U.S. patent application number 14/031919 was filed with the patent office on 2014-04-03 for screening method for the identification of agents capable of activating cd4+cd25+ regulatory t-cells through interactions with the hiv-1 gp120 binding site on cd4.
This patent application is currently assigned to TCF GMBH. The applicant listed for this patent is TCF GMBH. Invention is credited to Christian Becker, Tobias Bopp, Helmut Jonuleit, Edgar Schmitt, Franz-Josef Schneider.
Application Number | 20140093509 14/031919 |
Document ID | / |
Family ID | 39363828 |
Filed Date | 2014-04-03 |
United States Patent
Application |
20140093509 |
Kind Code |
A1 |
Schneider; Franz-Josef ; et
al. |
April 3, 2014 |
Screening Method for the Identification of Agents Capable of
Activating CD4+CD25+ Regulatory T-Cells Through Interactions with
the HIV-1 GP120 Binding Site on CD4
Abstract
The present invention relates specific activation of a
regulatory T cell via a specific CD4 epitope and uses thereof, e.g.
for the treatment of an autoimmune disease or an allergy or asthma
or graft rejection or tolerance induction.
Inventors: |
Schneider; Franz-Josef;
(Laupheim, DE) ; Becker; Christian; (Mainz,
DE) ; Bopp; Tobias; (Aabergen, DE) ; Jonuleit;
Helmut; (Russelsheim, DE) ; Schmitt; Edgar;
(Mainz, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TCF GMBH |
FRANKFURT |
|
DE |
|
|
Assignee: |
TCF GMBH
FRANKFURT
DE
|
Family ID: |
39363828 |
Appl. No.: |
14/031919 |
Filed: |
September 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12525142 |
Oct 7, 2009 |
8557533 |
|
|
PCT/EP2008/051144 |
Jan 30, 2008 |
|
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14031919 |
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Current U.S.
Class: |
424/139.1 ;
424/188.1; 530/324; 530/387.9 |
Current CPC
Class: |
A61P 11/02 20180101;
A61P 7/06 20180101; A61P 1/18 20180101; A61P 27/02 20180101; G01N
33/505 20130101; A61P 1/04 20180101; A61P 37/04 20180101; A61P 5/14
20180101; A61P 43/00 20180101; C12N 7/00 20130101; A61P 17/00
20180101; A61P 17/04 20180101; A61P 37/08 20180101; C12N 2740/16111
20130101; A61P 35/00 20180101; A61P 1/00 20180101; A61K 38/162
20130101; A61P 13/12 20180101; A61P 19/02 20180101; A61P 27/14
20180101; A61P 3/10 20180101; A61P 11/06 20180101; G01N 2333/162
20130101; A61P 21/04 20180101; A61P 37/02 20180101; A61P 35/02
20180101; G01N 2333/7051 20130101; A61P 1/16 20180101; C12N
2740/16122 20130101; A61P 17/06 20180101; A61P 21/02 20180101; A61P
25/00 20180101; C07K 16/18 20130101; A61P 37/06 20180101; A61P
11/00 20180101; A61P 11/08 20180101; A61P 11/16 20180101; A61P
29/00 20180101; C07K 14/005 20130101; A61K 38/00 20130101; A61P
17/02 20180101; A61P 1/14 20180101; C12N 2740/16033 20130101 |
Class at
Publication: |
424/139.1 ;
530/324; 530/387.9; 424/188.1 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C07K 14/005 20060101 C07K014/005 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2007 |
EP |
07101604.2 |
Dec 5, 2007 |
EP |
07122424.0 |
Claims
1. A CD4.sup.+CD25.sup.+ Treg cell activator which activates
CD4.sup.+CD25.sup.+ Treg cell via interaction with the
CD4.sup.+CD25.sup.+ Treg cell epitope as set forth in SEQ ID NO:1,
said CD4.sup.+CD25.sup.+ Treg cell activator being a peptide, a
polypeptide, or an antibody or a binding fragment thereof with the
proviso that the antibody or antibody fragment is not OKT4A, OKT4D,
OKTcdr4a, MAX.12H5 and not Leu3.
2. The CD4.sup.+CD25.sup.+ Treg cell activator according to claim 1
wherein said CD4.sup.+CD25.sup.+ Treg cell activator is a peptide
comprising the amino acid sequence as set forth in SEQ ID NO.: 3,
SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID
NO.: 8, SEQ ID NO.: 9, or SEQ ID NO.: 10.
3. A method of treating an individual in need thereof comprising
administering a CD4.sup.+CD25.sup.+ Treg cell activator which
activates CD4.sup.+CD25.sup.+ Treg cell via interaction with the
CD4.sup.+CD25.sup.+ Treg cell epitope as set forth in SEQ ID NO:1,
wherein said CD4.sup.+CD25.sup.+ Treg cell activator is a peptide,
a polypeptide, or an antibody or a binding fragment thereof with
the proviso that the antibody or antibody fragment is not OKT4A,
OKT4D, OKTcdr4a, MAX.12H5 and not Leu3.
4. The method of treating an individual in need thereof according
to claim 3 wherein said Treg cell activator is a peptide comprising
the amino acid sequence as set forth in SEQ ID NO.: 3, SEQ ID NO.:
4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ
ID NO.: 9, or SEQ ID NO.: 10.
5. The method of treating an individual in need thereof according
to claim 3 wherein said CD4.sup.+CD25.sup.+ Treg cell activator is
HIV-1 gp120.
6. The method of treating an individual in need thereof according
to claim 3 wherein said CD4.sup.+CD25.sup.+ Treg cell activator is
selected from a group consisting of: NSC 13778, peptide2 which
presents three HIV-1 gp120 fragments bound together through
comformationally flexible scaffolds, monoclonal antibody OKT4A,
monoclonal antibody OKT4D, monoclonal antibody OKTcdr4a, monoclonal
antibody MAX. 12H5, and monoclonal antibody Leu3.
7. Use of a CD4.sup.+CD25.sup.+ Treg cell activator which activates
CD4.sup.+CD25.sup.+ Treg cell via interaction with the
CD4.sup.+CD25.sup.+ Treg cell epitope as set forth in SEQ ID NO:1
for the preparation of a medicament for the treatment of a disease
selected from a group consisting of a non-autoimmune inflammatory
disease, an autoimmune inflammatory disease, an inflammatory
disease due to organ transplantation; a bone marrow
transplantation, and a disease due to exogenously administered self
or exogenously administered non-autologous recombinant polypeptide,
wherein said CD4.sup.+CD25.sup.+ Treg cell activator is an antibody
or an antibody fragment capable of binding to the peptide as set
forth in SEQ ID NO.:1 or a peptide comprising the amino acid
sequence as set forth in SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.:
5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, or
SEQ ID NO.: 10.
8. The use according to according to claim 7 wherein said
non-autoimmune inflammatory disease is selected from a group
consisting of: asthma, allergic asthma, respiratory allergy,
allergic rhinoconjunctivitis, allergic alveolitis, contact allergy,
atopic dermatitis, neurodermatitis, food allergy, graft-versus-host
disease, non-autoimmune inflammatory bowel disease, acute
respiratory distress syndrome, acute inflammatory pancreatitis,
burns, wound healing, skin scarring disorders, sarcoidosis,
Behcet's disease, Sweet's syndrome.
9. The use according to according to claim 7 wherein said an
autoimmune inflammatory disease is selected from a group consisting
of: rheumatoid arthritis, rheumatic fever, systemic lupus
erythematosus, ulcerative colitis, Crohn's disease, autoimmune
inflammatory bowel disease, diabetes type I, gastritis, autoimmune
atrophic gastritis, autoimmune hepatitis, Hashimoto's thyroiditis,
thyreoiditis, multiple sclerosis, myasthenia gravis, autoimmune
haemolytic anemia, Addison's disease, scleroderma, Goodpasture's
syndrome, Guillain-Barre syndrome, Graves' disease,
glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid,
vitiligo, idiopathic leukopenia, Sjogren's syndrome, Wegener's
granulomatosis.
10. The use according to according to claim 7 wherein said
CD4.sup.+CD25.sup.+ Treg cell activator is HIV-1 gp120.
11. The use according to according to claim 7 wherein said
CD4.sup.+CD25.sup.+ Treg cell activator is selected from a group
consisting of: NSC 13778, peptide2 which presents three HIV-1 gp120
fragments bound together through comformationally flexible
scaffolds, monoclonal antibody OKT4A, monoclonal antibody OKT4D,
monoclonal antibody OKTcdr4a, monoclonal antibody MAX. 12H5, and
monoclonal antibody Leu3.
12. A pharmaceutical composition comprising at least one
CD4.sup.+CD25.sup.+ Treg cell activator which activates
CD4.sup.+CD25.sup.+ Treg cell via interaction with the
CD4.sup.+CD25.sup.+ Treg cell epitope as set forth in SEQ ID NO:1
wherein said CD4.sup.+CD25.sup.+ Treg cell activator is a peptide,
a polypeptide, or an antibody or a binding fragment thereof with
the proviso that the antibody or antibody fragment is not OKT4A,
OKT4D, OKTcdr4a, MAX.12H5 and not Leu3.
13. The pharmaceutical composition according to claim 12 wherein
said CD4.sup.+CD25.sup.+ Treg cell activator is a peptide
comprising the amino acid sequence as set forth in SEQ ID NO.: 3,
SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID
NO.: 8, SEQ ID NO.: 9, or SEQ ID NO.: 10.
14. A method for reducing or preventing an unwanted immune reaction
due to a exogenously administered self or exogenously administered
non-autologous recombinant polypeptide comprising administering to
a being in need thereof a suitable amount of a pharmaceutical
composition comprising a CD4.sup.+CD25.sup.+ Treg cell activator
which is an antibody or an antibody fragment capable of binding to
the peptide as set forth in SEQ ID NO.:1 or is a peptide comprising
the amino acid sequence as set forth in SEQ ID NO.: 3, SEQ ID NO.:
4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ
ID NO.: 9, or SEQ ID NO.: 10.
15. The method according to claim 14 wherein said
CD4.sup.+CD25.sup.+ Treg cell activator is HIV-1 gp120.
16. The method according to claim 14 wherein said Treg cell
activator is selected from a group consisting of: NSC 13778,
peptide 2 which presents three HIV-1 gp120 fragments bound together
through comformationally flexible scaffolds, monoclonal antibody
OKT4A, monoclonal antibody OKT4D, monoclonal antibody OKTcdr4a,
monoclonal antibody MAX. 12H5, and monoclonal antibody Leu3.
Description
PRIORITY CLAIM
[0001] In accordance with 37 C.F.R. 1.76, a claim of priority is
included in an Application Data Sheet filed concurrently herewith.
Accordingly, the present invention claims priority as a divisional
of U.S. National Phase patent application Ser. No. 12/525,142;
entitled "SCREENING METHOD FOR THE IDENTIFICATION OF AGENTS CAPABLE
OF ACTIVATING CD4.sup.+CD25.sup.+REGULATORY T-CELLS THROUGH
INTERACTIONS WITH THE HIV-1 GP120 BINDING SITE ON CD4", filed Oct.
7, 2009; which claims priority to International PCT Patent
Application No. PCT/EP2008/051144, entitled "SPECIFIC ACTIVATION OF
A REGULATORY T CELL AND ITS USE FOR TREATMENT OF ASTHMA, ALLERGIC
DISEASE, AUTOIMMUNE DISEASE, GRAFT REJECTION AND FOR TOLERANCE
INDUCTION", filed Jan. 30, 2008, which claims priority to European
Patent Application No. 07101604.2, entitled "SPECIFIC ACTIVATION OF
A REGULATORY T CELL AND ITS USE FOR TREATMENT OF ASTHMA, ALLERGIC
DISEASE, AUTOIMMUNE DISEASE, GRAFT REJECTION AND FOR TOLERANCE
INDUCTION", filed Feb. 1, 2007 and 07122424.0, entitled "SPECIFIC
ACTIVATION OF A REGULATORY T CELL AND ITS USE FOR TREATMENT OF
ASTHMA, ALLERGIC DISEASE, AUTOIMMUNE DISEASE, GRAFT REJECTION AND
FOR TOLERANCE INDUCTION", filed Dec. 5, 2007. The contents of the
above referenced applications are incorporated herein by
reference.
INTRODUCTION
[0002] Asthma, an allergic disease, transplant rejection and an
autoimmune disease have one fundamental principle in common, they
all are triggered by an imbalanced immune system which reacts
hyperactive against a specific exogenic and/or endogenic challenge
and therewith contributes significantly to the disease status.
[0003] It is generally accepted that such aberrations of the immune
system have a common pathophysiological mechanism triggered by
hyper-responsive effector T cells playing a central role in immune
reactivity. Effector T cell-directed immunomodulation therefore is
the key to successful treatment of asthma, an autoimmune condition,
prevention of graft vs. host disease (GVHD) and prevention of graft
rejection.
[0004] T lymphocytes, designated as regulatory T cells ("Treg
cells") control immune responses by suppressing the effector
function of CD4+ T cells and CD8.sup.+ T cells (Shevach 2002).
Different subsets of Treg cells have been described. These include
but are not limited to (i) CD4.sup.+CD25.sup.+Treg cells--also
designated as "naturally occurring Treg cells" (Sakaguchi 2005),
(ii) Tr1 (Roncarolo et al. 2001) and (iii) Th3 (Weiner 2001). Tr1
and Th3 are induced in the periphery, whereas CD4.sup.+CD25.sup.+
Treg cells develop in the thymus and constitute 5-10% of peripheral
CD4.sup.+ T cells in healthy man. At least in vitro these cells are
anergic, produce minimal amounts of cytokines and exert their
suppressive effects only upon stimulation and in a strictly
cell-contact dependent manner. Tr1 and Th3 exert their suppressive
activity by production of IL-10 and TGF-beta, respectively (Shevach
2002).
[0005] Genetic defects that primarily affect Treg cell development
or function should cause autoimmune and inflammatory aberrations.
IPEX syndrome (immunodysregulation, polyendo-crinopathy and
enteropathy, X-linked), a rare recessive disorder in humans is
caused by a mutation in the gene of the transcription factor FOXP3
and subsequent absence of Treg cells. IPEX shows aggressive
autoimmunity, severe eczema, elevated IgE levels, eosinophilia and
food allergies and early death (Fontenot and Rudensky 2005).
[0006] Data from the literature show that Treg cells play an
important role in asthma and autoimmune diseases and have a
potential for treatment of GVHD and therewith transplantation
tolerance (Robinson 2004, Sakaguchi 2005).
[0007] Therefore, attempts have been started to use Treg cells as a
therapeutic agent for patients with established autoimmune disease
(Horwitz et al. 2003). It is believed that said patients lack
sufficient Treg cells or do have impaired Treg cell function
resulting in misdirected and uncontrolled effector T cell activity.
The previous thinking for solving this problem is administering
Treg cells to a said patient. Since Treg cells are rare in
peripheral blood clinical application of human Treg cells depends
on highly expensive ex vivo expansion of Treg cells (Hoffmann et
al. 2004, Horwitz et al. 2003, Tang et al. 2003, Zheng et al.
2004). Bluestone and Tang went one step further: They are trying to
solve the problem by not only increasing the amount of Treg cells
for therapy but enhancing suppressive activity of Treg cells by
activating Treg cells via triggering of the T cell receptor (TCR)
by an anti-CD3 antibody (Bluestone and Tang 2004). This approach
resembles by far no Treg cell specific activation as anti-CD3
activates all T cell receptor-expressing cells which bears the
obstacle that anti-CD3 treatment induces effector T cell function
probably leading to uncontrolled proliferation and non-specific
pro-inflammatory cytokine production and exaggeration of pathology.
To circumvent this unwanted triggering of effector T cells Treg
cells have to be highly purified and activated ex vivo with
anti-CD3 which again is a highly expensive and laborious procedure.
In addition, the absence of a Treg cell specific marker makes it
difficult to achieve high purity of Treg cells.
[0008] Therefore, it is a primary goal to identify a substance
which can activate Treg cells specifically without stimulating the
immune system any further and therefore would provide a basis for a
direct in vivo application without money consuming ex vivo
treatments.
[0009] There is some information in the prior art how Treg cells
possibly can be activated, e.g. nonspecifically via CD3 (Thornton
and Shevach 1998), or via CD28/B7 pathway (Shevach 2002, Bluestone
and Tang 2004, Hunig and Dennehy 2005), or via CD4 (WO04083247). So
far all this solutions do not result in a specific Treg cell
activation.
DESCRIPTION OF THE INVENTION
[0010] The present invention bases on the new finding that a
specific epitope of CD4 triggers activation of Treg cells. Said
epitope overlaps with the known HIV-1 gp120-binding site but
surprisingly binding to this site causes Treg cell activation. This
finding was totally unexpected for the following reasons: Until now
Treg cell activation via CD4 was attributed to a different epitope
to which the monoclonal antibody BF5 (WO04083247) binds.--Carriere
et al., 1995 have investigated that the binding site of BF5 on CD4
is completely independent of the HIV-1 gp120-binding
site.--Unexpectedly, despite reports in the literature on direct
anergizing and blocking of CD4 on T cell function by HIV-1 gp120
(Diamond et al. 1988), we found an activating property of HIV-1
gp120 on CD4 of Treg cells.
[0011] The present invention discloses a physiologically active CD4
epitope on a Treg cell which triggers suppressive activity of a
Treg cell. The epitope of the present invention has been identified
as a region on the human CD4 protein (SEQ ID NO.: 2) spanning amino
acid position No. 54 to 84 of SEQ ID NO.:2. Those 31 amino acids
are explicitly given in SEQ ID NO.:1.
[0012] A preferred peptide which is a CD4 fragment according to the
present invention is selected from a group consisting of an
isolated peptide spanning amino acid No. 1-31 of SEQ ID NO 1, No.
26 to 458 of SEQ ID NO.: 2, No. 26 to 419 of SEQ ID NO.:2, No. 26
to 207 of SEQ ID NO.: 2, No. 26 to 131 of SEQ ID NO.: 2 and No. 46
to 89 of SEQ ID NO.: 2. All said peptides are additionally
characterized in that they all do harbor the critical amino acid
Phenylalanine at residue 68 of SEQ ID NO.: 2.
[0013] The finding that the epitope given in SEQ ID NO.:1 is a key
to activate a Treg cell provides a basis for several uses,
e.g.:
[0014] Methods for identification of a substance which can activate
a regulatory T cell (Treg cell). Such a substance is designated
"Treg cell activator" of the present invention which is useful for
the treatment of a disease of the invention which is a disease in
which increase of activated regulatory T cells (Treg cells) can
improve clinical picture wherein such a disease is (i) a
non-autoimmune inflammatory disease: asthma, allergic asthma,
respiratory allergy, allergic rhinoconjunctivitis, allergic
alveolitis, contact allergy, atopic dermatitis, neurodermatitis,
food allergy, graft-versus-host disease, non-autoimmune
inflammatory bowel disease, acute respiratory distress syndrome,
acute inflammatory pancreatitis, burns, wound healing, skin
scarring disorders, sarcoidosis, Behcet's disease or Sweet's
syndrome; (ii) an autoimmune inflammatory disease: rheumatoid
arthritis, rheumatic fever, systemic lupus erythematosus,
ulcerative colitis, Crohn's disease, autoimmune inflammatory bowel
disease, diabetes type I, gastritis, autoimmune atrophic gastritis,
autoimmune hepatitis, Hashimoto's thyroiditis, thyreoiditis,
multiple sclerosis, myasthenia gravis, autoimmune haemolytic
anemia, Addison's disease, scleroderma, Goodpasture's syndrome,
Guillain-Barre syndrome, Graves' disease, glomerulonephritis,
psoriasis, pemphigus vulgaris, pemphigoid, vitiligo, idiopathic
leukopenia, Sjogren's syndrome or Wegener's granulomatosis; (iii)
an inflammatory disease due to organ transplantation; (iv) a bone
marrow transplantation; or (v) a disease due to exogenously
administered self or exogenously administered non-autologous
recombinant polypeptide.
[0015] A "substance" of the present invention can be used in a
method according to the present invention. The meaning of the term
substance according to the present invention includes but is not
limited to a peptide, a scaffolded peptide, an antibody, a fragment
of an antibody, a nucleic acid molecule, a ribozyme, an organic
compound or an inorganic compound.
[0016] In a second aspect the new epitope serves as the basis for
the synthesis of a new tool which can be used e.g. in a competition
assay or screening assay according to the present invention for
determining whether a substance can activate a regulatory T cell
(Treg cell) via interaction with the epitope as set forth in SEQ ID
NO.:1. Such a tool is a peptide of the present invention which is
an isolated peptide spanning amino acid No. 1-31 of SEQ ID NO 1, or
No. 26 to 458 of SEQ ID NO.: 2, or No. 26 to 419 of SEQ ID NO.:2,
or No. 26 to 207 of SEQ ID NO.: 2, or No. 26 to 131 of SEQ ID NO.:
2 or No. 46 to 89 of SEQ ID NO.: 2, or is an isolated peptide
spanning amino acid No. 1 to 31 of SEQ ID NO.: 1 and having
additional up-stream and/or downstream amino acids with the
prerequisite that the additional amino acids do not hinder binding
of a substance to the amino acid stretch as set forth in SEQ ID
NO.: 1. A preferred peptide according to the present invention
consist of the peptide as set forth in SEQ ID NO.:1 and
additionally consist of an additional up-stream amino acid or amino
acid stretch which is selected from a group consisting of the amino
acid or amino acid stretch as set forth in SEQ ID NO.: 2 at
position 53, at position 52-53, at position 51-53, at position
50-53, at position 49-53, at position 48-53, at position 47-53, at
position 46-53, at position 45-53, at position 44-53, at position
43-53, at position 42-53, at position 41-53, at position 40-53, at
position 39-53, at position 38-53, at position 37-53, at position
36-53, at position 35-53, at position 34-53, at position 33-53, at
position 32-53, at position 31-53, at position 30-53, at position
29-53, at position 28-53, at position 27-53, at position 26-53, at
position 25-53, at position 24-53, at position 23-53, at position
22-53, at position 21-53, at position 20-53, at position 19-53, at
position 18-53, at position 17-53, at position 16-53, at position
15-53, at position 14-53, at position 13-53, at position 12-53, at
position 11-53, at position 10-53, at position 9-53, at position
8-53, at position 7-53, at position 6-53, at position 5-53, at
position 4-53, at position 3-53, at position 2-53, and at position
1-53.
[0017] A further preferred peptide according to the present
invention consists of the peptide which is mentioned as "preferred
peptide" in the paragraph above and additionally consists of at
least one additional downstream amino acid as given in SEQ ID NO.:
2 at position 85, or additionally consists of amino acids as given
in SEQ ID NO.:2 at position 85 to n, wherein n is an integer
between 86-458, i.e. position 85 to 86, 85 to 87, 85 to 88, 85 to
89, 85 to 90, 85 to 91, 85 to 92, 85 to 93, 85 to 94, 85 to 95, 85
to 96, 85 to 97, 85 to 98, 85 to 99, 85 to 100, 85 to 101, 85 to
102, 85 to 103, 85 to 104, 85 to 105, 85 to 106, 85 to 107, 85 to
108, 85 to 109, 85 to 110, 85 to 111, 85 to 112, 85 to 113, 85 to
114, 85 to 115, 85 to 116, 85 to 117, 85 to 118, 85 to 119, 85 to
120, 85 to 121, 85 to 122, 85 to 123, 85 to 124, 85 to 125, 85 to
126, 85 to 127, 85 to 128, 85 to 129, 85 to 130, 85 to 131, 85 to
132, 85 to 133, 85 to 134, 85 to 135, 85 to 136, 85 to 137, 85 to
138, 85 to 139, 85 to 140, 85 to 141, 85 to 142, 85 to 143, 85 to
144, 85 to 145, 85 to 146, 85 to 147, 85 to 148, 85 to 149, 85 to
150, 85 to 151, 85 to 152, 85 to 153, 85 to 154, 85 to 155, 85 to
156, 85 to 157, 85 to 158, 85 to 159, 85 to 160, 85 to 161, 85 to
162, 85 to 163, 85 to 164, 85 to 165, 85 to 166, 85 to 167, 85 to
168, 85 to 169, 85 to 170, 85 to 171, 85 to 172, 85 to 173, 85 to
174, 85 to 175, 85 to 176, 85 to 177, 85 to 178, 85 to 179, 85 to
180, 85 to 181, 85 to 182, 85 to 183, 85 to 184, 85 to 185, 85 to
186, 85 to 187, 85 to 188, 85 to 189, 85 to 190, 85 to 191, 85 to
192, 85 to 193, 85 to 194, 85 to 195, 85 to 196, 85 to 197, 85 to
198, 85 to 199, 85 to 200, 85 to 201, 85 to 202, 85 to 203, 85 to
204, 85 to 205, 85 to 206, 85 to 207, 85 to 208, 85 to 209, 85 to
210, 85 to 211, 85 to 212, 85 to 213, 85 to 214, 85 to 215, 85 to
216, 85 to 217, 85 to 218, 85 to 219, 85 to 220, 85 to 221, 85 to
222, 85 to 223, 85 to 224, 85 to 225, 85 to 226, 85 to 227, 85 to
228, 85 to 229, 85 to 230, 85 to 231, 85 to 232, 85 to 233, 85 to
234, 85 to 235, 85 to 236, 85 to 237, 85 to 238, 85 to 239, 85 to
240, 85 to 241, 85 to 242, 85 to 243, 85 to 244, 85 to 245, 85 to
246, 85 to 247, 85 to 248, 85 to 249, 85 to 250, 85 to 251, 85 to
252, 85 to 253, 85 to 254, 85 to 255, 85 to 256, 85 to 257, 85 to
258, 85 to 259, 85 to 260, 85 to 261, 85 to 262, 85 to 263, 85 to
264, 85 to 265, 85 to 266, 85 to 267, 85 to 268, 85 to 269, 85 to
270, 85 to 271, 85 to 272, 85 to 273, 85 to 274, 85 to 275, 85 to
276, 85 to 277, 85 to 278, 85 to 279, 85 to 280, 85 to 281, 85 to
282, 85 to 283, 85 to 284, 85 to 285, 85 to 286, 85 to 287, 85 to
288, 85 to 289, 85 to 290, 85 to 291, 85 to 292, 85 to 293, 85 to
294, 85 to 295, 85 to 296, 85 to 297, 85 to 298, 85 to 299, 85 to
300, 85 to 301, 85 to 302, 85 to 303, 85 to 304, 85 to 305, 85 to
306, 85 to 307, 85 to 308, 85 to 309, 85 to 310, 85 to 311, 85 to
312, 85 to 313, 85 to 314, 85 to 315, 85 to 316, 85 to 317, 85 to
318, 85 to 319, 85 to 320, 85 to 321, 85 to 322, 85 to 323, 85 to
324, 85 to 325, 85 to 326, 85 to 327, 85 to 328, 85 to 329, 85 to
330, 85 to 331, 85 to 332, 85 to 333, 85 to 334, 85 to 335, 85 to
336, 85 to 337, 85 to 338, 85 to 339, 85 to 340, 85 to 341, 85 to
342, 85 to 343, 85 to 344, 85 to 345, 85 to 346, 85 to 347, 85 to
348, 85 to 349, 85 to 350, 85 to 351, 85 to 352, 85 to 353, 85 to
354, 85 to 355, 85 to 356, 85 to 357, 85 to 358, 85 to 359, 85 to
360, 85 to 361, 85 to 362, 85 to 363, 85 to 364, 85 to 365, 85 to
366, 85 to 367, 85 to 368, 85 to 369, 85 to 370, 85 to 371, 85 to
372, 85 to 373, 85 to 374, 85 to 375, 85 to 376, 85 to 377, 85 to
378, 85 to 379, 85 to 380, 85 to 381, 85 to 382, 85 to 383, 85 to
384, 85 to 385, 85 to 386, 85 to 387, 85 to 388, 85 to 389, 85 to
390, 85 to 391, 85 to 392, 85 to 393, 85 to 394, 85 to 395, 85 to
396, 85 to 397, 85 to 398, 85 to 399, 85 to 400, 85 to 401, 85 to
402, 85 to 403, 85 to 404, 85 to 405, 85 to 406, 85 to 407, 85 to
408, 85 to 409, 85 to 410, 85 to 411, 85 to 412, 85 to 413, 85 to
414, 85 to 415, 85 to 416, 85 to 417, 85 to 418, 85 to 419, 85 to
420, 85 to 421, 85 to 422, 85 to 423, 85 to 424, 85 to 425, 85 to
426, 85 to 427, 85 to 428, 85 to 429, 85 to 430, 85 to 431, 85 to
432, 85 to 433, 85 to 434, 85 to 435, 85 to 436, 85 to 437, 85 to
438, 85 to 439, 85 to 440, 85 to 441, 85 to 442, 85 to 443, 85 to
444, 85 to 445, 85 to 446, 85 to 447, 85 to 448, 85 to 449, 85 to
450, 85 to 451, 85 to 452, 85 to 453, 85 to 454, 85 to 455, 85 to
456, 85 to 457, or 85 to 458.
[0018] Additionally, the finding that the epitope given in SEQ ID
NO.:1 is a key to activate a Treg cell is a link between two
different up to now unrelated technical fields, namely that of (a)
HIV-1 related diseases with (b) diseases according to the present
invention e.g. autoimmune disease, allergy, asthma, graft rejection
and a diseases due to lacking immunotolerance caused by organ
transplantation or by therapeutical administration of a non-self or
self biological entity to a human in need thereof and therewith
allows a bundle of new uses as explained in the following:
[0019] The epitope given in SEQ ID NO.:1 is not only a further
epitope which can be used to activate Treg cells. HIV-1 gp120
interacts with CD4 of T cells and therewith enables virus entry
into a CD4.sup.+ cell (Klatzmann et al. 1984). The finding that
epitope given in SEQ ID NO.:1 harbors the high affinity binding
site on CD4 to which human immune deficiency virus 1 (HIV-1)
glycoprotein gp120 binds (Jameson et al. 1988, Arthos et al. 1989)
offers a further advantage. It provides the basis to bring together
the findings of two different unrelated technical fields that of
i.e. HIV-1 related diseases with diseases according to the present
invention.
[0020] To alleviate the worldwide HIV-1 problem many efforts have
been made to identify a substance which is able to inhibit HIV-1
entry into a CD4.sup.+ cell. As a result thereof, so called HIV-1
attachment or entry inhibitors are known in the art.
[0021] The keyhole which allows HIV-1 to enter the cell can be used
as the keyhole to activate Treg cells. Therefore, substances known
in the art to interfere with HIV-1 attachment and cell entry
(Markovic and Clouse 2004, Castagna et al. 2005), like e.g. HIV-1
gp120 itself, derivatives thereof, peptidomimetics, antibodies,
aptamers or any low molecular weight (LMW) compound directed
against the binding site of HIV-1 gp120 on CD4 could possibly be
useful to activate a Treg cell and therewith can be useful for the
treatment of a disease according to the present invention (HIV-1
gp120 is well-known in the art and its amino acid sequence as well
as the respective gene has been published since years (Muesing et
al. 1985, Starcich et al. 1986, Jeffs et al. 1996). Additionally,
methods for producing HIV-1 gp120 are known (Lasky et al. 1986,
Leonard et al. 1990, Culp et al. 1991, Jeffs et al. 1996).
[0022] A substance which can interfere with HIV-1 attachment and/or
cell entry is commonly named HIV-1 attachment inhibitor or entry
inhibitor. Such a substance, do either bind to (i) HIV-1, or (ii)
to CD4, or (iii) HIV-1 and CD4 or (iv) co-receptor e.g. CCR5 or
CXCR4. Such an inhibitor can be according to the present invention
useful for the treatment of diseases according to the present
invention like e.g. an autoimmune disease, an allergy, asthma or
GVHD if it exerts property (ii) or (iii) and activates a Treg cell.
To determine whether such an inhibitor can be useful for a said
disease the present invention discloses several assays which allow
to determine whether a substance identified in the technical field
of HIV-1 research to interfere with HIV-1 attachment and/or cell
entry can be useful in the other above-mentioned technical fields
like that of autoimmune diseases or allergies or asthma or organ
transplantation. Therefore, the present invention teaches a short
cut for identifying a substance which can be useful for the
treatment of a disease according to the present invention.
[0023] Identification of a substance which can be used as a
medicament in a specific disease usually depends on resource
consuming high through put screenings (HTS). Determination whether
a substance can activate a Treg cell currently depends on a
cellular assay comprising a Treg cell. Since Treg cells can only be
provided in small amounts large screening campaigns or even an HTS
therewith are not feasible today. The teaching of the present
invention allows to circumvent this obstacles since the present
invention allows to pre-select substances which possible can
activate a Treg cell. According to the present invention an
appropriate pre-selected substance is (i) proven to interact at
least with epitope (SEQ ID NO.:1), and/or is (ii) known from HIV-1
research as HIV-1 attachment inhibitor or entry inhibitor or
synthetic mimetics of the CD4 binding site of HIV-1 gp120.
[0024] It is state of the art to determine whether a substance can
interact with a given peptide and therewith with a given epitope
even in an HTS format. Concerning the present invention this can be
performed for example in an in vitro competition type assay
comprising a peptide spanning at least amino acids as set forth in
SEQ ID NO.: 1 mixed with an unlabeled substance to be tested and
subsequently with a labeled substance which is known to bind the
peptide (e.g. HIV-1 gp120) under conditions which allow binding of
the peptide with the labeled substance. A substance which interact
with the peptide will compete with the labeled substance and is
identifiable by a rendered readout, which can be performed e.g. by
measuring the bound or free labeled substance.
[0025] Such a type of assay for determining whether a substance can
interact with a specific peptide, i.e. epitope is not restricted to
in vitro assays since cellular assays for achieving such an
information on a substance or other in vitro formats are well known
in the art and broadly used.
[0026] In one embodiment the present invention concerns a method
for determining whether a substance which can interfere with the
interaction of HIV-1 gp120 with CD4 can be useful for positively
influence a disease in which increase of activated regulatory T
cells (Treg cells) can improve clinical picture comprising: [0027]
(a) providing a solution comprising a Treg cell, wherein a Treg
cell is preferably a CD4.sup.4CD25.sup.+ Treg cell or a Tr1 cell or
a Th3 cell. Said solution does more preferably comprise
additionally an inactivated syngenic CD3-depleted PBMC (peripheral
blood mononuclear cell which has preferably been inactivated via
irradiation or via mitomycin C) or a dendritic cell (DC) and an
allogeneic CD8.sup.+ T cell or an allogeneic CD4.sup.+ T cell,
[0028] (b) adding a substance to be tested under conditions which
allow interaction of the substance with a Treg cell, [0029] (c)
measuring whether a Treg cell has been activated, wherein an
activated Treg cell identifies the substance as a Treg cell
activator. [0030] Said measuring can be performed using a suitable
read-out system such as: [0031] (i) measuring whether a CD8.sup.+ T
cell has been suppressed--which preferably can be determined by
measuring inhibited proliferation of the CD8.sup.+ T cell or by
measuring reduced CD25 expression of the CD8.sup.+ T cell, or by
measuring inhibited cytokine production of the CD8.sup.+ T cell
wherein a suitable cytokine is IFN.gamma. or IL2, or
TNF.alpha.--wherein a suppressed CD8.sup.+ T cell identifies an
activated Treg cell and therewith identifies the substance as a
Treg cell activator, or by (ii) measuring whether a CD4.sup.+ T
cell has been suppressed--which preferably can be determined by
measuring inhibited proliferation of the CD4.sup.+ T cell or by
measuring reduced CD25 expression of the CD4.sup.+ T cell, or by
measuring inhibited cytokine production of the CD4.sup.+ T cell
wherein a suitable cytokine is IFN.gamma. or IL2, or
TNF.alpha.--wherein a suppressed CD4.sup.+ T cell identifies an
activated Treg cell and therewith identifies the substance as a
Treg cell activator, or by (iii) measuring the amount of
intracellular cAMP (i.e. cytosolic cAMP) and wherein an increased
amount of intracellular cAMP is indicative for an activated Treg
cell and therewith identifies the substance as a Treg cell
activator.
[0032] A disease according to the method above in which increase of
activated regulatory T cells (Treg cells) can improve clinical
picture is (i) a non-autoimmune inflammatory disease: asthma,
allergic asthma, respiratory allergy, allergic rhinoconjunctivitis,
allergic alveolitis, contact allergy, atopic dermatitis,
neurodermatitis, food allergy, graft-versus-host disease,
non-autoimmune inflammatory bowel disease, acute respiratory
distress syndrome, acute inflammatory pancreatitis, burns, wound
healing, skin scarring disorders, sarcoidosis, Behcet's disease or
Sweet's syndrome; (ii) an autoimmune inflammatory disease:
rheumatoid arthritis, rheumatic fever, systemic lupus
erythematosus, ulcerative colitis, Crohn's disease, autoimmune
inflammatory bowel disease, diabetes type I, gastritis, autoimmune
atrophic gastritis, autoimmune hepatitis, Hashimoto's thyroiditis,
thyreoiditis, multiple sclerosis, myasthenia gravis, autoimmune
haemolytic anemia, Addison's disease, scleroderma, Goodpasture's
syndrome, Guillain-Barre syndrome, Graves' disease,
glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid,
vitiligo, idiopathic leukopenia, Sjogren's syndrome or Wegener's
granulomatosis; (iii) an inflammatory disease due to organ
transplantation; (iv) a bone marrow transplantation; or (v) a
disease due to exogenously administered self or exogenously
administered non-autologous recombinant polypeptide.
[0033] As the readout systems as mentioned in (i) and (ii) above
are methods in which several steps have to be performed a specific
new test system has been invented to determine in only one step
whether a Treg cell has been activated which is mentioned in (iii)
above. Basing on the surprising finding that the activation status
of a Treg cell strongly correlates with the amount of intracellular
cAMP the present invention provides for a specific method for
determining whether a Treg cell has been activated which comprises:
[0034] (a) providing a first solution comprising a Treg cell,
[0035] (b) providing a second solution comprising a Treg cell,
[0036] (c) manipulation the first solution by adding at least a
test substance, [0037] (d) determining the amount of intracellular
cAMP of the first and the second solution [0038] wherein an
increased amount of intracellular cAMP of the first solution is
indicative for an activated Treg cell.
[0039] In a more preferred method the solution of (a) does not only
contain a Treg cell but also an inactivated syngenic CD3-depleted
PBMC (peripheral blood mononuclear cell) or a dendritic cell (DC)
and an allogeneic CD8.sup.+ T cell or an allogeneic CD4.sup.+ T
cell. These cells when combined in one solution with a activated
Treg cell do additionally increase intracellular cAMP amount and
therewith lead to a more sensitive readout system.
[0040] In a further embodiment the present invention concerns a
method for determining whether a substance can activate a Treg cell
via interaction with the HIV-1 gp120-binding site of CD4,
comprising: [0041] (a) pre-selecting a substance which can interact
with the HIV-1 gp120-binding site of CD4 (for a method for
pre-selection please see below), [0042] (b) providing a solution
comprising a Treg cell wherein a Treg cell is preferably a
CD4.sup.+CD25.sup.+ Treg cell or a Tr1 cell or a Th3 cell. Said
solution does more preferably comprise additionally an inactivated
syngenic CD3-depleted PBMC (peripheral blood mononuclear cell)
which has preferably been inactivated via irradiation or via
mitomycin C) or a dendritic cell (DC) and an allogeneic CD8.sup.+ T
cell or an allogeneic CD4.sup.+ T cell, [0043] (c) adding a
pre-selected substance according to (a) under conditions which
allow interaction of the substance with a Treg cell, [0044] (d)
measuring whether a Treg cell has been activated, wherein an
activated Treg cell identifies the substance as a Treg cell
activator. Said measuring can be performed using a suitable
read-out system such as: [0045] (i) measuring whether a CD8.sup.+ T
cell has been suppressed--which preferably can be determined by
measuring inhibited proliferation of the CD8.sup.+ T cell or by
measuring reduced CD25 expression of the CD8.sup.+ T cell, or by
measuring inhibited cytokine production of the CD8.sup.+ T cell
wherein a suitable cytokine is IFN.gamma. or IL2, or
TNF.alpha.--wherein a suppressed CD8.sup.+ T cell identifies an
activated Treg cell and therewith identifies the substance as a
Treg cell activator, or by [0046] (ii) measuring whether a
CD4.sup.+ T cell has been suppressed--which preferably can be
determined by measuring inhibited proliferation of the CD4.sup.+ T
cell or by measuring reduced CD25 expression of the CD4.sup.+ T
cell, or by measuring inhibited cytokine production of the
CD4.sup.+ T cell wherein a suitable cytokine is IFN.gamma. or IL2,
or TNF.alpha.--wherein a suppressed CD4.sup.+ T cell identifies an
activated Treg cell and therewith identifies the substance as a
Treg cell activator, or by [0047] (iii) measuring the amount of
intracellular cAMP and wherein an increased amount of intracellular
cAMP is indicative for an activated Treg cell and therewith
identifies the substance as a Treg cell activator.
[0048] The present invention also provides for a method which allow
to identify a substance which can interact with the HIV-1
gp120-binding site of CD4 and therewith can be used for
pre-selecting a substance which can interact with the HIV-1
gp120-binding site of CD4.
The method comprises: [0049] (a) providing a first solution
comprising CD4, [0050] (b) providing a second solution comprising
CD4, [0051] (c) adding to the first solution a substance to be
tested and HIV-1 gp120 under conditions which allow binding of
HIV-1 gp120 with CD4, [0052] (d) adding to the second solution
HIV-1 gp120 under conditions like (c) allowing binding of HIV-1
gp120 with CD4, [0053] (e) measuring in the first and in the second
solution whether the HIV-1 gp120 has bound to the CD4 wherein a
reduced amount of bound HIV-1 gp120 in the first solution indicates
that the substance can interact with HIV-1 gp120-binding site of
CD4.
[0054] This pre-selecting method can additionally be performed
according to the present invention if instead of CD4 a peptide is
used which comprises the amino acid stretch as set forth in SEQ ID
NO.1 or instead of CD4 a peptide is used as set forth in SEQ ID
NO.:1. In a more preferred method according to the present
invention instead of HIV-1 gp120 a peptide is used selected from a
group consisting of a peptide comprising the amino acid sequence as
set forth in SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID
NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, and SEQ ID
NO.: 10 or a peptide is used selected from a group consisting of a
peptide consisting of the amino acid sequence as set forth in SEQ
ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.:
7, SEQ ID NO.: 8, SEQ ID NO.: 9, and SEQ ID NO.: 10.
[0055] In a further embodiment the present invention concerns a
method for determining whether a substance can activate a
regulatory T cell (Treg) via interaction with the epitope as set
forth in SEQ ID NO: 1 comprising: [0056] (a) providing a first
solution comprising a Treg cell, wherein a Treg cell is preferably
a CD4.sup.+CD25.sup.+ Treg cell or a Tr1 cell or a Th3 cell. Said
solution does more preferably comprise additionally an inactivated
syngenic CD3-depleted PBMC (peripheral blood mononuclear cell which
has preferably been inactivated via irradiation or via mitomycin C)
or a dendritic cell (DC) and an allogeneic CD8.sup.+ T cell or an
allogeneic CD4.sup.+ T cell, [0057] (b) adding a substance to be
tested under conditions which allow interaction of the substance
with a Treg cell, [0058] (c) measuring whether a Treg cell of the
first solution has been activated, [0059] (d) providing a second
solution comprising a Treg cell, wherein a Treg cell is preferably
a CD4.sup.+CD25.sup.+Treg cell or a Tr1 cell or a Th3 cell. Said
solution does more preferably comprise additionally an inactivated
syngenic CD3-depleted PBMC (peripheral blood mononuclear cell which
has preferably been inactivated via irradiation or via mitomycin C)
or a dendritic cell (DC) and an allogeneic CD8.sup.+ T cell or an
allogeneic CD4.sup.+ T cell, (e) adding the substance to be tested
and a peptide comprising an amino acid sequence as set forth in SEQ
ID NO: 1 under conditions like (b), [0060] (f) measuring whether a
Treg cell of the second solution has been activated, [0061] Said
measuring can be performed using a suitable read-out system such
as: [0062] (i) measuring whether a CD8.sup.+ T cell has been
suppressed--which preferably can be determined by measuring
inhibited proliferation of the CD8.sup.+ T cell or by measuring
reduced CD25 expression of the CD8.sup.+ T cell, or by measuring
inhibited cytokine production of the CD8.sup.+ T cell wherein a
suitable cytokine is IFN.gamma. or IL2, or TNF.alpha. wherein a
suppressed CD8.sup.+ T cell identifies an activated Treg cell and
therewith identifies the substance as a Treg cell activator, or by
[0063] (ii) measuring whether a CD4.sup.+ T cell has been
suppressed--which preferably can be determined by measuring
inhibited proliferation of the CD4.sup.+ T cell or by measuring
reduced CD25 expression of the CD4.sup.+ T cell, or by measuring
inhibited cytokine production of the CD4.sup.+ T cell wherein a
suitable cytokine is IFN-.gamma. or IL2, or TNF.alpha. wherein a
suppressed CD4.sup.+ T cell identifies an activated Treg cell and
therewith identifies the substance as a Treg cell activator, or by
[0064] (iii) measuring the amount of intracellular cAMP and wherein
an increased amount of intracellular cAMP is indicative for an
activated Treg cell and therewith identifies the substance as a
Treg cell activator; [0065] (g) comparing results obtained from (c)
with those obtained from (0 wherein a reduced activation of (0
identifies the substance as a Treg cell activator which activates a
Treg cell via interaction with the epitope as set forth in SEQ ID
NO: 1.
[0066] In a more preferred method in step (e) of above mentioned
method the peptide used is selected from a group consisting of an
isolated peptide spanning amino acid No. 1-31 of SEQ ID NO 1, No.
26 to 458 of SEQ ID NO.: 2, No. 26 to 419 of SEQ ID NO.:2, No. 26
to 207 of SEQ ID NO.: 2, No. 26 to 131 of SEQ ID NO.: 2 and No. 46
to 89 of SEQ ID NO.: 2 or the peptide used is an isolated peptide
spanning amino acid No. 1 to 31 of SEQ ID NO.: 1 and having
additional up-stream and/or downstream amino acids with the
prerequisite that the additional amino acids do not hinder binding
of a substance to the amino acid stretch as set forth in SEQ ID
NO.:1. Said additional up-stream amino acid or amino acid stretch
is preferably selected from a group consisting of the amino acid or
amino acid stretch as set forth in SEQ ID NO.: 2 at position 53, at
position 52-53, at position 51-53, at position 50-53, at position
49-53, at position 48-53, at position 47-53, at position 46-53, at
position 45-53, at position 44-53, at position 43-53, at position
42-53, at position 41-53, at position 40-53, at position 39-53, at
position 38-53, at position 37-53, at position 36-53, at position
35-53, at position 34-53, at position 33-53, at position 32-53, at
position 31-53, at position 30-53, at position 29-53, at position
28-53, at position 27-53, at position 26-53, at position 25-53, at
position 24-53, at position 23-53, at position 22-53, at position
21-53, at position 20-53, at position 19-53, at position 18-53, at
position 17-53, at position 16-53, at position 15-53, at position
14-53, at position 13-53, at position 12-53, at position 11-53, at
position 10-53, at position 9-53, at position 8-53, at position
7-53, at position 6-53, at position 5-53, at position 4-53, at
position 3-53, at position 2-53, and at position 1-53. A more
preferred peptide does additionally comprise downstream one or more
amino acids as set forth in SEQ ID NO.: 2 at position 85, or at
position 85 to n, wherein n is an integer between 86-458, i.e.
position 85 to 86, 85 to 87, 85 to 88, 85 to 89, 85 to 90, 85 to
91, 85 to 92, 85 to 93, 85 to 94, 85 to 95, 85 to 96, 85 to 97, 85
to 98, 85 to 99, 85 to 100, 85 to 101, 85 to 102, 85 to 103, 85 to
104, 85 to 105, 85 to 106, 85 to 107, 85 to 108, 85 to 109, 85 to
110, 85 to 111, 85 to 112, 85 to 113, 85 to 114, 85 to 115, 85 to
116, 85 to 117, 85 to 118, 85 to 119, 85 to 120, 85 to 121, 85 to
122, 85 to 123, 85 to 124, 85 to 125, 85 to 126, 85 to 127, 85 to
128, 85 to 129, 85 to 130, 85 to 131, 85 to 132, 85 to 133, 85 to
134, 85 to 135, 85 to 136, 85 to 137, 85 to 138, 85 to 139, 85 to
140, 85 to 141, 85 to 142, 85 to 143, 85 to 144, 85 to 145, 85 to
146, 85 to 147, 85 to 148, 85 to 149, 85 to 150, 85 to 151, 85 to
152, 85 to 153, 85 to 154, 85 to 155, 85 to 156, 85 to 157, 85 to
158, 85 to 159, 85 to 160, 85 to 161, 85 to 162, 85 to 163, 85 to
164, 85 to 165, 85 to 166, 85 to 167, 85 to 168, 85 to 169, 85 to
170, 85 to 171, 85 to 172, 85 to 173, 85 to 174, 85 to 175, 85 to
176, 85 to 177, 85 to 178, 85 to 179, 85 to 180, 85 to 181, 85 to
182, 85 to 183, 85 to 184, 85 to 185, 85 to 186, 85 to 187, 85 to
188, 85 to 189, 85 to 190, 85 to 191, 85 to 192, 85 to 193, 85 to
194, 85 to 195, 85 to 196, 85 to 197, 85 to 198, 85 to 199, 85 to
200, 85 to 201, 85 to 202, 85 to 203, 85 to 204, 85 to 205, 85 to
206, 85 to 207, 85 to 208, 85 to 209, 85 to 210, 85 to 211, 85 to
212, 85 to 213, 85 to 214, 85 to 215, 85 to 216, 85 to 217, 85 to
218, 85 to 219, 85 to 220, 85 to 221, 85 to 222, 85 to 223, 85 to
224, 85 to 225, 85 to 226, 85 to 227, 85 to 228, 85 to 229, 85 to
230, 85 to 231, 85 to 232, 85 to 233, 85 to 234, 85 to 235, 85 to
236, 85 to 237, 85 to 238, 85 to 239, 85 to 240, 85 to 241, 85 to
242, 85 to 243, 85 to 244, 85 to 245, 85 to 246, 85 to 247, 85 to
248, 85 to 249, 85 to 250, 85 to 251, 85 to 252, 85 to 253, 85 to
254, 85 to 255, 85 to 256, 85 to 257, 85 to 258, 85 to 259, 85 to
260, 85 to 261, 85 to 262, 85 to 263, 85 to 264, 85 to 265, 85 to
266, 85 to 267, 85 to 268, 85 to 269, 85 to 270, 85 to 271, 85 to
272, 85 to 273, 85 to 274, 85 to 275, 85 to 276, 85 to 277, 85 to
278, 85 to 279, 85 to 280, 85 to 281, 85 to 282, 85 to 283, 85 to
284, 85 to 285, 85 to 286, 85 to 287, 85 to 288, 85 to 289, 85 to
290, 85 to 291, 85 to 292, 85 to 293, 85 to 294, 85 to 295, 85 to
296, 85 to 297, 85 to 298, 85 to 299, 85 to 300, 85 to 301, 85 to
302, 85 to 303, 85 to 304, 85 to 305, 85 to 306, 85 to 307, 85 to
308, 85 to 309, 85 to 310, 85 to 311, 85 to 312, 85 to 313, 85 to
314, 85 to 315, 85 to 316, 85 to 317, 85 to 318, 85 to 319, 85 to
320, 85 to 321, 85 to 322, 85 to 323, 85 to 324, 85 to 325, 85 to
326, 85 to 327, 85 to 328, 85 to 329, 85 to 330, 85 to 331, 85 to
332, 85 to 333, 85 to 334, 85 to 335, 85 to 336, 85 to 337, 85 to
338, 85 to 339, 85 to 340, 85 to 341, 85 to 342, 85 to 343, 85 to
344, 85 to 345, 85 to 346, 85 to 347, 85 to 348, 85 to 349, 85 to
350, 85 to 351, 85 to 352, 85 to 353, 85 to 354, 85 to 355, 85 to
356, 85 to 357, 85 to 358, 85 to 359, 85 to 360, 85 to 361, 85 to
362, 85 to 363, 85 to 364, 85 to 365, 85 to 366, 85 to 367, 85 to
368, 85 to 369, 85 to 370, 85 to 371, 85 to 372, 85 to 373, 85 to
374, 85 to 375, 85 to 376, 85 to 377, 85 to 378, 85 to 379, 85 to
380, 85 to 381, 85 to 382, 85 to 383, 85 to 384, 85 to 385, 85 to
386, 85 to 387, 85 to 388, 85 to 389, 85 to 390, 85 to 391, 85 to
392, 85 to 393, 85 to 394, 85 to 395, 85 to 396, 85 to 397, 85 to
398, 85 to 399, 85 to 400, 85 to 401, 85 to 402, 85 to 403, 85 to
404, 85 to 405, 85 to 406, 85 to 407, 85 to 408, 85 to 409, 85 to
410, 85 to 411, 85 to 412, 85 to 413, 85 to 414, 85 to 415, 85 to
416, 85 to 417, 85 to 418, 85 to 419, 85 to 420, 85 to 421, 85 to
422, 85 to 423, 85 to 424, 85 to 425, 85 to 426, 85 to 427, 85 to
428, 85 to 429, 85 to 430, 85 to 431, 85 to 432, 85 to 433, 85 to
434, 85 to 435, 85 to 436, 85 to 437, 85 to 438, 85 to 439, 85 to
440, 85 to 441, 85 to 442, 85 to 443, 85 to 444, 85 to 445, 85 to
446, 85 to 447, 85 to 448, 85 to 449, 85 to 450, 85 to 451, 85 to
452, 85 to 453, 85 to 454, 85 to 455, 85 to 456, 85 to 457, or 85
to 458.
[0067] In the context of the present invention new and known
substances have been examined in the assays according to the
present invention. As a result thereof substances could be
identified which can act as a Treg cell activator of the present
invention, i.e. such a substance is able to activate a Treg cell
via interaction with the Treg cell epitope as set forth in SEQ ID
NO:1 which could be proven in vivo. The present invention therefore
provides for Treg cell activators.
[0068] Disclosed are Treg cell activators according to the
invention which are structurally a peptide or a polypeptide,
preferably an antibody of a binding fragment thereof or a
scaffolded peptide.
[0069] The present invention further concerns a new antibody or a
binding fragment thereof capable of binding to the peptide as set
forth in SEQ ID NO.:1 with the proviso that the antibody or the
antibody fragment is not OKT4A, OKT4D4, OKTcdr4a and not Leu3. Said
disclaimed antibodies all relate to a totally different technical
filed, namely HIV-1 research which is not related to the activation
of Treg cells and disease according to the present invention. Even
the present invention combines for the first time the technical
field of (a) HIV-1 related diseases with (b) diseases according to
the present invention e.g. autoimmune disease, allergy, asthma,
graft rejection and a diseases due to lacking immunotolerance
caused by organ transplantation or by therapeutical administration
of a non-self or self biological entity to a human in need
thereof.
[0070] A further preferred Treg cell activator peptide is a peptide
selected from a group consisting of a peptide comprising the amino
acid sequence as set forth in SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID
NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9,
and SEQ ID NO.: 10, or is a peptide selected from a group
consisting of a peptide consisting of the amino acid sequence as
set forth in SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID
NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, and SEQ ID
NO.: 10.
[0071] A more preferred Treg cell activator polypeptide is selected
from a group consisting of: HIV-1 gp120, NSC 13778 which chemical
structure is disclosed in Yang et al. 2005 on page 6124 in FIG. 1,
peptide 2 which presents three HIV-1 gp120 fragments bound together
through comformationally flexible scaffolds which chemical
structure is disclosed in Franke et al. 2007 on page 4 at the
bottom of the right column, monoclonal antibody OKT4A which binds
to the HIV-1 gp120-binding region of CD4 as disclosed in Mizukami
et al. 1988 on page 9273 right column line 19, monoclonal antibody
OKT4D which binds to the HIV-1 gp120-binding region of CD4 as
disclosed in Mizukami et al. 1988 on page 9273 right column line
19, monoclonal antibody OKTcdr4a which derives from the murine
OKT4a as disclosed in Moreland et al. 1998 on page 222 right column
line 1, monoclonal antibody Leu3 which binds to epitope overlapping
the HIV-1 gp120-binding site of CD4 as disclosed in Lohmann et al.
1992 on page 3248 left column line 7, and monoclonal antibody
MAX.12H5 which binds to the CDR2-like region of CD4 as disclosed in
Repke et al. 1992 on page 1809 abstract line 11 and on page 1812
left column line 37.
[0072] Each of the above mentioned Treg cell activators according
to the present invention can be used as a medicament and for the
preparation of a medicament for the treatment of a disease selected
from a group consisting of (i) a non-autoimmune inflammatory
disease: asthma, allergic asthma, respiratory allergy, allergic
rhinoconjunctivitis, allergic alveolitis, contact allergy, atopic
dermatitis, neurodermatitis, food allergy, graft-versus-host
disease, non-autoimmune inflammatory bowel disease, acute
respiratory distress syndrome, acute inflammatory pancreatitis,
burns, wound healing, skin scarring disorders, sarcoidosis,
Behcet's disease, Sweet's syndrome; (ii) an autoimmune inflammatory
disease: rheumatoid arthritis, rheumatic fever, systemic lupus
erythematosus, ulcerative colitis, Crohn's disease, autoimmune
inflammatory bowel disease, diabetes type I, gastritis, autoimmune
atrophic gastritis, autoimmune hepatitis, Hashimoto's thyroiditis,
thyreoiditis, multiple sclerosis, myasthenia gravis, autoimmune
haemolytic anemia, Addison's disease, scleroderma, Goodpasture's
syndrome, Guillain-Barre syndrome, Graves' disease,
glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid,
vitiligo, idiopathic leukopenia, Sjogren's syndrome, Wegener's
granulomatosis; (iii) an inflammatory disease due to organ
transplantation; (iv) a bone marrow transplantation; and (v) a
disease due to exogenously administered self or exogenously
administered non-autologous recombinant polypeptide.
[0073] Another embodiment of the present invention relates to a
pharmaceutical composition comprising at least one Treg cell
activator according to the present invention--preferably HIV-1
gp120--as an active ingredient and can be formulated in
conventional manner. Methods for making such formulations can be
found in manuals, e.g. "Remington Pharmaceutical Science". Examples
for ingredients that are useful for formulating at least one
substance according to the present invention are also found in
WO99/18193, which is hereby incorporated by reference.
[0074] The composition may be manufactured in a manner that is
itself known, e.g. by mean of conventional mixing, dissolving,
granulating, dragee-making, levitating, powdering, emulsifying,
encapsulating, entrapping of lyophilizing processes.
[0075] In a further aspect the invention teaches a method for
treating a disease which is characterized in that its clinical
picture can be influenced positively by an increase of activated
Treg cells which method comprises administering to a being
preferably a human being in need of such a treatment a suitable
amount of a pharmaceutical composition comprising at least one Treg
cell activator according to the present invention, preferably HIV-1
gp120 or HIV-1 gp120 derived fragments and peptides thereof. The
present invention provides therefore for a method for treating (i)
a non autoimmune inflammatory disease: asthma, allergic asthma,
respiratory allergy, allergic rhinoconjunctivitis, allergic
alveolitis, contact allergy, atopic dermatitis, neurodermatitis,
food allergy, graft-versus-host disease, non-autoimmune
inflammatory bowel disease, acute respiratory distress syndrome,
acute inflammatory pancreatitis, burns, wound healing, skin
scarring disorders, sarcoidosis, Behcet's disease or Sweet's
syndrome; (ii) an autoimmune inflammatory disease: rheumatoid
arthritis, rheumatic fever, systemic lupus erythematosus,
ulcerative colitis, Crohn's disease, autoimmune inflammatory bowel
disease, diabetes type I, gastritis, autoimmune atrophic gastritis,
autoimmune hepatitis, Hashimoto's thyroiditis, thyreoiditis,
multiple sclerosis, myasthenia gravis, autoimmune haemolytic
anemia, Addison's disease, scleroderma, Goodpasture's syndrome,
Guillain-Barre syndrome, Graves' disease, glomerulonephritis,
psoriasis, pemphigus vulgaris, pemphigoid, vitiligo, idiopathic
leukopenia, Sjogren's syndrome or Wegener's granulomatosis; (iii)
an inflammatory disease due to organ transplantation; (iv) a bone
marrow transplantation; or (v) a disease due to exogenously
administered self or exogenously administered non-autologous
recombinant polypeptide which method comprises administering to a
being in need of such a treatment a suitable amount of a
pharmaceutical composition comprising at least one Treg cell
activator.
[0076] The present invention additionally, provides for a use of a
Treg cell activator according to the present invention for reducing
and/or preventing an unwanted immune reaction due to a exogenously
administered self or exogenously administered non-autologous
recombinant polypeptide and provides for a method for reducing or
preventing an unwanted immune reaction comprising: (a) adding a
sufficient amount of at least one Treg cell activator according to
the present invention to a non-human animal, preferably a non-human
primate.
[0077] In a further embodiment the present invention provides for a
test system for determining whether a substance is a Treg cell
activator according to the present invention comprising at
least
[0078] a) a Treg cell and
[0079] b) a peptide spanning at least the epitope as set forth in
SEQ ID NO.: 1.
[0080] In a preferred test system of the present invention a
peptide of b) is selected from a group consisting of an isolated
peptide spanning amino acid No. 26 to 458 of SEQ ID NO.: 2, amino
acid No. 26 to 419 of SEQ ID NO.: 2, amino acid No. 26 to 207 of
SEQ ID NO.: 2, amino acid No. 26 to 131 of SEQ ID NO.: 2, and amino
acid No. 46 to 89 of SEQ ID NO.: 2. or the peptide used is an
isolated peptide spanning amino acid No. 1 to 31 of SEQ ID NO.: 1
and having additional up-stream and/or downstream amino acids with
the prerequisite that the additional amino acids do not hinder
binding of a substance to the amino acid stretch as set forth in
SEQ ID NO.: 1. Said additional up-stream amino acid or amino acid
stretch is preferably selected from a group consisting of the amino
acid or amino acid stretch as set forth in SEQ ID NO.: 2 at
position 53, at position 52-53, at position 51-53, at position
50-53, at position 49-53, at position 48-53, at position 47-53, at
position 46-53, at position 45-53, at position 44-53, at position
43-53, at position 42-53, at position 41-53, at position 40-53, at
position 39-53, at position 38-53, at position 37-53, at position
36-53, at position 35-53, at position 34-53, at position 33-53, at
position 32-53, at position 31-53, at position 30-53, at position
29-53, at position 28-53, at position 27-53, at position 26-53, at
position 25-53, at position 24-53, at position 23-53, at position
22-53, at position 21-53, at position 20-53, at position 19-53, at
position 18-53, at position 17-53, at position 16-53, at position
15-53, at position 14-53, at position 13-53, at position 12-53, at
position 11-53, at position 10-53, at position 9-53, at position
8-53, at position 7-53, at position 6-53, at position 5-53, at
position 4-53, at position 3-53, at position 2-53, and at position
1-53. A more preferred peptide does additionally comprise
downstream one or more amino acids as set forth in SEQ ID NO.: 2 at
position 85, or at position 85 to n, wherein n is an integer
between 86-458, i.e. position 85 to 86, 85 to 87, 85 to 88, 85 to
89, 85 to 90, 85 to 91, 85 to 92, 85 to 93, 85 to 94, 85 to 95, 85
to 96, 85 to 97, 85 to 98, 85 to 99, 85 to 100, 85 to 101, 85 to
102, 85 to 103, 85 to 104, 85 to 105, 85 to 106, 85 to 107, 85 to
108, 85 to 109, 85 to 110, 85 to 111, 85 to 112, 85 to 113, 85 to
114, 85 to 115, 85 to 116, 85 to 117, 85 to 118, 85 to 119, 85 to
120, 85 to 121, 85 to 122, 85 to 123, 85 to 124, 85 to 125, 85 to
126, 85 to 127, 85 to 128, 85 to 129, 85 to 130, 85 to 131, 85 to
132, 85 to 133, 85 to 134, 85 to 135, 85 to 136, 85 to 137, 85 to
138, 85 to 139, 85 to 140, 85 to 141, 85 to 142, 85 to 143, 85 to
144, 85 to 145, 85 to 146, 85 to 147, 85 to 148, 85 to 149, 85 to
150, 85 to 151, 85 to 152, 85 to 153, 85 to 154, 85 to 155, 85 to
156, 85 to 157, 85 to 158, 85 to 159, 85 to 160, 85 to 161, 85 to
162, 85 to 163, 85 to 164, 85 to 165, 85 to 166, 85 to 167, 85 to
168, 85 to 169, 85 to 170, 85 to 171, 85 to 172, 85 to 173, 85 to
174, 85 to 175, 85 to 176, 85 to 177, 85 to 178, 85 to 179, 85 to
180, 85 to 181, 85 to 182, 85 to 183, 85 to 184, 85 to 185, 85 to
186, 85 to 187, 85 to 188, 85 to 189, 85 to 190, 85 to 191, 85 to
192, 85 to 193, 85 to 194, 85 to 195, 85 to 196, 85 to 197, 85 to
198, 85 to 199, 85 to 200, 85 to 201, 85 to 202, 85 to 203, 85 to
204, 85 to 205, 85 to 206, 85 to 207, 85 to 208, 85 to 209, 85 to
210, 85 to 211, 85 to 212, 85 to 213, 85 to 214, 85 to 215, 85 to
216, 85 to 217, 85 to 218, 85 to 219, 85 to 220, 85 to 221, 85 to
222, 85 to 223, 85 to 224, 85 to 225, 85 to 226, 85 to 227, 85 to
228, 85 to 229, 85 to 230, 85 to 231, 85 to 232, 85 to 233, 85 to
234, 85 to 235, 85 to 236, 85 to 237, 85 to 238, 85 to 239, 85 to
240, 85 to 241, 85 to 242, 85 to 243, 85 to 244, 85 to 245, 85 to
246, 85 to 247, 85 to 248, 85 to 249, 85 to 250, 85 to 251, 85 to
252, 85 to 253, 85 to 254, 85 to 255, 85 to 256, 85 to 257, 85 to
258, 85 to 259, 85 to 260, 85 to 261, 85 to 262, 85 to 263, 85 to
264, 85 to 265, 85 to 266, 85 to 267, 85 to 268, 85 to 269, 85 to
270, 85 to 271, 85 to 272, 85 to 273, 85 to 274, 85 to 275, 85 to
276, 85 to 277, 85 to 278, 85 to 279, 85 to 280, 85 to 281, 85 to
282, 85 to 283, 85 to 284, 85 to 285, 85 to 286, 85 to 287, 85 to
288, 85 to 289, 85 to 290, 85 to 291, 85 to 292, 85 to 293, 85 to
294, 85 to 295, 85 to 296, 85 to 297, 85 to 298, 85 to 299, 85 to
300, 85 to 301, 85 to 302, 85 to 303, 85 to 304, 85 to 305, 85 to
306, 85 to 307, 85 to 308, 85 to 309, 85 to 310, 85 to 311, 85 to
312, 85 to 313, 85 to 314, 85 to 315, 85 to 316, 85 to 317, 85 to
318, 85 to 319, 85 to 320, 85 to 321, 85 to 322, 85 to 323, 85 to
324, 85 to 325, 85 to 326, 85 to 327, 85 to 328, 85 to 329, 85 to
330, 85 to 331, 85 to 332, 85 to 333, 85 to 334, 85 to 335, 85 to
336, 85 to 337, 85 to 338, 85 to 339, 85 to 340, 85 to 341, 85 to
342, 85 to 343, 85 to 344, 85 to 345, 85 to 346, 85 to 347, 85 to
348, 85 to 349, 85 to 350, 85 to 351, 85 to 352, 85 to 353, 85 to
354, 85 to 355, 85 to 356, 85 to 357, 85 to 358, 85 to 359, 85 to
360, 85 to 361, 85 to 362, 85 to 363, 85 to 364, 85 to 365, 85 to
366, 85 to 367, 85 to 368, 85 to 369, 85 to 370, 85 to 371, 85 to
372, 85 to 373, 85 to 374, 85 to 375, 85 to 376, 85 to 377, 85 to
378, 85 to 379, 85 to 380, 85 to 381, 85 to 382, 85 to 383, 85 to
384, 85 to 385, 85 to 386, 85 to 387, 85 to 388, 85 to 389, 85 to
390, 85 to 391, 85 to 392, 85 to 393, 85 to 394, 85 to 395, 85 to
396, 85 to 397, 85 to 398, 85 to 399, 85 to 400, 85 to 401, 85 to
402, 85 to 403, 85 to 404, 85 to 405, 85 to 406, 85 to 407, 85 to
408, 85 to 409, 85 to 410, 85 to 411, 85 to 412, 85 to 413, 85 to
414, 85 to 415, 85 to 416, 85 to 417, 85 to 418, 85 to 419, 85 to
420, 85 to 421, 85 to 422, 85 to 423, 85 to 424, 85 to 425, 85 to
426, 85 to 427, 85 to 428, 85 to 429, 85 to 430, 85 to 431, 85 to
432, 85 to 433, 85 to 434, 85 to 435, 85 to 436, 85 to 437, 85 to
438, 85 to 439, 85 to 440, 85 to 441, 85 to 442, 85 to 443, 85 to
444, 85 to 445, 85 to 446, 85 to 447, 85 to 448, 85 to 449, 85 to
450, 85 to 451, 85 to 452, 85 to 453, 85 to 454, 85 to 455, 85 to
456, 85 to 457, or 85 to 458.
[0081] The following examples are meant to illustrate the present
invention, however, shall not be construed as limitation. However,
the Examples describe most preferred embodiments of the
invention.
EXAMPLES
[0082] (1) CD4/HIV-1 gp120 competition assay for determining
whether a substance can bind at least to a peptide spanning epitope
as set fourth in SEQ ID NO.: 1. 96 well assay plates (Nunc,
Germany) are coated overnight at 4.degree. C. with CD4 (sCD4,
Immunodiagnostics, USA) 100 ng per well in PBS, pH 7.4. Coated
plates are saturated with PBS/3% BSA buffer and washed three times.
To determine binding of a test substance sample is added for 1 hour
in different concentrations. No test substance is added to control
wells. After washing three times HIV-1 gp120-peroxidase conjugate
(Immunodiagnostics, USA) is added to the plate for 1 h. Unbound
HIV-1 gp120-peroxidase conjugate is removed by washing three times.
After washing, 3,3,5,5-tetramethylbenzidine chromogen substrate
(Pierce, USA) for peroxidase is added and the optical density is
read at 450 nm. A substance which interacts with the CD4 HIV-1
gp120-binding site will block the binding of labeled HIV-1 gp120
and is identifiable by a reduced signal compared to the
controls.
[0083] (2) Assay for determining whether e.g. HIV-1 gp120 or a
substance which can interfere with HIV-1 gp120-binding to a
CD4.sup.+CD25.sup.+ Treg can activate a CD4.sup.+CD25.sup.+ Treg
and therewith can be useful in the treatment an autoimmune disease
(e.g. Inflammatory Bowel Disease, Multiple Sclerosis, Rheumatoid
Arthritis, Psoriasis, Diabetes Type I, Lupus Erythematosus,
Phemphigus vulgaris, Thyreoiditis), other diseases with autoimmune
aspects in their pathogenesis such as vitiligo, atopic dermatitis,
an allergy (e.g. Allergic rhinitis,), asthma (e.g. allergic
asthma), GVHD (graft-versus-host disease), graft rejection.
[0084] (2.1) Method for isolation of cells P (2.1.1) Isolation of
PBMC
[0085] The isolation procedure starts with PBMC (peripheral blood
mononuclear cells) isolated by standard density gradient
centrifugation from normal buffy coat preparations of healthy
volunteers. Alternatively, PBMC isolated from whole peripheral
blood or leukapheresis products can be used.
[0086] Blood from buffy coats is diluted 1:1 with PBS (phosphate
buffered saline) containing 0.2% Liquemine (Sodium-Heparin) and 2
mM EDTA at room temperature. The diluted blood is thoroughly
pipetted onto prepared Ficoll layers (30 ml diluted blood per 15 ml
Ficoll layer per 50 ml tube) and centrifuged for 15 min. (minute)
at 200.times.g (with brake on) at room temperature. 8-10 ml of the
upper fluid are carefully removed and the tubes centrifuged at
450.times.g for 15 min. at room temperature (with brake off). PBMC
are collected from the interphase of each gradient, washed three
times with 50 ml PBS/1 mM EDTA separately, than pooled and wash two
more times. Finally, PBMC are re-diluted in X-VIVO-15 (Cambrex,
Verviers, Belgium) in cell culture medium and counted.
[0087] (2.1.2) Isolation of CD4.sup.+ T helper cells
[0088] For determining suppressive activity of Treg cells highly
purified cell populations are needed. Therefore, antibody-coated
magnetic beads are used (Miltenyi, Germany and or Dynal, Norway).
Magnetic beads in this context are paramagnetic particles that are
coupled to specific monoclonal antibodies. They are used to
magnetically label the target cell population. The antibody-coated
magnetic beads bind to the target cells. This labeled cell fraction
is retained by magnetic force and can be recovered subsequently
highly purified (positive selection). Before positive isolation of
CD4.sup.+CD25.sup.- T helper cells PBMC are washed two times with
50 ml washing buffer according to the manufacturer's instructions
(Miltenyi Germany). For isolation of CD4.sup.+ T cells, CD4
microbeads (Miltenyi Germany, 2-4 .mu.L microbeads/10.sup.7 PBMC)
are used according to the manufacturer's instructions. The
CD4.sup.+ fraction is isolated using a MACS separator (Miltenyi)
according to the instructions of the manufacturer. MACS separator
retains magnetic bead-labeled cells by magnetic force.
Contaminating CD4.sup.+CD25.sup.+ Treg cells are depleted in a
second step by using CD25 Dynabeads, (Dynal, Norway) according to
the instructions of the manufacturer (details see 2.1.4) using (0.5
beads/cell). This depletion procedure results in highly purified
CD4.sup.+CD25.sup.- T helper cells (negative selection).
[0089] To circumvent CD4 antibodies binding to CD4.sup.+ cells (to
avoid CD4.sup.- dependent pre activation) in the isolation
procedure of CD4.sup.+ T cells, alternatively untouched CD4.sup.+ T
helper cells are generated by using the negative isolation kit
(Miltenyi Germany) according to the manufacturer's instructions.
Before negative isolation of CD4.sup.+ T helper cells PBMC are
washed two times with 50 ml washing buffer according to the
manufacturer's instructions (Miltenyi Germany). For isolation of
CD4.sup.+ T helper cells, PBMC are incubated with a cocktail of
biotinylated CD45RO, CD8, CD14, CD16, CD19, CD56, CD36, CD123,
anti-TCR.gamma./.delta., and CD235a antibodies. These cells are
subsequently magnetically labeled with Anti-Biotin Microbeads for
depletion.
[0090] The CD4.sup.+ fraction is isolated using a MACS separator
(Miltenyi) according to the instructions of the manufacturer. MACS
separator retains magnetic bead-labeled cells by magnetic force.
Contaminating CD4.sup.+CD25.sup.+ Treg cells are depleted in a
second step by using CD25 Dynabeads, (Dynal, Norway) according to
the instructions of the manufacturer (details see 2.1.4) using 0.5
beads/cell.
[0091] (2.1.3) Isolation of CD8.sup.+ T effector cells
[0092] Before positive isolation of CD8.sup.+ T effector cells PBMC
are washed two times with 50 ml washing buffer according to the
manufacturer's instructions (Miltenyi Germany). For isolation of
CD8.sup.+CD25.sup.- T cells, CD8 microbeads (Miltenyi Germany, 2-4
.mu.L microbeads/10.sup.7 PBMC) are used according to the
manufacturer's instructions. The CD8.sup.+ fraction is isolated
using a MACS separator (Miltenyi) according to the instructions of
the manufacturer. Contaminating CD8.sup.+CD25.sup.+ T cells are
depleted in a second step by using CD25 Dynabeads, (Dynal, Norway)
according to the instructions of the manufacturer (details see
2.1.4) using (0.5 beads/cell). This depletion procedure results in
highly purified CD8.sup.+CD25.sup.- T effector cells (negative
selection).
[0093] (2.1.4) Isolation of CD4.sup.+CD25.sup.+ Treg cells
[0094] For Isolation of CD4.sup.+CD25.sup.+ Treg cells positive and
negative selection is combined. PBMC are washed with washing buffer
according to the manufacturer's instructions (Miltenyi Germany) and
subsequently incubated with CD25 microbeads (2 .mu.L
microbeads/10.sup.7 PBMC) for 20 min. at 4.degree. C. in isolation
buffer (1.times.10.sup.8/ml) according to the manufacturer's
instructions. Afterwards, the cells are washed two times in PBS.
The CD25.sup.+ fraction is isolated using a MACS separator
(Miltenyi) according to the instructions of the manufacturer. The
positively selected CD25.sup.+ fraction contains 65-80% CD4.sup.+ T
cells and 20-35% contaminating CD19.sup.+ B cells, CD8.sup.+ T
cells, and few CD14.sup.+ monocytes. The contaminating cells are
depleted with Dynabeads (Dynal, Norway). The following amounts of
beads are used: CD19 Dynabeads: 2 beads/cell, CD8 Dynabeads: 3
beads/cell, CD14 Dynabeads: 1 bead/cell. Collected Dynabeads are
washed two times in 15 ml tubes with depletion buffer using the
magnetic particle concentrator (Dynal) according to the
manufacturer's instructions. The CD25.sup.+ PBMC fraction
(5.times.10.sup.7/ml) is added in depletion buffer and incubated
for 20 min. at 4.degree. C. on a shaker (sample mixer, Dynal).
Contaminating cells are depleted according to the manufacturer's
instructions by the use of the magnetic particle concentrator. For
higher purity of CD4.sup.+CD25.sup.+ Treg cells Dynabeads depletion
is repeated once (>98% after two rounds of depletion).
[0095] (2.1.5) Generation of monocyte-derived dendritic cells
[0096] Dendritic cells (DC) are generated from buffy coats of
healthy volunteers. PBMC (2.1.6) are plated in 6-well tissue
culture plates at a density of 15.times.10.sup.6 cells/well in 3 ml
X-VIVO-15 (Cambrex, Verviers, Belgium) plus 1.5% heat-inactivated
autologous plasma containing 800 U/ml GM-CSF (Leukomax; Novartis,
Basel, Switzerland) and 1,000 U/ml IL-4 (Strathmann Biotec,
Hamburg, Germany). Cultures are fed every other day (days 2, 4 and
6) by removing 1 ml of the medium and adding back 1 ml fresh medium
with cytokines. On day 7, non-adherent cells were harvested and
transferred to new 6 well plates and cultured further on in the
presence of 10 ng/ml IL-1.beta., 10 ng/ml TNF-.alpha., 1,000 U/ml
IL-6 (all from Strathmann, Biotech, Germany) and 1 .mu.g/ml
PGE.sub.2 (Pharmacia-Upjohn, Uppsala, Sweden). Mature CD83.sup.+ DC
are harvested at day 9 of culture.
[0097] (2.1.6) Isolation of CD3-depleted PBMC
[0098] T cells are depleted from PBMC with CD3 Dynabeads (Dynal,
Norway) by using 0.5 beads/cell. Collected Dynabeads are washed two
times in 15 ml tubes with depletion buffer using the magnetic
particle concentrator (Dynal) according to the manufacturer's
instructions. PBMC (5.times.10.sup.7/ml) are added in depletion
buffer and incubated for 20 min. at 4.degree. C. on a shaker
(sample mixer, Dynal). CD3.sup.+ cells are depleted according to
the manufacturer's instructions by the use of the magnetic particle
concentrator resulting in a purity of >98% CD3.sup.- PBMC.
[0099] (2.1.7) Isolation of human CD25-depleted PBMC
[0100] PBMC are isolated according to (2.1.1). CD25-expressing
regulatory T cells in the PBMC preparation are depleted with CD25
Dynabeads (Dynal, Norway) according to the instructions of the
manufacturer (details see 2.1.4) by using 0.5 beads/cell. Collected
Dynabeads are washed two times in 15 ml tubes with depletion buffer
using the magnetic particle concentrator (Dynal) according to the
manufacturer's instructions. PBMC (5.times.10.sup.7/ml) are added
in depletion buffer and incubated for 20 min. at 4.degree. C. on a
shaker (sample mixer, Dynal). CD25.sup.+ cells are depleted
according to the manufacturer's instructions by the use of the
magnetic particle concentrator resulting in a purity of >99%
CD25-negative PBMC.
[0101] (2.2) Method for testing suppressive activity of
CD4.sup.+CD25.sup.+ Treg cells
[0102] (2.2.1) Coculture suppression assay A: Mixed leukocyte
reaction (MLR) Cocultures of CD4.sup.+ T helper cells or CD8.sup.+
T effector cells with CD4.sup.+CD25.sup.+ Treg cells and allogeneic
DC have to be performed to analyze the suppressive activity of
CD4.sup.+CD25.sup.+ Treg cells on CD4.sup.+ T helper cells or
CD8.sup.+ T effector cells. Therefore, 1.times.10.sup.5/well
CD4.sup.+ T helper cells (2.1.2) or CD8.sup.+ T effector cells
(2.1.3) are cocultured with different numbers of
CD4.sup.+CD25.sup.+ Treg cells (2.1.4; ratio 1:1 to 1:4) and
1.times.10.sup.4/well DC in 96 well flat bottom culture plates in
X-VIVO 15 (Cambrex, Verviers, Belgium) in the presence or absence
of a CD4-binding compound e.g. HIV-1 gp120 (0.1-10 .mu.g/ml).
Mature dendritic cells (DC) generated as described (2.1.5) are from
the same donor as CD4.sup.+CD25.sup.+ Treg cells (syngenic) but
allogeneic to the CD4.sup.+ T helper cells or CD8.sup.+ T effector
cells are used for T cell stimulation. In this assay, only
CD4.sup.+ T helper cells or CD8.sup.+ T effector cells are
activated by the allogeneic DC (MLR) resulting in a strong
proliferation of the T cell subset. Non-activated
CD4.sup.+CD25.sup.+ Treg cells did not suppress this proliferation
in absence of a Treg cell activating compound. A functional
activation of CD4.sup.+CD25.sup.+ Treg cells by a CD4.sup.- binding
compound resulted in a reduced proliferation of CD4.sup.+T helper
cells or CD8.sup.+ T effector cells.
[0103] Proliferation is determined after 4 days of culture by
adding 37 kBq 3H-Thymindine (.sup.3H-Tdr) for additional 16 h.
[0104] (2.2.2) Coculture Suppression Assay B: Stimulation of
CD8.sup.+ T effector cells with allogeneic PBMC and
CD4.sup.+CD25.sup.+ Treg cells from the same healthy volunteer.
[0105] In order to study the influence of a substance, e.g. HIV-1
gp120 which can bind at least to the epitope given in SEQ ID NO.:1
exclusively on the suppressive function of CD4.sup.+CD25.sup.+ Treg
cells, we developed a co-culture assay which contained CD8.sup.+ T
cells as effectors to exclude any influence of this substance on
the latter (CD8.sup.+ T cells don't express CD4). In this setting,
activation of alloreactive CD8.sup.+ T effector cells is only
suppressed by activated CD4.sup.+CD25.sup.+ Treg cells such as upon
additional anti-CD3 mAb stimulation (positive control). To evaluate
the influence of e.g. HIV-1 gp120 on the function of
CD4.sup.+CD25.sup.+ Treg cells, isolated CD4.sup.+CD25.sup.+ Treg
cells (2.1.4) are co-cultured with syngenic, T cell-depleted and
irradiated (50 Gy) PBMC 2.1.6) and allogeneic CD8.sup.+ T effector
cells (2.1.3) in presence of varying concentrations (0.1-10
.mu.g/ml) of different HIV-1 gp 120 preparations. Briefly,
1.times.10.sup.5 CD4.sup.+CD25.sup.+ Treg cells are incubated with
3.times.10.sup.5 syngenic T cell-depleted PBMC in the presence or
absence of varying amounts of HIV-1 gp120. Stimulation with 0.5
.mu.g/ml anti-CD3 monoclonal antibody (OKT-3, ebioscience, USA)
serves as positive control. No additional stimulation represents
negative control. Either immediately or 24 h. later,
1.times.10.sup.5 allogeneic CD8.sup.+ T effector cells are added to
the cultures and proliferation is determined after additional 72 h.
by .sup.3H-Tdr incorporation (37 kBq/well). Functional activation
of CD4.sup.+CD25.sup.+ Treg cells via interaction with the epitope
as set forth in SEQ ID NO:1 result in suppressed proliferation of
CD8.sup.+ T effector cells and therewith identifies a substance as
a Treg cell activator (Data see FIG. 1).
[0106] To show in parallel whether a substance can interfere with
HIV-1 gp120 binding to CD4 epitope of a CD4.sup.+CD25.sup.+ Treg
cell, CD4 is added in different concentrations (0.1-10 .mu.g/ml) to
isolated CD4.sup.+CD25.sup.- Treg cells (2.1.4) cocultured with
syngenic T cell-depleted PBMC (2.1.6) and allogeneic CD8.sup.+ T
effector cells (2.1.3) in the presence of varying amounts of the
substance. Enhanced proliferation of CD8.sup.+ effector cells
resembles competitive binding to CD4 and blocked activation of a
Treg cell via interaction with the epitope as set forth in SEQ ID
NO:1. All cultures are performed in serum free X-VIVO-15 (Cambrex,
Verviers, Belgium).
[0107] Alternatively to irradiation-induced inactivation and block
of proliferation PBMC can be treated with Mitomycin C (Sigma,
Germany). Briefly, 3.times.10.sup.7 PBMC are incubated in 3 ml
MEM/10% FCS/180 .mu.g Mitomycin C for 30 min at 37.degree. C.
Afterwards cells are washed 5.times. using MEM/10% FCS.
Subsequently cells are subjected to the assay.
[0108] (2.2.3) Coculture suppression assay C: Stimulation of T
cells in presence of pre-activated CD4.sup.+CD25.sup.+ Treg cells
and allogeneic PBMC
[0109] To evaluate the direct Treg cell activating potential of a
compound in the absence of antigen-presenting cells such as PBMC,
isolated CD4.sup.+CD25.sup.+ Treg cells (according to 2.1.4) are
pre-cultured in X-VIVO-15 for 16-48 h. alone, in presence of 0.5
.mu.g/ml anti-CD3 monoclonal antibody (OKT-3) as positive control,
or in presence of different concentrations of HIV-1 gp120.
Afterwards, cells are washed intensively and added to cocultures of
syngenic, irradiated (50 Gy) PBMC and allogeneic CD4.sup.+ T helper
cells or CD8.sup.+ T effector cells. Proliferation is determined
after additional 72 h. by .sup.3H-Tdr incorporation (37 kBq/well).
Functional activation of CD4.sup.+CD25.sup.+ Treg cells via
interaction with the epitope as set forth in SEQ ID NO.: 1 result
in suppressed proliferation of CD4.sup.+ T helper cells or
CD8.sup.+ T effector cells and therewith identifies a substance as
a Treg cell activator.
[0110] A reduced incorporated radioactivity resembles suppressed
proliferation and identifies CD8.sup.+ inhibition and therewith
identifies a substance to activate a Treg cell via interaction with
the epitope as set forth in SEQ ID NO.: 1.
[0111] (2.3) Method for readout of suppressive activity of
CD4.sup.+CD25.sup.+ Treg cells
[0112] (2.3.1) Analysis of proliferation
[0113] After 3-4 days of incubation of cells in assays provided
under (2.2.1), (2.2.2) and (2.2.3) T cell proliferation is
measured. For additional 16 h., cells are pulsed with .sup.3H-Tdr
(37 kBq/well, MB Biomedicals), and incorporated radioactivity is
measured by using a liquid scintillation counter (Betaplate,
Wallac/PerkinElmer). In the absence of a Treg cell activating
compound, resting Treg cells cannot inhibit the proliferation of
CD4.sup.+ T helper cells or CD8.sup.+ T effector cells (negative
control). In contrast, in presence of an activating CD4-binding
compound or anti-CD3 monoclonal antibody (positive control) the
proliferation of CD4.sup.+ T helper cells or CD8.sup.+ T effector
cells is suppressed by CD4.sup.+CD25.sup.+ Treg cells. A reduced
incorporated radioactivity resembles suppressed proliferation and
identifies CD4.sup.+ T helper cell or CD8.sup.+ T effector cell
inhibition and therewith identifies that a CD4.sup.+CD25.sup.+ Treg
cell has been activated.
[0114] For some experiments the proliferation of either
CD4.sup.+CD25.sup.+ Treg cells, CD4.sup.+ T helper cells or
CD8.sup.+ T effector cells is selectively followed by flow
cytometry after labeling of these population with Vibrant CFDA SE
Cell Tracer Kit (Invitrogen Life Technologies, San Diego, USA)
according to the manufacturer's protocol. For CFDA labeling, T
cells (1.times.10.sup.7 cells/ml) are incubated in PBS with 1 .mu.M
CFDA Vibrant CFDA SE Cell Tracer Kit (Invitrogen Life Technologies,
San Diego, USA) at 37.degree. C. for 30 min. Thereafter, cells are
washed with X-VIVO-15 and incubated for 30 min. at 37.degree. C. in
the dark. After an additional wash the cells are counted and added
to the cocultures. In addition, proliferation of cells is measured
by flow cytometry after 4-6 days of culture.
[0115] (2.3.2) Analysis of cytokine production
[0116] Activated Treg cells not only inhibit the proliferation of
cocultured CD4.sup.+ T helper cells or CD8.sup.+ T effector cells,
they suppress also the cytokine synthesis of these T cells.
Therefore, detection of cytokines produced by cocultured T cells is
an alternative method to analyze the Treg cell activating potential
of a reagent. In this assay, isolated CD4.sup.+CD25.sup.+ Treg
cells (according to 2.1.4), syngenic CD3-depleted PBMC (according
to 2.1.6) and allogeneic CD8.sup.+ T effector cells (according to
2.1.3) are cocultured as described (2.2) before. After 7 days,
alloreactive CD8.sup.+ T effector cells are polyclonal restimulated
with 2.4 .mu.g/ml phytohemagglutinin (PHA, Sigma, Germany) and 1
ng/ml PMA for 5 h. in presence of monensin (BD GolgiStop.TM., BD
Biosciences Pharmingen 1.3 .mu.M). Thereafter, cells are collected,
washed with PBS, fixed and permeabilized according to the
manufacturer's instruction (perm/fix solution, BD PharMingen,
Germany) and stained with 0.5 .mu.g/test of a cytokine-specific
monoclonal antibody (anti-IFN-.gamma., anti-IL-2, anti-TNF-.alpha.,
all from BD PharMingen). Subsequently, production of cytokines by
CD4.sup.+ T helper cells or CD8.sup.+ T effector cells is analyzed
by flow cytometry. In the absence of a Treg cell activating
compound Treg cells cannot inhibit the cytokine production of
CD4.sup.+ T helper cells or CD8.sup.+ T effector cells (negative
control). In contrast, in presence of an activating CD4-binding
compound or anti-CD3 monoclonal antibodies (positive control) the
cytokine production of CD4.sup.+ T helper cells or CD8.sup.+ T
effector cells is suppressed by functional activated Treg cells. A
reduced cytokine production identifies inhibition and therewith
identifies that a CD4.sup.+CD25.sup.+ Treg cell has been
activated.
[0117] (2.3.3) Analysis of CD25 expression
[0118] Activated Treg cells inhibit the ability of CD8.sup.+ T
effector cells and CD4.sup.+ T helper cells to express the
.alpha.-chain of the IL-2 receptor, CD25. Therefore, the analysis
of CD25 expression by flow cytometry is an additional method to
evaluate the Treg cell activating potential of a reagent. In this
assay, isolated CD4.sup.+CD25.sup.+ Treg cells (according to 2.1.4)
and syngenic CD3-depleted PBMC (according to 2.1.6) are cocultured
with allogeneic CD8.sup.+ T effector cells or allogeneic CD4.sup.+
T helper cells (according to 2.1.3, 2.1.2 and 2.2.1, 2.2.2 and
2.2.3). After 7 days, alloreactive CD8.sup.+ T effector cells or
allogeneic CD4.sup.+ T helper cells are stimulated with allogeneic
PBMC from the same donor or DC as used in primary culture and
expression of CD25 on alloreactive CD8.sup.+ T effector or
allogeneic CD4.sup.+ T helper cells cells are analyzed 24 h.
thereafter by flow cytometry. Activation of Treg cells by the test
compound results directly in an inhibited CD25 expression on the
re-stimulated CD8.sup.+ T effector cells or CD4.sup.+ T helper
cells. In the absence of a Treg cell activating compound, Treg
cells cannot inhibit the CD25 expression on re-stimulated CD8.sup.+
T effector cells or CD4.sup.+ T helper cells (negative control). In
contrast, in presence of an activating reagent or an anti-CD3
monoclonal antibody (positive control) the CD25 expression of
CD8.sup.+ T effector cells or CD4+ T helper cells is suppressed by
Treg cells. A reduced CD25 expression identifies inhibition and
therewith identifies that a CD4.sup.+CD25.sup.+ Treg cell has been
activated.
[0119] (2.3.4) Analysis of cyclic AMP production
[0120] Stimulation of Treg cells results in strong increase of
cytosolic (i.e. intracellular) cAMP. Therefore, the analysis of
cAMP in Treg cells is an additional method to determine whether a
substance which can interfere with HIV-1 gp120 binding to CD4
epitope of a CD4-positive cell can activate a Treg cell. In this
assay freshly isolated CD4.sup.+CD25.sup.+ Treg cells (according to
2.1.4) (1.times.10.sup.5-1.times.10.sup.6/well) are incubated with
anti-CD3 monoclonal antibody (OKT-3; 0.5 .mu.g/ml) or HIV-1 gp120
(0.1-10 m/ml; Protein Science Corp., Meriden, Conn., USA) or left
untreated for 16 hours. To assess cytosolic cAMP-concentrations a
cAMP-specific ELISA (Parameter.TM. Cyclic AMP Assay, Cat. No.
KGE002; R&D Systems, Wiesbaden, Germany) is applied. Treg cells
are washed three times in ice-cold PBS and subsequently lysed
(1.times.10.sup.7/ml) using lysis buffer supplied by the
manufacturer and applied to the ELISA according to the
manufacturer's recommendations.
[0121] Activation of Treg cells by the test compound results
directly in an increase of cytosolic cAMP. In the absence of a Treg
cell activating compound no increase of cAMP in CD4.sup.+CD25.sup.+
Treg cells is detected (negative control). In contrast, in presence
of an activating CD4-binding compound or an anti-CD3 monoclonal
antibody (positive control) CD4.sup.+CD25.sup.+ Treg cell show a
strong increase of cytosolic cAMP. An increase of cytosolic cAMP
identifies that a CD4.sup.+CD25.sup.+ Treg cell has been activated.
Data see FIG. 2.
[0122] Alternatively, additional analysis of assay supernatant cAMP
concentration and use of phoshodiesterase inhibitors e.g.
Roflumilast (1-50 .mu.M) enhance the cAMP signal in the assay.
[0123] (3) In vivo assay for determining whether HIV-1 gp120 or a
substance which can interfere with HIV-1 gp120-binding to a
CD4.sup.+CD25.sup.+ Treg can activate a CD4.sup.+CD25.sup.+ Treg in
a disease-related model and therewith can be useful in the
treatment of an autoimmune disease (e.g. Inflammatory Bowel
Disease, Multiple Sclerosis, Rheumatoid Arthritis, Psoriasis,
Diabetes Type I, Lupus Erythematosus, Phemphigus vulgaris,
Thyreoiditis), other diseases with autoimmune aspects in their
pathogenesis such as vitiligo, atopic dermatitis, an allergy (e.g.
Allergic rhinitis,), asthma (e.g. allergic asthma), GVHD
(graft-versus-host disease), graft rejection.
[0124] (3.1) Method for induction of GVHD by transfer of human PBMC
into NOD-Scid mice and measurement of disease severity.
[0125] NOD-Scid mice, non-obese diabetic/severe combined
immunodeficient mice (mutant allele: Prkdc.sup.Scid, strain:
NOD.CB17-Prkdc.sup.Scid, (Shultz et al. 1995)) from the Central
Laboratory Animal Facility of the University of Mainz (also
purchasable from The Jackson Laboratory via Charles River
Laboratories, Germany) are used as an animal model for transfer of
peripheral human immune cells and thus for the in vivo analysis of
immuno-regulatory interactions (Hesselton et al. 1995). Human
peripheral blood mononuclear cells (PBMC) transferred into NOD-Scid
mice induce an accelerating age-dependent graft-versus-host disease
(GVHD). While the number of cells transferred determines the onset
of this disease, the co-transfer of human Tregs can gradually delay
or prevent the disease. This model system further allows the study
of human Treg function. To induce a GVHD NOD-Scid mice at the age
of three to six days after birth are intraperitoneally injected
with 1.times.10.sup.7 to 3.times.10.sup.7 human PBMC (isolation of
human PBMC according to 2.1.1). GVHD is induced by immunological
(xenogenic) activation of human effector T cells in the PBMC
fraction injected into the mice. NOD-Scid mice, however, are not
able to react against the grafted human PBMC. The human
PBMC-induced disease is characterized by lack of weight increase
and weight loss, decreased mobility, hunched posture, ruffled fur
and organ inflammation in the treated animals (Kizilisik and
Al-Sebayel 1997). Transfer of human PBMC results in growth arrest
or weight loss within 30 to 40 days (depending on the number of
cells transferred) after transfer compared to untreated mice. Lack
of weight increase/weight loss is used as a parameter to score GVHD
severity. Control mice did not receive any PBMC. Data see FIG.
3.
[0126] (3.2) Method for induction of GVHD by transfer of human PBMC
into NOD-Scid mice and prevention of disease by additional transfer
of human regulatory T cells and measurement of disease
severity.
[0127] To prevent a GVHD disease in NOD-Scid mice at the age of
three to six days after birth induced by intraperitoneally
injection of 1.times.10.sup.7 to 3.times.10.sup.7 human PBMC
(isolation of human PBMC according to 2.1.1) additional
2.5.times.10.sup.6 human regulatory T cells (isolation of human
regulatory T cells according to 2.1.4) are injected
intraperitoneally (ratio Tregs to PBMC: 1:4). Co-transfer of human
regulatory T cells along with human PBMC (enhancement of Treg
ratio) results in prevention of development of GVHD and weight loss
similar development as untreated mice. Lack of weight
increase/weight loss is used as a parameter to score GVHD severity.
Control mice did not receive any PBMC. Data see FIG. 3.
[0128] (3.3) Method for induction of GVHD by transfer of human PBMC
into NOD-Scid mice and prevention of disease by additional
administration of HIV-1 gp120 or a substance which can interfere
with HIV-1 gp120-binding and measurement of disease severity.
[0129] To prevent a GVHD disease induced in NOD-Scid mice at the
age of three to six days after birth induced by intraperitoneally
injection of 1.times.10.sup.7 to 3.times.10.sup.7 human PBMC
(isolation of human PBMC according to 2.1.1) HIV-1 gp120 or a
substance which can interfere with HIV-1 gp120-binding is
administered in addition to the injection of human PBMC.
Administration of HIV-1 gp120 or a substance which can interfere
with HIV-1 gp120-binding induces an activation of human regulatory
T cells in the PBMC fraction resulting in prevention of organ
inflammation and prevention of growth arrest/weight loss similar to
mice treated with human PBMC and additional human regulatory T
cells or untreated mice. Lack of weight increase/weight loss is
used as a parameter to score GVHD severity. Control mice did not
receive any PBMC. Data see FIG. 4.
[0130] (3.4) Method for induction of GVHD by transfer of human PBMC
into NOD-Scid mice and to demonstrate that prevention of disease by
administration of HIV-1 gp120 or a substance which can interfere
with HIV-1 gp120-binding is mediated by human regulatory T cells in
the PBMC fraction injected into NOD-Scid mice and measurement of
disease severity.
[0131] NOD-Scid mice at the age of three to six days after birth
are intraperitoneally injected with 1.times.10.sup.7 human
CD25-depleted PBMC (isolation of human CD25-depleted PBMC according
to 2.1.7). GVHD is induced by 1.times.10.sup.7 human CD25-depleted
PBMC at the same magnitude as by transfer of 1.times.10.sup.7 human
non CD25-depleted PBMC into NOD-Scid mice. (Data see FIG. 5 and
FIG. 3 and FIG. 4) Mice receiving CD25-depleted PBMC and an
additional administration of HIV gp120 are not protected from
development of GVHD and weight loss. Prevention of GVHD by HIV
gp120 or a substance which can interfere with HIV-1 gp120-binding
depends on regulatory T cells. Lack of weight increase/weight loss
is used as a parameter to score GVHD severity. Data see FIG. 5.
DESCRIPTION OF FIGURES
[0132] FIG. 1: HIV-1 gp120 treatment activates human
CD4.sup.+CD25.sup.+ Treg cells
[0133] In order to study the influence of HIV-1 gp120 exclusively
on the function of CD4.sup.+CD25.sup.+ Treg cells, we developed a
coculture assay which contained CD8.sup.+ T cells as effectors to
exclude any influence of HIV-1 gp120 on the latter. Therefore,
isolated CD4.sup.+CD25.sup.+ Treg cells are cocultured with T
cell-depleted syngenic PBMC and allogeneic CD8.sup.+ T effector
cells. In this setting, proliferation of alloreactive CD8.sup.+ T
effector cells is only suppressed by activated CD4.sup.+CD25.sup.+
Treg cells such as upon additional anti-CD3 mAb stimulation or an
activating HIV-1 gp120-mediated signal. (All HIV-1 gp120 proteins:
Protein Sciences Corp. Meriden, USA).
[0134] 1.times.10.sup.5 isolated CD25.sup.+ Treg cells were
incubated with 3.times.10.sup.5 irradiated (50 Gy) syngenic T
cell-depleted PBMC and 1.times.10.sup.5 allogeneic CD8.sup.+ T
effector cells in presence/absence of different HIV-1 gp120
preparations, addition of 0.5 .mu.g/ml anti-CD3 (OKT-3) served as
positive control. Proliferation was determined by
.sup.3H-Tdr-Incorporation 4 days later.
[0135] Grey bars represent proliferation of CD8.sup.+ T cells and
inactivated syngenic CD3-depleted PBMC, white bars represent the
proliferation of CD4.sup.+CD25.sup.+ Tregs and the black bars
represent the proliferation of CD8.sup.+ T cells co-cultured with
CD4.sup.+CD25.sup.+ Tregs cells and inactivated syngenic
CD3-depleted PBMC. Due to inactivation PBMC don't contribute to
proliferation of the samples.
[0136] As can be seen by the low white bars, Tregs don't show a
significant proliferation under all conditions (without additional
stimulus, or anti-CD3, or HIV-1 gp120 MN, or HIV-1 gp120 LAV, or
HIV-1 gp120 CM). CD8.sup.+ T cells (grey bars) and CD8.sup.+ T
cells co-cultured with Treg cells (black bars) show the same
magnitude of proliferation under the condition without additional
stimulus representing the controls. Upon anti-CD3 stimulation
CD8.sup.+ T cells (grey bars) show strong increased proliferation
but CD8.sup.+ T cells co-cultured with Treg cells (black bars) show
a reduced proliferation indicating suppressed proliferation of
CD8.sup.+ T cells by anti-CD3 activated and therefore suppressive
Tregs. The effect is anti-CD3 dose-dependent. Upon stimulation with
HIV-1 gp120 MN, or HIV-1 gp120 LAV, or HIV-1 gp120 CM CD8.sup.+ T
cells co-cultured with Treg cells (black bars) in contrast to
CD8.sup.+ T cells without Tregs (grey bars) reduced proliferation
indicating HIV-1 gp120 activated Tregs. This demonstrates clearly
that HIV-1 gp120 activates Tregs which subsequently exert
suppressive activity on CD8.sup.+ T cells by reducing their
proliferation. The suppressive activity of Treg cells and therefore
the reduced proliferation is HIV-1 gp120 dose-dependent.
[0137] FIG. 2: Cyclic AMP measurement in CD4+CD25+ Treg cells.
[0138] HIV-1 gp120 treatment augments cytosolic cAMP in human
CD4.sup.+CD25.sup.+ Treg cells.
[0139] CD4.sup.+CD25.sup.+ regulatory T cells (CD25) are left
untreated (O) or stimulated with an anti-CD3 monoclonal Antibody
(OKT-3; 1 .mu.g/ml) or HIV-1 gp120 (14 ml). Upon 16 hours of
stimulation the CD4.sup.+CD25.sup.+ regulatory T cells are lysed
(1.times.10.sup.7/ml) and the cytosolic cAMP concentration of
1.times.10.sup.6 T cells is assessed using a cAMP-specific
ELISA.
[0140] CD4.sup.+CD25.sup.+ regulatory T cells (CD25) are left
untreated (O) or stimulated with an anti-CD3 monoclonal Antibody
(OKT-3; 1 .mu.g/ml) or HIV-1 gp120 (1 .mu.g/ml). Upon 16 hours of
stimulation the CD4.sup.+CD25.sup.+ regulatory T cells are lysed
(1.times.10.sup.7/ml) and the cytosolic cAMP concentration of
1.times.10.sup.6 T cells is assessed using a cAMP-specific
ELISA.
[0141] Bars represent the amount of cytosolic (intracellular)
cyclic AMP (cAMP) in untreated Treg cells or Treg cells treated
with different stimuli. The upper bar represent Tregs left
untreated (O) and shows the basal level of intracellular cAMP. Upon
anti-CD3 stimulation (OKT-3) Tregs are activated and show an
increase of intracellular cAMP compared to the untreated control
(upper bar) as demonstrated with the second bar. Treatment of Tregs
with HIV-1 gp120 also induces an increase of intracellular cAMP as
demonstrated with the lower bar. This demonstrated that activation
of Treg cells with different stimuli induces an increase of
cytosolic cAMP which can be used as readout for activation.
[0142] FIG. 3: Induction of GVHD by transfer of PBMC into NOD-Scid
mice and prevention of GVHD by additional transfer of regulatory T
cells (Tregs).
[0143] Three to six days old NOD-Scid mice are intraperitoneally
injected with 1.times.10.sup.7 human PBMC without (circles) or
together with 2.5.times.10.sup.6 human regulatory T cells
(triangles). Control mice (rhombi) did not receive any PBMC. Mice
having received PBMC develop a fatal GVHD, do not grow and die.
Animals having additionally received regulatory T cells are
protected from development of GVHD and develop normally (3 mice per
group). Lack of weight increase/weight loss is used as a parameter
to score GVHD severity. The diagram shows the relative body weight
at different time points after transfer.
[0144] FIG. 4: Induction of GVHD by transfer of PBMC into NOD-Scid
mice and prevention of GVHD by additional injection of HIV
gp120.
[0145] Three to six days old NOD-Scid mice are intraperitoneally
injected with 1.times.10.sup.7 human PBMC without (circles) or
together with 5 .mu.g HIV gp120 (triangles). Control mice (rhombs)
did not receive any PBMC. Mice having received PBMC develop a fatal
GVHD, do not grow and die. Animals having additionally received HIV
gp120 are protected from development of GVHD and develop normally
(3 mice per group). Lack of weight increase/weight loss is used as
a parameter to score GVHD severity. The diagram shows the relative
body weight at different time points after transfer.
[0146] FIG. 5: Prevention of GVHD in NOD-Scid mice transferred with
PBMC by additional injection of HIV gp120 depends on the presence
of regulatory T cells.
[0147] Three to six days old NOD-Scid mice are intraperitoneally
injected with 1.times.10.sup.7 human CD25-depleted PBMC without
(circles) or together with 5 .mu.g HIV gp120 (triangles). Control
mice (rhombi) do not receive any PBMC (3 mice per group). Mice
having received CD25-depleted PBMC develop a fatal GVHD, do not
grow and die. Animals having received 1.times.10.sup.7 human
CD25-depleted PBMC and additionally HIV gp120 are not protected
from development of GVHD, also do not grow and die. Lack of weight
increase/weight loss is used as a parameter to score GVHD severity.
The diagram shows the relative body weight at different time points
after transfer.
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Sequence CWU 1
1
10131PRThuman 1Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly
Ser Phe Leu 1 5 10 15 Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala
Asp Ser Arg Arg 20 25 30 2458PRThuman 2Met Asn Arg Gly Val Pro Phe
Arg His Leu Leu Leu Val Leu Gln Leu 1 5 10 15 Ala Leu Leu Pro Ala
Ala Thr Gln Gly Lys Lys Val Val Leu Gly Lys 20 25 30 Lys Gly Asp
Thr Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser 35 40 45 Ile
Gln Phe His Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn 50 55
60 Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala
65 70 75 80 Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly Asn Phe Arg Leu
Ile Ile 85 90 95 Lys Asn Leu Lys Ile Glu Asp Ser Asp Thr Tyr Ile
Cys Glu Val Glu 100 105 110 Asp Gln Lys Glu Glu Val Gln Leu Leu Val
Phe Gly Leu Thr Ala Asn 115 120 125 Ser Asp Thr His Leu Leu Gln Gly
Gln Ser Leu Thr Leu Thr Leu Glu 130 135 140 Ser Pro Pro Gly Ser Ser
Pro Ser Val Gln Cys Arg Ser Pro Arg Gly 145 150 155 160 Lys Asn Ile
Gln Gly Gly Lys Thr Leu Ser Val Ser Gln Leu Glu Leu 165 170 175 Gln
Asp Ser Gly Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys 180 185
190 Val Glu Phe Lys Ile Asp Ile Val Val Leu Ala Phe Gln Lys Ala Ser
195 200 205 Ser Ile Val Tyr Lys Lys Glu Gly Glu Gln Val Glu Phe Ser
Phe Pro 210 215 220 Leu Ala Phe Thr Val Glu Lys Leu Thr Gly Ser Gly
Glu Leu Trp Trp 225 230 235 240 Gln Ala Glu Arg Ala Ser Ser Ser Lys
Ser Trp Ile Thr Phe Asp Leu 245 250 255 Lys Asn Lys Glu Val Ser Val
Lys Arg Val Thr Gln Asp Pro Lys Leu 260 265 270 Gln Met Gly Lys Lys
Leu Pro Leu His Leu Thr Leu Pro Gln Ala Leu 275 280 285 Pro Gln Tyr
Ala Gly Ser Gly Asn Leu Thr Leu Ala Leu Glu Ala Lys 290 295 300 Thr
Gly Lys Leu His Gln Glu Val Asn Leu Val Val Met Arg Ala Thr 305 310
315 320 Gln Leu Gln Lys Asn Leu Thr Cys Glu Val Trp Gly Pro Thr Ser
Pro 325 330 335 Lys Leu Met Leu Ser Leu Lys Leu Glu Asn Lys Glu Ala
Lys Val Ser 340 345 350 Lys Arg Glu Lys Ala Val Trp Val Leu Asn Pro
Glu Ala Gly Met Trp 355 360 365 Gln Cys Leu Leu Ser Asp Ser Gly Gln
Val Leu Leu Glu Ser Asn Ile 370 375 380 Lys Val Leu Pro Thr Trp Ser
Thr Pro Val Gln Pro Met Ala Leu Ile 385 390 395 400 Val Leu Gly Gly
Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile 405 410 415 Phe Phe
Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met 420 425 430
Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro 435
440 445 His Arg Phe Gln Lys Thr Cys Ser Pro Ile 450 455
331PRTHIV-1MISC_FEATURE(3)..(4)Xaa can be any naturally occurring
amino acid 3Trp Gln Xaa Xaa Gly Xaa Ala Xaa Tyr Ala Xaa Pro Ile Xaa
Gly Xaa 1 5 10 15 Xaa Xaa Cys Xaa Ser Asn Ile Thr Gly Xaa Xaa Leu
Thr Xaa Asp 20 25 30 431PRTHIV-1 4Trp Gln Gly Ala Gly Gln Ala Met
Tyr Ala Pro Pro Ile Ser Gly Lys 1 5 10 15 Ile Asn Cys Val Ser Asn
Ile Thr Gly Ile Leu Leu Thr Arg Asp 20 25 30 531PRTHIV-1 5Trp Gln
Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln 1 5 10 15
Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp 20 25
30 631PRTHIV-1 6Trp Gln Lys Val Gly Lys Ala Met Tyr Ala Pro Pro Ile
Glu Gly Gln 1 5 10 15 Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu
Leu Thr Arg Asp 20 25 30 731PRTHIV-1MISC_FEATURE(3)..(4)Xaa can be
any naturally occurring amino acid 7Trp Gln Xaa Xaa Gly Xaa Ala Xaa
Tyr Ala Xaa Pro Ile Xaa Gly Xaa 1 5 10 15 Xaa Xaa Cys Xaa Ser Lys
Ile Thr Gly Xaa Xaa Leu Thr Xaa Asp 20 25 30
831PRTHIV-1MISC_FEATURE(3)..(4)Xaa can be any naturally occurring
amino acid 8Trp Gln Xaa Xaa Gly Xaa Ala Xaa Tyr Ala Xaa Pro Thr Xaa
Gly Xaa 1 5 10 15 Xaa Xaa Cys Xaa Ser Asn Ile Thr Gly Xaa Xaa Leu
Thr Xaa Asp 20 25 30 931PRTHIV-1MISC_FEATURE(3)..(4)Xaa can be any
naturally occurring amino acid 9Trp Gln Xaa Xaa Gly Xaa Ala Xaa Tyr
Thr Xaa Pro Ile Xaa Gly Xaa 1 5 10 15 Xaa Xaa Cys Xaa Ser Asn Ile
Thr Gly Xaa Xaa Leu Thr Xaa Asp 20 25 30
1031PRTHIV-1MISC_FEATURE(3)..(4)Xaa can be any naturally occurring
amino acid 10Val Ala Xaa Xaa Arg Xaa Ala Xaa Tyr Ala Xaa Pro Ile
Xaa Arg Xaa 1 5 10 15 Xaa Xaa Cys Xaa Ser Asn Ile Thr Gly Xaa Xaa
Leu Thr Xaa Asp 20 25 30
* * * * *