:500),
anti T-bet (diluted 1:1000), anti-GATA-3 (diluted 1:1000),
anti-Lamin B (diluted 1:1000). Immunoreactive protein bands were
visualized using labeled secondary antibodies and the enhanced ECL
system.
[0338] Induction and Evaluation of Liver Damage.
[0339] BALB/c mice were maintained at the Animal Breeding Facility
of the E. Wolfson Medical Center. Treatment of the animals was in
accordance with institutional guidelines. Acute liver injury was
induced by injecting 6-8-week-old male mice with concanavalin A
(Con-A) (12 mg/ml; Sigma-Aldrich, Rehovot, Israel) in 250 ml of
phosphate buffered saline (PBS) via the tail vein. HSP60, p277 or
MTp277 were administered (330 .mu.l, 500 ng/ml) i.p. 18 and 1 hr
prior to ConA. After 24 hrs, the mice were bled, euthanized with
chloral hydrate anesthesia, their abdomens opened by a midline
incision and sections from the left liver lobe were excised for
histopathologic examination. Liver sections were fixed in a 5%
neutral formol solution and stained with hematoxylin and eosin.
[0340] Enzymatic Assessment of Liver Injury and Determination of
Serum Levels of Cytokines.
[0341] In addition to a histopathological examination, the extent
of the liver damage was estimated, by determining the serum levels
of alanine aminotransferase (ALT) and aspartate aminotransferase
(AST), with an automated Monarch Monoanalyzer 2000 (Allied,
Lexington, Mass.) 24 hr after ConA administration. For
determination of serum levels of cytokines, blood was drawn from
mice 2 hr after administration of Con A. The levels of TNF-.alpha.,
IL-6, and IL-10 were determined by ELISA, using the appropriate
mAb, according to the manufacturers' instructions. Anti mouse
TNF-.alpha. CytoSets.TM. was purchased from Biosource (CA, USA).
Anti murine IL-6 Eli-pair and anti murine IL-10 Eli-pair were
purchased from Diaclone Research (France).
Example 27
HSP60 Inhibits T-Cell IFN.gamma. and TNF.alpha. Secretion and
Up-Regulates IL-10 Secretion Via TLR2
[0342] It was previously shown that treatment of human patients or
diabetic NOD mice with HSP60 or its bio-active peptide p277
inhibited Th1 autoimmunity and enhanced Th2-like immune responses
(Raz et al., 2001; Quintana et al., 2002; Elias et al., 1997). This
shift in the cytokine profile was associated with the arrest of
inflammatory .beta.-cell destruction in newly diagnosed human
subjects with type I diabetes (Raz et al., 2001; Elias et al.,
1997). The effects of HSP60 on the cytokine secretion profile of
freshly isolated and purified human T cells that were activated by
anti-CD3 mAb, were analyzed. HSP60 alone did not induce cytokine
secretion from the T cells. However, when the T cells were
activated by immobilized anti-CD3, the secretion of the Th1-related
cytokines IFN-.gamma. and INF-.alpha. was inhibited by HSP60 (FIGS.
24A and 24B), while IL-10 secretion was enhanced (FIG. 24C).
Recently, the inventors have shown that the biological effects of
HSP60 on T cells manifested a bell-shaped dose-response curve
(Zanin-Zhorov et al., 2003). Interestingly, both the inhibitory
effect of HSP60 on IFN-.gamma. and TNF-.alpha. secretion and its
activation of IL-10 secretion also manifested a bell-shaped
dose-response. Significant effects were achieved with relatively
low concentrations of HSP60 (0.1-1.0 ng/ml; P<0.05), while
higher doses (in the order of 10 ng/ml) did not appear to affect
cytokine secretion. However, cytokine secretion was again affected
at higher concentrations of HSP60 (0.1-1 .mu.g/ml; P<0.05).
Thus, HSP60 can modulate the profiles of Th1 and Th2-associated
cytokine secretion in human T cells.
[0343] HSP60 has been reported to activate responsive cells via
TLR4 or TLR2 signaling (Ohashi et al., 2000; Kol et al., 1998). The
inventors have previously reported that HSP60 by TLR2 signaling can
induce T-cell adhesion to the extracellular matrix glycoprotein,
fibronectin, and inhibit T-cell chemotaxis through fibronectin
toward ELC and SDF-1.alpha. (CXCL12) (Zanin-Zhorov et al., 2003).
To test whether TLR2 or TLR4 was functionally involved in the
effects of HSP60 on T-cell cytokines, human T cells were
pre-incubated with anti-TLR2 or TLR4 mAb, and their cytokine
secretion profile was assessed. The inhibition of IFN-.gamma. and
TNF-.alpha.c secretion (FIG. 25, A and B), or activation of IL-10
secretion (FIG. 25C) by HSP60 was blocked by treating the T cells
with a mAb to TLR2, but not by a mAb to TLR4. In addition, the
effects of HSP60 on T-cell cytokine secretion were blocked by
anti-HSP60 mAb, but not by an isotype-matched control mAb. Thus,
TLR2 appears to play a role in mediating the effects of HSP60 on
activated human T cells.
Example 28
The Effects of HSP60 on T-Cell Cytokine Secretion are not Due to
Contaminating LPS
[0344] LPS-TLR2 interactions can transmit intracellular activation
signals in various types of leukocytes. Using a
kinetic-turbidimetric test method, it is hereby shown that the
recombinant human HSP60 used in this study contained less than
0.001 EU/.mu.g protein (0.1 pg/.mu.g) of bacterial endotoxin. The
following studies were performed to further exclude the possibility
that even minute amounts of LPS might affect T-cell cytokine
secretion using the LPS inhibitor polymyxin B (PMB), and by boiling
the HSP60. FIG. 26 shows that the effects of HSP60 on cytokine
secretion were completely inhibited by boiling (which denatures
proteins, but not LPS), but not by PMB (an inhibitor of LPS).
Moreover, in contrast to its effects on macrophages, it is herein
demonstrated that purified LPS did not modify T-cell cytokine
secretion. Thus, the effects of HSP60 on T-cell cytokine secretion
could not be attributed to LPS.
[0345] Recently, lipoproteins extracted from the Escherichia coli
were shown to interact with macrophages via TLR-2 (Gao et al.,
2003). Such lipoproteins can be removed from LPS by passage over
PMB-coupled agarose beads (Gao et al., 2003). The HSP60 was
therefore incubated with PMB-coupled agarose beads, the unbound
material was collected, and the protein content assayed, and the
effect of the PMB-treated HSP60 on TNF.alpha. secretion was tested.
Pre-incubation with PMB-conjugated agarose beads did not block the
efficacy of our HSP60 preparation on TNF.alpha. secretion, but the
efficacy of the bacterial preparation was completely abolished.
Thus, the effects of HSP60 on cytokine secretion by T cells could
be attributed to HSP60 itself.
Example 29
HSP60 Inhibits Nuclear Translocation of NF-.kappa.B in T Cells
[0346] Activation and nuclear translocation of NF-.kappa.B is an
essential step in the regulation of gene expression and secretion
of various pro-inflammatory cytokines in leukocytes, including T
cells. To address the modulatory role of HSP60 on the signal
transduction cascade leading to regulation of T-cell secretion of
TNF-.alpha., IFN-.gamma., and IL-10, the nuclear translocation of
NF-.kappa.B was examined by probing nuclear and cytoplasmic T-cell
extracts using mAb specific for the p65 subunit of NF-.kappa.B. The
nuclear protein Lamin B and the cytoplasmic ERK. were used as
continuatively expressed control proteins, for the quantification
of protein amounts (FIG. 27). Treatment of T cells with HSP60 alone
did not affect the nuclear translocation of NF-.kappa.B. However,
pre-treatment of T cells with HSP60, followed by their exposure to
anti-CD3, caused a significant down-regulation of NF-.kappa.B
activation and translocation to the nucleus (FIG. 27A); the p65
subunit of this nuclear factor remained in the cytoplasmic
compartment (FIG. 27B). Similar to the results in the cytokine
secretion assays (FIG. 24), maximal effects on NF-.kappa.B
(P<0.01) were observed with 1 ng/ml or 1 .mu.g/ml HSP60 (FIG.
27A). At 1 ng/ml of HSP60, inhibition was already apparent after 60
min (P<0.01), and reached its peak after 120 to 240 min (FIG.
27D).
Example 30
HSP60 Inhibits NFATp Activation in T Cells
[0347] NFAT is a critical regulator of TCR-mediated signals
involved in early events associated with gene transcription.
Currently, four NFAT genes encoding the cytoplasmic subunits have
been characterized: NFATc, NFATp, NFAT3, and NFAT4 (also called
NFATc1, NFATc2, NFATc4, and NFATc3, respectively). Several studies
indicate that NFATp negatively controls Th2 development. The
activation of NFATp and its entry into the nucleus depend on the
de-phosphorylation of serine/threonine residues, which leads to a
10- to 20-kDa decrease in its apparent molecular mass, as
visualized by immunoblots. To study the effects of HSP60 on NFATp
activation, the T cells were incubated with HSP60 and immobilized
anti-CD3 antibodies, prepared their nuclear extracts, and analyzed
the products of activation using immunoblots. Cells that were
pre-treated with 1 ng/ml of HSP60 and then exposed to immobilized
anti-CD3 antibodies, exhibited a significant down-regulation of
their activation and de-phosphorylation of NFATp (FIG. 28A). At 1
ng/ml of HSP60, inhibition was apparent after 60 min, and reached
its peak after 120 to 240 min (FIG. 28B). In the absence of
anti-CD3, HSP60 alone did not affect NFATp phosphorylation.
[0348] FIG. 28C shows that the inhibitory effect of HSP60 on NFATp
dephosphorylation and activation was blocked by the protein
synthesis inhibitor cycloheximide (CHX). This is compatible with
the conclusion that inhibition of NFATp mediated by HSP60 is an
active process.
Example 31
HSP60 Inhibits T-Bet and Up-Regulates GATA-3 Expression Via TLR2 in
Human T Cells
[0349] HSP60 inhibited the secretion of Th1-associated cytokines
IFN.gamma. and TNF.alpha., and increases the secretion of the
Th2-associated cytokines IL-10 (FIG. 24), IL-4, and IL-13. Based on
these results, it was examined whether HSP60 differentially
regulates transcription factors associated with the Th1 and Th2
phenotypes, T-bet and GATA-3. As is demonstrated in FIG. 29A, 1
ng/ml and 1 .mu.g/ml HSP60 significantly (P<0.01) up-regulated
the protein level of GATA-3, but not of T-bet. To rule out the
possibility that the differences in T-bet and GATA-3 expression
were due to unequal amounts of protein loaded on the gel, the same
blot was incubated with anti-T-bet and anti-GATA-3 Ab (separated by
a stripping procedure). The results confirm that the differences
between GATA-3 and T-bet expression were not a result of a
difference in protein loading of the gels.
[0350] The up-regulation of GATA-3 was TLR2-dependent, since
anti-TLR2, but not anti-TLR4 mAb, abrogated the activation by HSP60
(FIG. 29B).
[0351] It has been recently reported that the level of T-bet is
augmented by TCR-mediated signals. It was also shown that the
maximal expression of GATA-3 in human T cells requires stimulatory
signals from both TCR and CD28 (Th2-inducing conditions); signaling
via TCR alone induces a lesser degree of expression of GATA-3. To
analyze the effect of HSP60 on CD3-induced T-bet and GATA-3
expression, the cells were pre-treated with different
concentrations of HSP60 (1 hr). The cells were then washed and
seeded on immobilized anti-CD3. In the absence of HSP60, expression
levels of T-bet were markedly up-regulated in anti-CD3-treated T
cells. However, at 1 ng/ml and 1 .mu.g/ml, HSP60 significantly
(P<0.05) inhibited the T-bet expression induced by the anti-CD3
(FIG. 29C). As expected, anti-CD3 alone up-regulated the expression
of GATA-3 to a much lesser extent, compared to that of T-bet.
Furthermore, pre-incubation of T cells with HSP60 significantly
up-regulated the expression level of GATA-3. Thus, when the T cells
were pre-activated by anti-CD3, HSP60 inhibited the expression of
T-bet, but up-regulated that of GATA-3.
Example 32
HSP60 Modulates Expression of Th1/Th2 Transcription Factors in
Activated Mouse T-Cells
[0352] The above findings indicated that HSP60 can down-regulate
the molecular events leading to Th1 expression and up-regulate
those leading to Th2 expression in human T cells in vitro. To test
whether HSP60 might exert similar effects in vivo, mouse models
were used. The present inventors have previously found that HSP60
abrogated the ability of antigen-reactive T cells to adoptively
transfer DTH response to oxazolone in BALB/c mice (WO 03/070761);
the DTH reaction is a classical Th1-type immune response that
involves the SDF-1.alpha.-CXCR4 interaction. This finding suggested
that the inhibitory effect of HSP60 on T cell-mediated inflammation
might be explained by the ability of HSP60 to down-regulate both
T-cell chemotaxis and the secretion of the Th1-type cytokines,
TNF.alpha. and IFN.gamma. (FIG. 24, A and B). To test this
hypothesis, the ability of HSP60 to modulate the expression levels
of T-bet and GATA-3 in mouse T cells was examined. Treatment of
oxazolone-reactive mouse T cells with 1 ng/ml of HSP60 for 2 hr
resulted in down-regulation of T-bet and up-regulation of GATA-3
expression (FIG. 30D). Thus, the inhibitory effect of HSP60 on DTH
may be explained not only by its ability to down-regulate the
SDF-1.alpha.-CXCR4 interactions required for T-cell chemotaxis to
inflammatory sites, but also by the ability of HSP60 to shift the
transcription factor expression from a Th1 to Th2 pattern.
Example 33
HSP60 Suppresses Th1-Mediated ConA-Induced Hepatic Injury in Mice,
Associated with Modulation of Expression of Th1/Th2 Transcription
Factors and Cytokines
[0353] To test whether HSP60 administration could affect
inflammatory disease in an in vivo model, the effect of HSP60 on
BALB/c mice with acute liver injury induced by intravenous ConA was
examined; the liver injury has been shown to be caused by
pro-inflammatory CD4.sup.+ T cells (Tiegs et al., 1992). It has
also been shown that the level of expression of TNF.alpha. and IL-4
are increased following ConA injection, while that of IL-10 is
decreased. Down-regulation of SOCS3 is also involved in hepatitis,
and SDF-1.alpha.-CXCR4 interactions were found to play a major role
in the disease.
[0354] Here, HSP60 was administered intraperitoneally 1 hr and 18
hr prior to their IV injection with ConA. Control mice were not
treated with HSP60. Changes in the secretion profiles of cytokines
and a rise in serum levels of liver enzymes were used as markers
for the severity of hepatitis; serum cytokines were measured at 2
hr and liver enzymes at 24 hr. Histological analysis of hepatic
tissues was also performed 24 hr after ConA administration. HSP60
significantly (P<0.05) inhibited the secretion of AST and ALT
from the injured livers of the ConA-treated mice (FIG. 30A).
[0355] Histopathologic examination of liver sections confirmed that
HSP60 administration reduced liver damage. ConA induced a marked
inflammatory-cell infiltrate around the central veins and large
areas of necrosis in the liver lobules. In contrast, mice treated
with HSP60 manifested only minimal liver damage; there were no
areas of necrosis and leukocyte infiltration was almost absent
(FIG. 30C).
[0356] HSP60 also reduced the levels of TNF.alpha. in the sera of
the mice (FIGS. 30B and 9A), while increasing the level of IL-10
(FIG. 30C).
[0357] To assay the effects of HSP60 on the T cells of the mice, T
cells were purified from the spleens and the expression of SOCS3,
GATA-3, and T-bet in lysates of the T cells was measured. T-cell
donor mice were either healthy, treated with HSP60, treated with
ConA, or treated with both HSP60 and ConA. SOCS3 expression was
minimal in healthy mice, but became elevated following HSP60
treatment, and was even more elevated in the mice treated with both
ConA and HSP60 (FIG. 30D). Thus, the suppression of ConA-induced
hepatitis was associated with augmentation of SOCS3 expression.
[0358] The expression of GATA and T-bet in these mouse T cells was
also measured. HSP60 suppressed the ConA-induced expression of the
Th1-associated transcription factor T-bet, while significantly
augmenting the expression of the Th2-associated factor GATA-3
(P<0.05) (FIG. 30E). Thus, HSP60 down-regulates hepatitis in
mice by down-regulating the expression of T-bet and up-regulating
the expression of GATA-3 and SOCS3, thereby inducing a Th1 to Th2
cytokine shift. These in vivo and ex vivo results in mice confirmed
the findings induced by HSP60 in human T cells obtained in
vitro.
Example 34
p277 Suppresses Th1-Mediated ConA-Induced Hepatic Injury in Mice
Associated with Modulation of Expression of Th1 and Th2
Cytokines
[0359] The p277 peptide, or a control peptide, MTp277 were injected
(330 .mu.l, 500 ng/ml) to BALB/c mice 1 hr and 18 hr prior to ConA
treatment. Liver enzymes ALT and AST were measured in sera obtained
from the mice 24 hr after ConA administration. p277, but not the
control peptide MTp277 significantly inhibited ALT and AST
secretion from injured livers of the ConA-treated mice (FIG.
31).
[0360] The serum levels of TNF-.alpha. and IL-10 were also
examined, 2 hr following ConA administration. The levels of
TNF-.alpha. in the sera were significantly decreased in mice
treated with p277, while the level of IL-10 was significantly
increased (FIGS. 32A and 32C). The control peptide, MTp277, did not
seem to have a significant effect on the levels of cytokines in the
sera of ConA-treated mice.
[0361] Thus, p277 retains the down-regulating effect of HSP60 on
hepatitis in mice, as well as its ability to induce a Th1 to Th2
cytokine shift in ConA-treated mice.
[0362] IL-6 is a cytokine secreted by both lymphoid and
non-lymphoid cells, which has been implicated in various
physiological and pathological processes. IL-6 was previously
demonstrated to serve a protective role in ConA-induced hepatitis
and other models of liver injury, and to induce a Th1 to Th2 shift
by regulating Th1/Th2 transcription factors. Thus, the level of
IL-6 in the sera of the mice was assayed. As can be seen in FIG.
32B, p277, and to a lesser extent, HSP60, increased the level of
IL-6 in the sera of ConA treated mice.
Example 35
Preparation of T Cell Vaccines
[0363] Human T cells are isolated on a Ficoll gradient, washed, and
incubated (2 h, 37.degree. C., 7.5% CO.sub.2, humidified
atmosphere) on petri dishes. The nonadherent cells are then
collected and incubated (1 h, 37.degree. C., 7.5% CO.sub.2,
humidified atmosphere) on nylon wool columns (Novamed, Jerusalem,
Israel). Unbound cells are eluted from the columns by extensive
washings.
[0364] A portion of the cells are activated on anti-CD3 mAb
pre-coated 24-well plates (0.5 .mu.g/ml; non tissue culture grade
plates), irradiated (5000 rads), and plated in round bottom 96-well
microplates 2.times.10.sup.5 cells per well in the presence of
10-50 .mu.g/ml p277 and 2.times.10.sup.5 untreated T cells per
well. The cells are cultured in medium RPMI-1640 (Gibco)
supplemented with 10% fetal bovine serum (HyClone), 100 U/ml
penicillin, 100 .mu.g/ml streptomycin, and 0.1% glutamine in
37.degree. C., 5% CO.sub.2 incubator. Following cultivation for
7-14 days, the cultures are split and subcultures are prepared and
re-stimulated with p277-primed T cells as described above. The
index of cell stimulation (SI) in response to p277 is examined
after additional 72 h in culture using .sup.3H-thymidine
incorporation assays (Amersham, Arlington Heights, Ill.). Wells
exhibiting a minimal SI>3 (threefold increase in
.sup.3H-thymidine incorporation relative to the average
incorporation in reference control wells not stimulated with
peptide) are selected for line propagation and expanded with IL-2
(50 IU/ml, Roche).
Example 36
Anti-CD3-Induced T-Cell Proliferation and IFN.gamma. Secretion are
Inhibited by p277 Analogs
[0365] Mice:
[0366] Female C57BL/6J were purchased from Harlan Olac (Bicester,
U.K). The mice were maintained in a specific pathogen-free (SPF)
facility in the Weizmann institute, and were used at the age of 5-8
weeks.
[0367] Purification of Naive T Cells:
[0368] Mouse spleen-cell suspensions were depleted of red blood
cells by treatment with red blood lysis buffer (Sigma, St Louis,
USA). T cells were then purified by negative selection with a
T-cell isolation kit containing biotin-conjugated monoclonal
antibodies to CD11b, CD45R, DX5 and Ter-119 (Miltenyi Biotec,
Bergisch Gladbach, Germany). This procedure routinely yielded T
cell preparations that were >95% positive for the CD3 marker as
determined by FACS analysis.
[0369] Cells and Medium:
[0370] The T-cells cultures were set in RPMI-1640 supplemented with
5% FCS (Hyclon Logan, Utah,) 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, 50 .mu.M 213-ME, 12.5 mM hepes, 2 mM L-glutamine, 1
mM sodium pyruvate and non essential amino acids (biological
industries 01-340-1 diluted 1:100).
[0371] Reagents:
[0372] The peptides used in this Example (VLGGGVALLRVIPALDSLTPANED,
p277(Val.sup.6Val.sup.11), SEQ ID NO:2; and
VLGGGSALLRSIPALDSLTPANED, p277(Ser.sup.6Ser.sup.11), SEQ ID NO:3)
were synthesized using the F-MOC technique with an automatic
multiple peptide synthesizer (AMS 422, ABIMED, Langenfeld,
Germany). The purity of the peptides was analyzed by HPLC.
[0373] The hamster anti-mouse anti-CD3 antibody was collected by
TCA precipitation from 2011 hybridoma supernatant. For the
proliferation assay, non-tissue culture-treated polystyrene
flat-bottom 96-well microtiter plates (Nunc 269787) were incubated
overnight at 4.degree. C. with 0.5-2 .mu.g/ml anti-CD3
antibody.
[0374] T-Cell Proliferation:
[0375] Mouse splenic purified T cells (1.times.10.sup.5) were
pre-incubated with the p277 analogs p277(Val.sup.6Val.sup.11) (SEQ
ID NO:2) or p277(Ser.sup.6Ser.sup.11) (SEQ ID NO:3) at the
indicated concentrations for 30-60 min, and transferred to anti-CD3
coated wells in triplicate or quadruplicate in 200 .mu.l culture
medium. After 72 h of incubation (37.degree. C., 5% CO.sub.2) the
cells were pulsed with 1 .mu.Ci [.sup.3H]-thymidine for 7 h, and
[.sup.3H] thymidine incorporation was measured using a 96-well
plate beta-counter. The mean cpm.+-.Standard Error were calculated
for each triplicate or quadruplicate.
[0376] Interferon-gamma (IFN.gamma.) secretion to the culture media
was determined by using an ELISA assay (OptiEIA kits, BD Pharmingen
and Pierce Endogen) following the manufacturer's instructions.
Standard curves were established using mouse recombinant cytokines.
The assay detection limit was 16-32 pg/ml.
[0377] The results are presented in Table 1 herein:
TABLE-US-00002 TABLE 1 anti-CD3-induced T-cell proliferation and
IFN.gamma. secretion in the presence of p277(Val.sup.6Val.sup.11)
or p277(Ser.sup.6Ser.sup.11). Added peptide Peptide
p277(Val.sup.6Val.sup.11) p277(Ser.sup.6Ser.sup.11) concen- Percent
Percent Percent Percent tration Inhibition of Inhibition of
Inhibition of Inhibition of (ng/ml) proliferation IFN.gamma.
secretion proliferation IFN.gamma. secretion 0 0% .+-. 11% 0% .+-.
15.2% 0% .+-. 9.6% 0% .+-. 15% 0.1 50% .+-. 19% 94% .+-. 0.81% 80%
.+-. 2.2% 86.6% .+-. 11.3% 1 73% .+-. 7.5% 88.9% .+-. 3.4% 76% .+-.
8% 86.4% .+-. 1.3% 10 69% .+-. 14% 86.7% .+-. 8.8% 68% .+-. 7.8%
82.6% .+-. 4.75%
[0378] The values for anti-CD3-induced T-cell proliferation and
IFN.gamma. secretion without the presence of
p277(Val.sup.6Val.sup.11) or p277(Ser.sup.6Ser.sup.11) were 5879
cpm and 5 ng/ml, respectively.
[0379] As can be seen in Table 1, both p277(Val.sup.6Val.sup.11)
and p277(Ser.sup.6Ser.sup.11) inhibited T-cell proliferation and
IFN.gamma. secretion induced by the anti-CD3 antibody.
[0380] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without undue
experimentation and without departing from the generic concept,
and, therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. Although the invention
has been described in conjunction with specific embodiments
thereof, it is evident that many alternatives, modifications and
variations will be apparent to those skilled in the art.
Accordingly, it is intended to embrace all such alternatives,
modifications and variations that fall within the spirit and broad
scope of the appended claims.
REFERENCES
[0381] 1. Quintana et al., 2000. J Immunol 165:6148-6155. [0382] 2.
Quintana et al., 2003. J Immunol 171:3533-3541. [0383] 3. Anderton
et al., 1994. J Immunol 152:3656-3664. [0384] 4. van Eden et al.,
1988. Nature 331:171-173. [0385] 5. Mor et al., 1992. J Clin Invest
90:2447-2455. [0386] 6. Quintana et al., 2002. J Immunol
169:6030-6035. [0387] 7. Quintana et al., 2002b. J Immunol
169:3422-3428. [0388] 8. Ragno et al., 1997. Arthritis Rheum
40:277-283. [0389] 9. Wang et al., 2002. Clin. Immunol.
105:199-207. [0390] 10. Ferris et al., 1988. Proc Natl Acad Sci USA
85:3850-3854. [0391] 11. Reizis et al., 1996. Int Immunol
8:1825-1832. [0392] 12. Zanin-Zhorov et al., 2003. Faseb J 17:1567.
[0393] 13. Zanin-Zhorov et al., 2003b. J Immunol 171:5882. [0394]
14. Kollet Oet al., 2001. Blood 97:3283. [0395] 15. Kol et al.,
1999. J Clin Invest 103:571. [0396] 16. Kol et al., 1998.
Circulation 98: 300-307. [0397] 17. Ohashi et al., 2000. J Immunol
164:558. [0398] 18. Wallin et al., 2002. Trends Immunol 23:130.
[0399] 19. Yokota et al., 2000. Cell Stress Chaperones 5:337.
[0400] 20. Xu et al., 2000. Circulation 102:14. [0401] 21. Laplante
et al., 1998. J Histochem Cytochem 46:1291. [0402] 22. Elias et
al., 1997. Diabetes 46:758. [0403] 23. Raz et al., 2001. Lancet
358:1749. [0404] 24. Mosmann, 1983. J Immunol Methods 65:55. [0405]
25. Gao et al., 2003. J Biol Chem 278:22523. [0406] 26. Ben-Nun et
al., 1987. Nature 292:60. [0407] 27. Holoshitz et. al., 1983.
Science 219:56. [0408] 28. Stewart, J. M. and Young, J. D., 1963.
Solid Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco);
[0409] 29. Meienhofer, 1973. Hormonal Proteins and Peptides, vol.
2, p. 46, Academic Press (New York). [0410] 30. Schroder and Lupke,
1965. The Peptides, vol. 1, Academic Press (New York). [0411] 31.
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Labs Press, 1989. [0412] 32. Wolff et al., 1990.
Science 247, 1465-1468. [0413] 33. Stribling et al., 1992. Proc.
Natl. Acad. Sci. USA 189:11277-11281. [0414] 34. Achiron et al.,
2004. Clin. Immunol:113 155160. [0415] 35. Tiegs et al., 1992. J
Clinic. Invest. 90: 196-203. [0416] 36. Heneghan et al., 2002.
Hepatology 35:7. [0417] 37. Chedid et al, 1993. Gastroenterology
105:254. [0418] 38. Rosen et al., 2002. Hepatology 35:190. [0419]
39. Hu et al., 1998. Eur. J. Immunol. 28: 2444-2455.
Sequence CWU 1
1
16124PRTArtificialSYNTHETIC PEPTIDE 1Val Leu Gly Gly Gly Cys Ala
Leu Leu Arg Cys Ile Pro Ala Leu Asp 1 5 10 15 Ser Leu Thr Pro Ala
Asn Glu Asp 20 224PRTArtificialsynthetic peptide 2Val Leu Gly Gly
Gly Val Ala Leu Leu Arg Val Ile Pro Ala Leu Asp 1 5 10 15 Ser Leu
Thr Pro Ala Asn Glu Asp 20 324PRTArtificialsynthetic peptide 3Val
Leu Gly Gly Gly Ser Ala Leu Leu Arg Ser Ile Pro Ala Leu Asp 1 5 10
15 Ser Leu Thr Pro Ala Asn Glu Asp 20 4573PRTHomo sapiens 4Met Leu
Arg Leu Pro Thr Val Phe Arg Gln Met Arg Pro Val Ser Arg 1 5 10 15
Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 20
25 30 Gly Ala Asp Ala Arg Ala Leu Met Leu Gln Gly Val Asp Leu Leu
Ala 35 40 45 Asp Ala Val Ala Val Thr Met Gly Pro Lys Gly Arg Thr
Val Ile Ile 50 55 60 Glu Gln Ser Trp Gly Ser Pro Lys Val Thr Lys
Asp Gly Val Thr Val 65 70 75 80 Ala Lys Ser Ile Asp Leu Lys Asp Lys
Tyr Lys Asn Ile Gly Ala Lys 85 90 95 Leu Val Gln Asp Val Ala Asn
Asn Thr Asn Glu Glu Ala Gly Asp Gly 100 105 110 Thr Thr Thr Ala Thr
Val Leu Ala Arg Ser Ile Ala Lys Glu Gly Phe 115 120 125 Glu Lys Ile
Ser Lys Gly Ala Asn Pro Val Glu Ile Arg Arg Gly Val 130 135 140 Met
Leu Ala Val Asp Ala Val Ile Ala Glu Leu Lys Lys Gln Ser Lys 145 150
155 160 Pro Val Thr Thr Pro Glu Glu Ile Ala Gln Val Ala Thr Ile Ser
Ala 165 170 175 Asn Gly Asp Lys Glu Ile Gly Asn Ile Ile Ser Asp Ala
Met Lys Lys 180 185 190 Val Gly Arg Lys Gly Val Ile Thr Val Lys Asp
Gly Lys Thr Leu Asn 195 200 205 Asp Glu Leu Glu Ile Ile Glu Gly Met
Lys Phe Asp Arg Gly Tyr Ile 210 215 220 Ser Pro Tyr Phe Ile Asn Thr
Ser Lys Gly Gln Lys Cys Glu Phe Gln 225 230 235 240 Asp Ala Tyr Val
Leu Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln Ser 245 250 255 Ile Val
Pro Ala Leu Glu Ile Ala Asn Ala His Arg Lys Pro Leu Val 260 265 270
Ile Ile Ala Glu Asp Val Asp Gly Glu Ala Leu Ser Thr Leu Val Leu 275
280 285 Asn Arg Leu Lys Val Gly Leu Gln Val Val Ala Val Lys Ala Pro
Gly 290 295 300 Phe Gly Asp Asn Arg Lys Asn Gln Leu Lys Asp Met Ala
Ile Ala Thr 305 310 315 320 Gly Gly Ala Val Phe Gly Glu Glu Gly Leu
Thr Leu Asn Leu Glu Asp 325 330 335 Val Gln Pro His Asp Leu Gly Lys
Val Gly Glu Val Ile Val Thr Lys 340 345 350 Asp Asp Ala Met Leu Leu
Lys Gly Lys Gly Asp Lys Ala Gln Ile Glu 355 360 365 Lys Arg Ile Gln
Glu Ile Ile Glu Gln Leu Asp Val Thr Thr Ser Glu 370 375 380 Tyr Glu
Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly 385 390 395
400 Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp Val Glu Val Asn Glu
405 410 415 Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala
Ala Val 420 425 430 Glu Glu Gly Ile Val Leu Gly Gly Gly Cys Ala Leu
Leu Arg Cys Ile 435 440 445 Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn
Glu Asp Gln Lys Ile Gly 450 455 460 Ile Glu Ile Ile Lys Arg Thr Leu
Lys Ile Pro Ala Met Thr Ile Ala 465 470 475 480 Lys Asn Ala Gly Val
Glu Gly Ser Leu Ile Val Glu Lys Ile Met Gln 485 490 495 Ser Ser Ser
Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe Val Asn 500 505 510 Met
Val Glu Lys Gly Ile Ile Asp Pro Thr Lys Val Val Arg Thr Ala 515 520
525 Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr Thr Ala Glu Val
530 535 540 Val Val Thr Glu Ile Pro Lys Glu Glu Lys Asp Pro Gly Met
Gly Ala 545 550 555 560 Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly
Met Phe 565 570 572DNAArtificialsynthetic oligonucleotide
5gttttgggag ggggttgtgc cctccttcga tgcattccag ccttggactc attgactcca
60gctaatgaag at 72672DNAArtificialsynthetic oligonucleotide
6gttttgggag ggggtgttgc cctccttcga gtcattccag ccttggactc attgactcca
60gctaatgaag at 72772DNAArtificialsynthetic loigonucleotide
7gttttgggag ggggttctgc cctccttcga tccattccag ccttggactc attgactcca
60gctaatgaag at 7282202DNAHomo sapiens 8cacgcttgcc gccgccccgc
agaaatgctt cggttaccca cagtctttcg ccagatgaga 60ccggtgtcca gggtactggc
tcctcatctc actcgggctt atgccaaaga tgtaaaattt 120ggtgcagatg
cccgagcctt aatgcttcaa ggtgtagacc ttttagccga tgctgtggcc
180gttacaatgg ggccaaaggg aagaacagtg attattgagc agggttgggg
aagtcccaaa 240gtaacaaaag atggtgtgac tgttgcaaag tcaattgact
taaaagataa atacaagaac 300attggagcta aacttgttca agatgttgcc
aataacacaa atgaagaagc tggggatggc 360actaccactg ctactgtact
ggcacgctct atagccaagg aaggcttcga gaagattagc 420aaaggtgcta
atccagtgga aatcaggaga ggtgtgatgt tagctgttga tgctgtaatt
480gctgaactta aaaagcagtc taaacctgtg accacccctg aagaaattgc
acaggttgct 540acgatttctg caaacggaga caaagaaatt ggcaatatca
tctctgatgc aatgaaaaaa 600gttggaagaa agggtgtcat cacagtaaag
gatggaaaaa cactgaatga tgaattagaa 660attattgaag gcatgaagtt
tgatcgaggc tatatttctc catactttat taatacatca 720aaaggtcaga
aatgtgaatt ccaggatgcc tatgttctgt tgagtgaaaa gaaaatttct
780agtatccagt ccattgtacc tgctcttgaa attgccaatg ctcaccgtaa
gcctttggtc 840ataatcgctg aagatgttga tggagaagct ctaagtacac
tcgtcttgaa taggctaaag 900gttggtcttc aggttgtggc agtcaaggct
ccagggtttg gtgacaatag aaagaaccag 960cttaaagata tggctattgc
tactggtggt gcagtgtttg gagaagaggg attgaccctg 1020aatcttgaag
acgttcagcc tcatgactta ggaaaagttg gagaggtcat tgtgaccaaa
1080gacgatgcca tgctcttaaa aggaaaaggt gacaaggctc aaattgaaaa
acgtattcaa 1140gaaatcattg agcagttaga tgtcacaact agtgaatatg
aaaaggaaaa actgaatgaa 1200cggcttgcaa aactttcaga tggagtggct
gtgctgaagg ttggtgggac aagtgatgtt 1260gaagtgaatg aaaagaaaga
cagagttaca gatgccctta atgctacaag agctgctgtt 1320gaagaaggca
ttgttttggg agggggttgt gccctccttc gatgcattcc agccttggac
1380tcattgactc cagctaatga agatcaaaaa attggtatag aaattattaa
aagaacactc 1440aaaattccag caatgaccat tgctaagaat gcaggtgttg
aaggatcttt gatagttgag 1500aaaattatgc aaagttcctc agaagttggt
tatgatgcta tggctggaga ttttgtgaat 1560atggtggaaa aaggaatcat
tgacccaaca aaggttgtga gaactgcttt attggatgct 1620gctggtgtgg
cctctctgtt aactacagca gaagttgtag tcacagaaat tcctaaagaa
1680gagaaggacc ctggaatggg tgcaatgggt ggaatgggag gtggtatggg
aggtggcatg 1740ttctaactcc tagactagtg ctttaccttt attaatgaac
tgtgacagga agcccaaggc 1800agtgttcctc accaataact tcagagaagt
cagttggaga aaatgaagaa aaaggctggc 1860tgaaaatcac tataaccatc
agttactggt ttcagttgac aaaatatata atggtttact 1920gctgtcattg
tccatgccta cagataattt attttgtatt tttgaataaa aaacatttgt
1980acattcctga tactgggtac aagagccatg taccagtgta ctgctttcaa
cttaaatcac 2040tgaggcattt ttactactat tctgttaaaa tcaggatttt
agtgcttgcc accaccagat 2100gagaagttaa gcagcctttc tgtggagagt
gagaataatt gtgtacaaag tagagaagta 2160tccaattatg tgacaacctt
tgtgtaataa aaatttgttt aa 22029548PRTEscherichia coli 9Met Ala Ala
Lys Asp Val Lys Phe Gly Asn Asp Ala Arg Val Lys Met 1 5 10 15 Leu
Arg Gly Val Asn Val Leu Ala Asp Ala Val Lys Val Thr Leu Gly 20 25
30 Pro Lys Gly Arg Asn Val Val Leu Asp Lys Ser Phe Gly Ala Pro Thr
35 40 45 Ile Thr Lys Asp Gly Val Ser Val Ala Arg Glu Ile Glu Leu
Glu Asp 50 55 60 Lys Phe Glu Asn Met Gly Ala Gln Met Val Lys Glu
Val Ala Ser Lys 65 70 75 80 Ala Asn Asp Ala Ala Gly Asp Gly Thr Thr
Thr Ala Thr Val Leu Ala 85 90 95 Gln Ala Ile Ile Thr Glu Gly Leu
Lys Ala Val Ala Ala Gly Met Asn 100 105 110 Pro Met Asp Leu Lys Arg
Gly Ile Asp Lys Ala Val Thr Val Ala Val 115 120 125 Glu Glu Leu Lys
Ala Leu Ser Val Pro Cys Ser Asp Ser Lys Ala Ile 130 135 140 Ala Gln
Val Gly Thr Ile Ser Ala Asn Ser Asp Glu Thr Val Gly Lys 145 150 155
160 Leu Ile Ala Glu Ala Met Asp Lys Val Gly Lys Glu Gly Val Ile Thr
165 170 175 Val Glu Asp Gly Thr Gly Leu Gln Asp Glu Leu Asp Val Val
Glu Gly 180 185 190 Met Gln Phe Asp Arg Gly Tyr Leu Ser Pro Tyr Phe
Ile Asn Lys Pro 195 200 205 Glu Thr Gly Ala Val Glu Leu Glu Ser Pro
Phe Ile Leu Leu Ala Asp 210 215 220 Lys Lys Ile Ser Asn Ile Arg Glu
Met Leu Pro Val Leu Glu Ala Val 225 230 235 240 Ala Lys Ala Gly Lys
Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly 245 250 255 Glu Ala Leu
Ala Thr Leu Val Val Asn Thr Met Arg Gly Ile Val Lys 260 265 270 Val
Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met 275 280
285 Leu Gln Asp Ile Ala Thr Leu Thr Gly Gly Thr Val Ile Ser Glu Glu
290 295 300 Ile Gly Met Glu Leu Glu Lys Ala Thr Leu Glu Asp Leu Gly
Gln Ala 305 310 315 320 Lys Arg Val Val Ile Asn Lys Asp Thr Thr Thr
Ile Ile Asp Gly Val 325 330 335 Gly Glu Glu Ala Ala Ile Gln Gly Arg
Val Ala Gln Ile Arg Gln Gln 340 345 350 Ile Glu Glu Ala Thr Ser Asp
Tyr Asp Arg Glu Lys Leu Gln Glu Arg 355 360 365 Val Ala Lys Leu Ala
Gly Gly Val Ala Val Ile Lys Val Gly Ala Ala 370 375 380 Thr Glu Val
Glu Met Lys Glu Lys Lys Ala Arg Val Glu Asp Ala Leu 385 390 395 400
His Ala Thr Arg Ala Ala Val Glu Glu Gly Val Val Ala Gly Gly Gly 405
410 415 Val Ala Leu Ile Arg Val Ala Ser Lys Leu Ala Asp Leu Arg Gly
Gln 420 425 430 Asn Glu Asp Gln Asn Val Gly Ile Lys Val Ala Leu Arg
Ala Met Glu 435 440 445 Ala Pro Leu Arg Gln Ile Val Leu Asn Cys Gly
Glu Glu Pro Ser Val 450 455 460 Val Ala Asn Thr Val Lys Gly Gly Asp
Gly Asn Tyr Gly Tyr Asn Ala 465 470 475 480 Ala Thr Glu Glu Tyr Gly
Asn Met Ile Asp Met Gly Ile Leu Asp Pro 485 490 495 Thr Lys Val Thr
Arg Ser Ala Leu Gln Tyr Ala Ala Ser Val Ala Gly 500 505 510 Leu Met
Ile Thr Thr Glu Cys Met Val Thr Asp Leu Pro Lys Asn Asp 515 520 525
Ala Ala Asp Leu Gly Ala Ala Gly Gly Met Gly Gly Met Gly Gly Met 530
535 540 Gly Gly Met Met 545 1022PRTArtificialsynthetic peptide
10Val Ala Gly Gly Gly Val Thr Leu Leu Gln Ala Ala Pro Thr Leu Asp 1
5 10 15 Glu Leu Lys Leu Glu Gly 20 11573PRTHomo sapiens 11Met Leu
Arg Leu Pro Thr Val Phe Arg Gln Met Arg Pro Val Ser Arg 1 5 10 15
Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 20
25 30 Gly Ala Asp Ala Arg Ala Leu Met Leu Gln Gly Val Asp Leu Leu
Ala 35 40 45 Asp Ala Val Ala Val Thr Met Gly Pro Lys Gly Arg Thr
Val Ile Ile 50 55 60 Glu Gln Gly Trp Gly Ser Pro Lys Val Thr Lys
Asp Gly Val Thr Val 65 70 75 80 Ala Lys Ser Ile Asp Leu Lys Asp Lys
Tyr Lys Asn Ile Gly Ala Lys 85 90 95 Leu Val Gln Asp Val Ala Asn
Asn Thr Asn Glu Glu Ala Gly Asp Gly 100 105 110 Thr Thr Thr Ala Thr
Val Leu Ala Arg Ser Ile Ala Lys Glu Gly Phe 115 120 125 Glu Lys Ile
Ser Lys Gly Ala Asn Pro Val Glu Ile Arg Arg Gly Val 130 135 140 Met
Leu Ala Val Asp Ala Val Ile Ala Glu Leu Lys Lys Gln Ser Lys 145 150
155 160 Pro Val Thr Thr Pro Glu Glu Ile Ala Gln Val Ala Thr Ile Ser
Ala 165 170 175 Asn Gly Asp Lys Glu Ile Gly Asn Ile Ile Ser Asp Ala
Met Lys Lys 180 185 190 Val Gly Arg Lys Gly Val Ile Thr Val Lys Asp
Gly Lys Thr Leu Asn 195 200 205 Asp Glu Leu Glu Ile Ile Glu Gly Met
Lys Phe Asp Arg Gly Tyr Ile 210 215 220 Ser Pro Tyr Phe Ile Asn Thr
Ser Lys Gly Gln Lys Cys Glu Phe Gln 225 230 235 240 Asp Ala Tyr Val
Leu Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln Ser 245 250 255 Ile Val
Pro Ala Leu Glu Ile Ala Asn Ala His Arg Lys Pro Leu Val 260 265 270
Ile Ile Ala Glu Asp Val Asp Gly Glu Ala Leu Ser Thr Leu Val Leu 275
280 285 Asn Arg Leu Lys Val Gly Leu Gln Val Val Ala Val Lys Ala Pro
Gly 290 295 300 Phe Gly Asp Asn Arg Lys Asn Gln Leu Lys Asp Met Ala
Ile Ala Thr 305 310 315 320 Gly Gly Ala Val Phe Gly Glu Glu Gly Leu
Thr Leu Asn Leu Glu Asp 325 330 335 Val Gln Pro His Asp Leu Gly Lys
Val Gly Glu Val Ile Val Thr Lys 340 345 350 Asp Asp Ala Met Leu Leu
Lys Gly Lys Gly Asp Lys Ala Gln Ile Glu 355 360 365 Lys Arg Ile Gln
Glu Ile Ile Glu Gln Leu Asp Val Thr Thr Ser Glu 370 375 380 Tyr Glu
Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly 385 390 395
400 Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp Val Glu Val Asn Glu
405 410 415 Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala
Ala Val 420 425 430 Glu Glu Gly Ile Val Leu Gly Gly Gly Cys Ala Leu
Leu Arg Cys Ile 435 440 445 Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn
Glu Asp Gln Lys Ile Gly 450 455 460 Ile Glu Ile Ile Lys Arg Thr Leu
Lys Ile Pro Ala Met Thr Ile Ala 465 470 475 480 Lys Asn Ala Gly Val
Glu Gly Ser Leu Ile Val Glu Lys Ile Met Gln 485 490 495 Ser Ser Ser
Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe Val Asn 500 505 510 Met
Val Glu Lys Gly Ile Ile Asp Pro Thr Lys Val Val Arg Thr Ala 515 520
525 Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr Thr Ala Glu Val
530 535 540 Val Val Thr Glu Ile Pro Lys Glu Glu Lys Asp Pro Gly Met
Gly Ala 545 550 555 560 Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly
Met Phe 565 570 1221PRTArtificialsynthetic peptide 12Phe Asn Glu
Glu Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser 1 5 10 15 Ala
Ser His Leu Glu 20 1321DNAArtificialsynthetic oligonucleotide
13aagagcgagt accagctggt g 211415PRTArtificialsynthetic peptide
14Glu Glu Ser Asn Thr Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly 1 5
10 15 1520PRTArtificialsynthetic peptide 15Ala Tyr Asp Glu Glu Ala
Arg Arg Gly
Leu Glu Arg Gly Leu Asn Ala 1 5 10 15 Leu Ala Asp Ala 20
1620PRTArtificialsynthetic peptide 16Lys Phe Gly Ala Asp Ala Arg
Ala Leu Met Leu Gln Gly Val Asp Leu 1 5 10 15 Leu Ala Asp Ala
20
* * * * *