U.S. patent application number 16/588234 was filed with the patent office on 2020-03-05 for methods and compositions of treating autoimmune diseases.
This patent application is currently assigned to THE BRIGHAM AND WOMEN'S HOSPITAL, INC.. The applicant listed for this patent is THE BRIGHAM AND WOMEN'S HOSPITAL, INC.. Invention is credited to Ivan D. MASCANFRONI, Francisco J. QUINTANA.
Application Number | 20200069731 16/588234 |
Document ID | / |
Family ID | 51690049 |
Filed Date | 2020-03-05 |
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United States Patent
Application |
20200069731 |
Kind Code |
A1 |
QUINTANA; Francisco J. ; et
al. |
March 5, 2020 |
METHODS AND COMPOSITIONS OF TREATING AUTOIMMUNE DISEASES
Abstract
Embodiments of various aspects described herein are directed to
methods and compositions for producing a tolerogenic or
immunosuppressive dendritic cell. In particular, an
immunosuppressive dendritic cell can be produced by contacting a
dendritic cell with an agent that stimulates the
IL-27/ectonucleotidase CD39 axis signaling. In some embodiments,
the methods and/or compositions described herein can be used for
treating an autoimmune disease or disorder, e.g., but not limited
to multiple sclerosis (MS) and type 1 diabetes.
Inventors: |
QUINTANA; Francisco J.;
(Brookline, MA) ; MASCANFRONI; Ivan D.; (Brighton,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BRIGHAM AND WOMEN'S HOSPITAL, INC. |
Boston |
MA |
US |
|
|
Assignee: |
THE BRIGHAM AND WOMEN'S HOSPITAL,
INC.
Boston
MA
|
Family ID: |
51690049 |
Appl. No.: |
16/588234 |
Filed: |
September 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14783679 |
Oct 9, 2015 |
10426794 |
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PCT/US14/33872 |
Apr 11, 2014 |
|
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16588234 |
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61853745 |
Apr 11, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2851 20130101;
C07K 2319/33 20130101; A61P 37/06 20180101; A61K 35/15 20130101;
A61K 39/00 20130101; A61K 39/0008 20130101; A61K 2039/577 20130101;
C12Y 306/01005 20130101; A61K 47/6929 20170801; A61K 2039/5154
20130101; C12N 9/14 20130101; C07K 14/54 20130101; A61K 45/06
20130101 |
International
Class: |
A61K 35/15 20060101
A61K035/15; A61K 45/06 20060101 A61K045/06; A61K 39/00 20060101
A61K039/00; C07K 14/54 20060101 C07K014/54; C07K 16/28 20060101
C07K016/28; C12N 9/14 20060101 C12N009/14; A61K 47/69 20060101
A61K047/69 |
Claims
1. A method of treating an autoimmune disease or disorder
comprising administering to a patient in need thereof a dendritic
cell (DC)-targeting composition comprising a nanoparticle
containing: (a) an agent that stimulates IL-27/ectonucleotidase
CD39 axis signaling; and (b) an autoimmune antigen, thereby
treating the autoimmune disease or disorder in the patient in need
thereof.
2. The method of claim 1, wherein the agent comprises an IL-27
agonist, a CD39 agonist, and/or an ATP degrading agonist.
3. The method of claim 1, wherein the nanoparticle is a
liposome.
4. The method of claim 1, wherein the autoimmune disease or
disorder is multiple sclerosis, encephalomyelitis, or type I
diabetes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisonal application of U.S.
application Ser. No. 14/783,679 filed Oct. 9, 2015, now U.S. Pat.
No. 10,426,794, which is a 35 U.S.C. .sctn. 371 National Phase
Entry Application of International Application No.
PCT/US2014/033872 filed Apr. 11, 2014, which designates the U.S.,
and which claims benefit under 35 U.S.C. .sctn. 119(e) of the U.S.
Provisional Application No. 61/853,745 filed Apr. 11, 2013, the
contents of each of which are incorporated herein by reference in
their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 30, 2019, is named 043214-077852USD1_SL-14783679.txt, and
is 2,358 bytes in size.
TECHNICAL FIELD
[0003] Described herein generally relates to methods and
compositions for producing a tolerogenic or immunosuppressive
dendritic cell. The methods and/or compositions described herein
can be used for treating an autoimmune disease or disorder, e.g.,
but not limited to multiple sclerosis (MS), encephalomyelitis, and
type 1 diabetes.
BACKGROUND
[0004] The dysregulated activity of effector cells of the TH1 and
TH17 subsets of helper T cells results in the development of tissue
inflammation and autoimmunity. Myelin-specific TH1 and TH17 cells,
for example, contribute to disease pathogenesis in multiple
sclerosis (MS) and its animal model, experimental autoimmune
encephalomyelitis (EAE). Nylander and Hafler, J. Clin. Invest
(2012) 122:1180-188; and Pierson et al. Immunol. Rev. (2012) 248:
205-215. During EAE, dendritic cells (DCs) control the activation
and differentiation of myelin-specific effector T cells and
regulatory T cells (Treg cells). Bailey et al. Nat. Immunol. (2007)
8:172-180; and Yogev et al. Immunity (2012)37: 264-275. Moreover,
DCs isolated from patients with MS generally produce large amounts
of TH1- and TH17-polarizing cytokines. Comabella et al. Nat. Rev.
Nephrol. (2010) 6: 499-507. DCs control several pathogenic
mechanisms associated with the development of central nervous
system (CNS) autoimmunity. DCs promote the entry of T cells into
the CNS, the activation and differentiation of pathogenic T cells
in the CNS, and the spreading of the autoimmune response to new CNS
epitopes. Greter et al. Nat. Med. (2005) 11: 328-334; Bailey et al.
Nat. Immunol. (2007) 8:172-180; and McMahon et al. Nat. Med. (2005)
11:335-339. Accordingly, there is a need to identify pathways that
regulate DC activity during the course of autoimmunity, to identify
mechanisms of disease pathogenesis and also to develop new
approaches for therapeutic intervention to treat an autoimmune
disease.
SUMMARY
[0005] Embodiments of various aspects described herein are, in
part, based on the discovery that interleukin 27 (IL-27) acts on
dendritic cells (DCs) to expand regulatory T cells (Tregs) and/or
suppress T cell response (including, e.g., by limiting the
generation of effector cells of the Th1 and/or Th17 subsets of
helper T cells), which in turn inhibits development of an
autoimmune response. The inventors have also discovered that the
immunosuppressive effects of IL-27 on DCs are mediated at least in
part through induction of the immunoregulatory molecule
ectonucleotidase CD39 expression in DCs. Further, the inventors
have discovered that CD39 expressed by conventional DCs (cDCs)
reduced the extracellular concentration of ATP (eATP) and decreased
ATP-triggered activation of the NLRP3 inflammasome. The inventors
have also discovered that therapeutic vaccination with
IL-27-conditioned DCs can suppress established relapsing-remitting
experimental autoimmune encephalomyelitis (EAE). Thus, not only can
agents that modulate the activity and/or expression/level of IL-27,
IL-27RA, CD39 (or ectonucleotidase CD39), and/or pro-inflammatory
eATP be targeted to DCs for treatment of immune-related diseases or
disorders such as autoimmune diseases, but IL-27-conditioned DCs
can also be administered to a subject for treatment of
immune-related diseases or disorders. Accordingly, various aspects
described herein provide for methods for generating an
immunosuppressive dendritic cell, as well as methods and
compositions for treating an immune-related disease or disorder,
including, e.g., autoimmune disease.
[0006] One aspect provided herein relates to a method of generating
an immunosuppressive dendritic cell. The method comprises
contacting a dendritic cell with a composition comprising an
effective amount of an agent that stimulates or activates an
IL-27/ectonucleotidase CD39 axis signaling. The dendritic cell can
be obtained or derived from any source. For example, the dendritic
cell can be derived from a spleen, lymph node, blood, monocyte,
and/or hematopoietic progenitor cell.
[0007] The IL-27/ectonucleotidase CD39 axis or IL-27/CD39 axis
suppresses proinflammatory immune responses, e.g., via limiting the
generation of effector cells of the Th1 and Th17 subsets of helper
T cells. As defined herein and throughout the specification, the
terms "IL-27/ectonucleotidase CD39 axis" and "IL-27/CD39 axis," as
used interchangeably herein, refer to an immunosuppressive pathway
of DCs to regulate their antigen presenting function. The
immunosuppressive pathway includes IL-27 and ectonucleotidase CD39
(where the terms "ectonucleotidase CD39" and "CD39" are used
interchangeably herein), where the immunosuppressive effects of
IL-27 on DCs are mediated at least in part through induction of the
immunoregulatory molecule ectonucleotidase CD39 expression in DCs.
As noted above, the inventors have discovered that CD39 expressed
by DCs decreases the extracellular concentration of ATP (eATP) and
thus reduces ATP-triggered activation of the NLRP3 inflammasome.
Accordingly, in some embodiments, the IL-27/ectonucleotidase CD39
axis can further include an ATP-degrading enzyme, and thus the
"IL-27/ectonucleotidase CD39 axis" can refer to an
immunosuppressive pathway including IL-27, CD39, and an
ATP-degrading enzyme, including, e.g., apyrase.
[0008] In some embodiments, the agent that stimulates or activates
the IL-27/ectonucleotidase CD39 axis signaling (referred to as
"IL-27/CD39 agonistic agent" herein) is an IL-27 agonist. For
example, an IL-27 agonist can comprise a recombinant IL-27 protein
or peptide. In some embodiments, the IL-27/CD39 agonistic agent is
a CD39 agonist. In some embodiments, the IL-27/CD39 agonistic agent
is an ATP-degrading enzyme, including, e.g., apyrase.
[0009] An IL-27/CD39 agonistic agent can be present in any amount
sufficient to generate an immunosuppressive dendritic cell. For
example, the effective amount of an IL-27/CD39 agonistic agent can
be sufficient to upregulate the expression of CD39, phosphorylate
STAT3, and/or express one or more anti-inflammatory genes
(including, e.g., IDO1, IDO2, IL-10, IL-27, A20, TGF.beta.1, IL-10,
and/or IFN-.beta.) in DCs. Methods for detecting and/or measuring
these biological molecules or cytokines are known in the art. For
example, CD39 or anti-inflammatory gene and/or protein expressions
in DCs can be analyzed by quantitative PCR and/or FACS; while
phosphorylated STAT3 can be determined by FACS and/or western blot.
In some embodiments, the effective amount of the IL-27/CD39
agonistic agent can range from about 1 ng/mL to about 100
ng/mL.
[0010] In some embodiments, the method can further comprise
contacting the dendritic cell with an autoimmune antigen. The
dendritic cell can be contacted with an amount of an autoimmune
antigen sufficient to establish tolerance to a specific antigen. In
some embodiments, the autoimmune antigen to be contacted with a
dendritic cell can have a concentration of about 1 .mu.g/mL to
about 100 .mu.g/mL. Non-limiting examples of an autoimmune antigen
include myelin basic protein (MBP); proteolipid protein (PLP);
myelin oligodendrocyte glycoprotein (MOG), myelin-associated
oligodendrocytic basic protein cardiac myosin; outer surface
protein (OSP); myelin associated glycoprotein (MAG);
neurofilaments; interferon omega; transglutaminase; aromatic acid
carboxylase; 17-hydroxylase; 21-hydroxylase, cardiolipin; pyruvate
dehydrogenase; .beta.2 glycoprotein I; phosphatidylserine; apoH;
Annexin A5; LKM-1; soluble liver antigen; carbonic anhydrase;
gpIIb-IIIa or Ib-IX; type XVII collagen; tissue transglutaminase;
gliadin; GD1a; GQ1b; BP-1; BP-2; epidermal transglutaminase;
histidine-tRNA; signal recognition peptide; Mi-2; Jo1; Glutamic
acid decarboxylase, HSP60; HSP70; HSP90; IGRP; insulin;
carboxypeptidase H; insulinoma antigen-2; IA-2beta; ICA69; ZnT8;
chromogranin A; IAPP; scl70; topoisomerase; histones; Basement
Membrane Collagen Type IV; enolase; thyroid peroxidase;
thyroglobulin; complement component 3; voltage-gated calcium
channels; Q-type calcium channel, synaptogagmin, muscarinic
acetylcholine receptor M1; SMA; LKM-1; LKM-2; LKM-3; soluble liver
antigen; SLA; LP; major peripheral myelin protein P0;
myeloperoxidase; GQ1b; U1-RNP; Kir4.1; nicotinic acetylcholine
receptor; MuSK protein; hypocretin; orexin; keratin; AQP4; Yo; Hu;
glutamate receptor; Desmoglein 3; p62; sp100,; Ro; LA;
glycoproteins IIb-IIIa or Ib-IX; ADAMTS13; cardiolipin;
.beta..sub.2 glycoprotein I; HPA-Ia; HPA-5b; IFN-gamma, IL-1,
TNF-alpha; GMCSF, portions thereof, and combinations thereof.
[0011] The methods of generating an immunosuppressive dendritic
cell as described herein can be performed in a subject, ex vivo or
in vitro. Accordingly, in some embodiments, a dendritic cell can be
contacted ex vivo or in vitro with a composition comprising an
IL-27/CD39 agonistic agent. In alternative embodiments, a dendritic
cell can be contacted in vivo with a composition comprising an
IL-27/CD39 agonistic agent.
[0012] The immunosuppressive dendritic cells generated by the
methods described herein are distinct and can be identified from
non-treated dendritic cells or other tolerogenic dendritic cells.
In some embodiments, the immunosuppressive dendritic cells
described herein can comprise an increased expression of IL-27, as
compared to dendritic cells not contacted with an IL-27/CD39
agonistic agent (including, e.g., an IL-27 agonist). In some
embodiments, the immunosuppressive dendritic cells described herein
can comprise an increased expression of CD39, as compared to
dendritic cells not contacted with an IL-27/CD39 agonistic agent
(including, e.g., an IL-27 agonist). In some embodiments, the
immunosuppressive dendritic cells can comprise a reduced production
of an effector polarizing cytokine and/or an increased production
of an anti-inflammatory cytokine, as compared to dendritic cells
not contacted with an IL-27/CD39 agonistic agent (including, e.g.,
an IL-27 agonist). Exemplary effector polarizing cytokines include,
but are not limited to IL-12 and/or IL-6. Exemplary
anti-inflammatory cytokine include, but are not limited to
TGF.beta.1, IL-10, IFN-.beta., or any combinations thereof.
[0013] Accordingly, in another aspect, immunosuppressive dendritic
cell produced by the methods described herein involving an
IL-27/CD39 agonistic agent are also provided herein.
[0014] As noted earlier, the inventors have discovered, among other
things that, IL-27 acts on DCs to expand Tregs, limit Teffs and
suppress autoimmune diseases, e.g., but not limited to type 1
diabetes, multiple sclerosis (MS) and encephalomyelitis in animal
models. The anti-inflammatory effects of IL-27 on DCs are mediated
at least in part by the up-regulation of the ectonucleotidase CD39
(encoded by ENTPD1) and the consequent decrease in the levels of
pro-inflammatory extracellular ATP (eATP). In some immune-related
diseases or disorders, e.g., but not limited to autoimmune
diseases, it can be desirable to suppress pro-inflammatory
responses, e.g., via Th1 and/or Th17 responses for a therapeutic
effect. Accordingly, these immune-related diseases or disorders can
be treated by targeting the IL-27/CD39 axis in DCs to generate
tolerogenic or immunosuppressive DCs.
[0015] In some aspects, provided herein are methods of treating an
autoimmune disease or disorder. In some embodiments, the methods
can be adapted to treat multiple sclerosis. In some embodiments,
the methods can be adapted to treat encephalomyelitis. In some
embodiments, the methods can be adapted to treat type 1 diabetes.
The method of treatment comprises administering to a patient in
need thereof a dendritic cell (DC)-targeting composition comprising
(i) an agent that stimulates or activates IL-27/ectonucleotidase
CD39 axis signaling, and (ii) a DC-binding agent.
[0016] In some embodiments, the agent that stimulates or activates
the IL-27/ectonucleotidase CD39 axis signaling (referred to as
"IL-27/CD39 agonistic agent" herein) is an IL-27 agonist. For
example, an IL-27 agonist can comprise a recombinant IL-27 protein
or peptide. In some embodiments, the IL-27/CD39 agonistic agent is
a CD39 agonist. In some embodiments, the IL-27/CD39 agonistic agent
is an ATP-degrading enzyme, including, e.g., apyrase.
[0017] An IL-27/CD39 agonistic agent can be present in any amount
sufficient to generate an immunosuppressive dendritic cell. For
example, the effective amount of an IL-27/CD39 agonistic agent can
be sufficient to upregulate the expression of CD39, phosphorylate
STAT3, and/or express one or more anti-inflammatory genes
(including, e.g., IDO1, IDO2, IL-10, IL-27, A20, TGF.beta.1, IL-10,
and/or IFN-.beta.) in DCs. In some embodiments, the effective
amount of the IL-27/CD39 agonistic agent can range from about 1
ng/mL to about 100 ng/mL. In some embodiments, the effective amount
of the IL-27/CD39 agonistic agent can range from about 1 ng/kg to
about 100 mg/kg, or from about 0.1 mg/kg to about 50 mg/kg.
[0018] A DC-binding agent can be any agent or moiety that can
target or bind to DCs. In some embodiments, a DC-binding agent is
an agent or moiety that specifically targets or binds to DCs.
DC-binding agents are known in the art, including, e.g., agents
that bind to a DC surface protein or receptor. An exemplary
DC-binding agent includes, but is not limited to an antibody
against Clec9A and/or DEC205.
[0019] In some embodiments, the DC-targeting composition can
further comprise at least one or more autoimmune antigens. The
amount of an autoimmune antigen present in the DC-targeting
composition can be sufficient to establish immune tolerance to a
specific antigen in a subject in need thereof. For example, the
amount of an autoimmune antigen in the DC-targeting composition can
range from about 1 .mu.g/mL to about 100 .mu.g/mL. In some
embodiments, the amount of an autoimmune antigen in the
DC-targeting composition can range from about 0.1 .mu.g/kg to about
500 mg/kg, or from about 0.5 mg/kg to about 250 mg/kg. Non-limiting
examples of an autoimmune antigen include myelin basic protein
(MBP); proteolipid protein (PLP); myelin oligodendrocyte
glycoprotein (MOG), myelin-associated oligodendrocytic basic
protein cardiac myosin; outer surface protein (OSP); myelin
associated glycoprotein (MAG); neurofilaments; interferon omega;
transglutaminase; aromatic acid carboxylase; 17-hydroxylase;
21-hydroxylase, cardiolipin; pyruvate dehydrogenase; .beta.2
glycoprotein I; phosphatidylserine; apoH; Annexin A5; LKM-1;
soluble liver antigen; carbonic anhydrase; gpIIb-IIIa or lb-IX;
type XVII collagen; tissue transglutaminase; gliadin; GD1a; GQ1b;
BP-1; BP-2; epidermal transglutaminase; histidine-tRNA; signal
recognition peptide; Mi-2; Jo1; Glutamic acid decarboxylase, HSP60;
HSP70; HSP90; IGRP; insulin; carboxypeptidase H; insulinoma
antigen-2; IA-2beta; ICA69; ZnT8; chromogranin A; IAPP; scl70;
topoisomerase; histones; Basement Membrane Collagen Type IV;
enolase; thyroid peroxidase; thyroglobulin; complement component 3;
voltage-gated calcium channels; Q-type calcium channel,
synaptogagmin, muscarinic acetylcholine receptor M1; SMA; LKM-1;
LKM-2; LKM-3; soluble liver antigen; SLA; LP; major peripheral
myelin protein P0; myeloperoxidase; GQ1b; U1-RNP; Kir4.1; nicotinic
acetylcholine receptor; MuSK protein; hypocretin; orexin; keratin;
AQP4; Yo; Hu; glutamate receptor; Desmoglein 3; p62; sp100,; Ro;
LA; glycoproteins IIb-IIIa or Ib-IX; ADAMTS13; cardiolipin;
.beta..sub.2 glycoprotein I; HPA-1a; HPA-5b; IFN-gamma, IL-1,
TNF-alpha; GMCSF, portions thereof, and combinations thereof.
[0020] The DC-targeting composition acts as an immunomodulator that
preferentially or specifically targets or binds DCs, and can be
present in any appropriate format. For example, a DC-targeting
composition can be a fusion protein comprising a DC-binding agent
and at least one or more IL-27/CD39 agonistic agents described
herein.
[0021] Additionally or alternatively, the DC-targeting composition
can be formulated in the form of nanoparticle(s). The IL-27/CD39
agonistic agent, DC-binding agent, and/or optional autoimmune
antigen can distribute on a surface of the nanoparticle(s) or be
encapsulated in the nanoparticle(s). In some embodiments, the
DC-binding agent can form on the surface of the nanoparticle(s)
while one or more IL-27/CD39 agonistic agents and optional
autoimmune antigen(s) can be encapsulated in the nanoparticle(s),
which can be released therefrom to DCs.
[0022] In some embodiments, the nanoparticle(s) can further
comprise on its surface a biocompatible layer. The biocompatible
layer can prolong the half-time of the nanoparticles in a subject.
In one embodiment, the nanoparticle(s) can further comprise on its
surface a PEG layer.
[0023] Generally, nanoparticles administered to a subject can be
made of any biocompatible material. In one embodiment, the
nanoparticles are gold nanoparticles.
[0024] In another aspect, dendritic cells can be pre-treated with
one or more IL-27/CD39 agonistic agents as described herein
(including, e.g., IL-27 agonists) to generate immunosuppressive
dendritic cells, which can then be administered or transplanted to
a subject in need thereof, e.g., a subject diagnosed with an
autoimmune disease or disorder. Accordingly, also provided herein
is a method of treating an autoimmune disease or disorder
comprising administering to or placing in a subject in need thereof
a composition comprising a population of immunosuppressive
dendritic cells that are generated by contacting dendritic cells
with at least one or more IL-27/CD39 agonistic agents (including,
e.g., IL-27 agonists).
[0025] In some embodiments, the population of immunosuppressive
dendritic cells are autologous dendritic cells. Thus, in some
embodiments, the method can further comprise obtaining dendritic
cells from a sample of a subject. The sample can be a tissue biopsy
from a spleen or lymph node, or a blood sample. The autologous
dendritic cells can then be pre-treated ex vivo with at least one
or more IL-27/CD39 agonistic agents (including, e.g., IL-27
agonists), followed by transplantation into the subject.
[0026] In some embodiments, the composition comprising
immunosuppressive dendritic cells can further comprise an
autoimmune antigen as described herein. The autoimmune antigen can
be administered prior to, concurrently with, or after the
administration or placement of the composition comprising
immunosuppressive dendritic cells at a target tissue or organ site
of a subject.
[0027] It is contemplated that in other immune-related diseases or
disorder, including, e.g., cancer, it can be desirable to induce
proinflammatory responses, e.g., Th1/Th17 responses, at a target
site (e.g., a tumor) for a therapeutic effect. Accordingly, these
immune-related diseases or disorders, e.g., but not limited to
cancer, where upregulation of immune response is desirable, can be
treated by suppressing the IL-27/CD39 axis signaling. For example,
in some embodiments, a subject who is diagnosed with cancer can be
administered with a DC-targeting composition comprising a
DC-binding agent and an agent that suppresses IL-27/CD39 axis
signaling (also referred to as "IL-27/CD39 antagonistic
agent").
[0028] It is also contemplated that other inflammatory diseases or
disorders, including, e.g., allergy and asthma, where a dampening
Th2 response is desirable could be treated by downregulating or
suppressing the IL-27/CD39 axis signaling.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows fluorescence-activated cell sorting of DCs.
Splenic DCs were stained for F4/80, CD11b, CD11c, B220, MHCII and
Ly6c and sorted by flow cytometry into F4/80.sup.- CD11b.sup.-
CD11c.sup.low B220.sup.+ Ly6c.sup.+ pDCs and F4/80.sup.-
CD11b.sup.+ CD11c.sup.+ B220.sup.- MHC-II.sup.+ Ly6c.sup.- cDCs.
Numbers adjacent to outlined areas indicate percentage of positive
cells. Data are from one of more than 3 independent experiments
with similar results.
[0030] FIGS. 2A-2C show IL-27RA expression in DCs. Flow cytometry
(FIG. 2A), quantitative PCR (FIG. 2B) and immunoblot analysis (FIG.
2C) of IL-27RA expression in sorted cDCs and pDCs. Numbers above
bracketed line (FIG. 2A) indicate percent IL-27RA+ cDCs (red) and
pDCs (blue); dotted line, isotype-matched control antibody. Actin
serves as a loading control throughout. WT, wild-type. *P<0.05
(Student's t-test). Data are representative of more than three
independent experiments with similar results (error bars (FIG. 2B),
s.e.m.).
[0031] FIGS. 3A-3I show that IL-27 modulates the antigen-presenting
function of cDCs. (FIG. 3A) Flow cytometry of wild-type cDCs left
untreated (None) or treated with IL-27 (20 ng/ml) or ecLPS (100
ng/ml) alone or sequentially, presented as mean fluorescence
intensity (MFI). MHCII, MHC class II. (FIG. 3B) Enzyme-linked
immunosorbent assay of cytokines in culture supernatants of cDCs
treated as in a. (FIG. 3C) Quantitative PCR analysis of Il27 mRNA
in cDCs treated as in a, presented relative to that of the control
gene Gapdh. ND, not detected. (FIGS. 3D-3F) Proliferation (FIG.
3D), cytokines in culture supernatants (FIG. 3E) and frequency of
CD4+ IFN-.gamma.+, IL-17+, IL-10+ and Foxp3+ cells (FIG. 3F) among
naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus cDCs treated
as in FIG. 3A. (FIGS. 3G-3H) Cytokine secretion (FIG. 3G) and
frequency of Foxp3+ CD4+ T cells (FIG. 3H) among naive 2D2 CD4+ T
cells stimulated with MOG(35-55) plus cDCs treated as in FIG. 3A,
in the presence of exogenous cytokines to promote the
differentiation of TH1, TH17 and Tr1 cells (FIG. 3G) or Foxp3+ T
cells (FIG. 3H). Numbers in outlined areas (FIG. 3H, left) indicate
percent Foxp3+ CD4+ T cells. *P<0.05 and **P<0.01 (one-way
analysis of variance (ANOVA)). Data are from three independent
experiments (FIG. 3A-3H, right; mean and s.e.m.) or are
representative of three independent experiments (FIG. 3H, left).
(FIG. 3I) IL-27 signaling in DCs modulates MHC-II and
co-stimulatory molecule expression in DCs. Flow cytometry analysis
of ecLPS-treated cDC in the presence or absence of IL-27.
Representative histograms of three independent experiments, the
staining obtained with isotype control antibodies is shown in
gray.
[0032] FIGS. 4A-4E show that IL-27 limits effector T-cell
differentiation and EAE development. (FIG. 4A) Development of EAE
in WT and Il-27ra.sup.-/- mice, clinical score (left panel) and
linear-regression curves of disease for each group (dashed lines
indicate 95% confidence intervals). (FIG. 4B) CNS-infiltrating CD4+
T cells analyzed for the expression of IFN-.gamma., IL-17, IL-10
and Foxp3 by flow cytometry. (FIG. 4C) Recall response to MOG
(35-55) in splenocytes from WT and Il-27ra.sup.-/- mice isolated 21
days after EAE induction. (FIG. 4D) Frequency of
CD4+CD44+CD40L.sup.hi splenic IFN-.gamma.+, IL-17+, IFN-.gamma.+
IL-17+ (DP), IL-10+ and Foxp3+ CD4+ T cells in WT and
Il-27ra.sup.-/- mice 21 days after EAE induction. (FIG. 4E) Naive
CFSE labeled 2D2+ CD4+ T cells were stimulated with MOG (35-55) and
cDCs sorted from WT and Il-27ra.sup.-/- mice 21 days after
immunization, and T-cell proliferation was analyzed. The frequency
of proliferated cells is shown in the histogram and the
proliferation index is shown in the right (FIG. 4E). Numbers within
histograms show the percentage of positive cells. Shown is a
representative experiment (of three) with n.gtoreq.5 mice/group.
*P<0.05 and **P<0.01 (Student's t-test).
[0033] FIGS. 5A-5H show generation of mice lacking IL-27RA
expression in DCs. (FIG. 5A) Lethally irradiated WT mice were
reconstituted with bone marrow (BM) from mice expressing the
diphtheria toxin receptor (DTR) under the control of the CD11c
(itgax) promoter (CD11c-DTR mice). Following reconstitution, DCs
were depleted by the administration of diphtheria toxin (DTx) and
DCs compartment was reconstituted with DC precursors from WT
(Cx3Cr1.sup.-GFP.sup.+/-WT) or Il.sup.-27ra.sup.-/-
(Cx3Cr1.sup.-GFP.sup.+/-Il-27ra.sup.-/-) mice. (FIG. 5B)
Representative flow cytometry analysis of DCs precursors (CDPs).
(FIG. 5C) Antibodies against Diphtheria toxin (DT) in serum from DC
(WT) and DC (IL-27RA-KO) mice. (FIG. 5D) Expression of IL27ra in
cDCs, Ly6C.sup.lo and Ly6C.sup.hi monocytes sorted from naive DC
(WT) and DC (IL-27RA-KO) mice, analyzed by qPCR. (FIG. 5E)
Frequency (left panel) and absolute numbers of cDCs and pDCs in
spleens from DC (WT) and DC (IL-27RA-KO) mice. (FIGS. 5F-5H)
Passive transfer EAE in DC (WT) and DC (IL-27RA-KO) recipients. 2D2
mice were immunized with MOG (35-55) and 7 d after immunization T
cells were cultured with MOG (35-55) in the presence of IL-12 or
IL-23 and 48 h after re-stimulation IL-17 and IFN-.gamma. secreted
into the cell culture medium were determined by ELISA (FIG. 5F).
Following transfer of TH1 or TH17 polarized T cells into in DC (WT)
and DC (IL-27RA-KO) mice, the development of EAE was monitored in
the recipient mice. Clinical score (left panel) and
linear-regression curves of disease for each group (dashed lines
indicate 95% confidence intervals) (FIG. 5G). CNS-infiltrating CD4+
T cells analyzed for the expression of IFN-.gamma. and IL-17, IL-10
by flow cytometry (FIG. 5H). **P<0.01 (One-way ANOVA and
student's t-test) versus DC (WT). Data are representative of at
least three independent experiments.
[0034] FIGS. 6A-6I show that IL-27RA signaling in cDCs controls T
cell differentiation and EAE development. (FIG. 6A) Flow cytometry
of IL-27RA in splenic cDCs sorted from naive DC(WT) or
DC(IL-27RA-KO) mice. Numbers above bracketed line indicate percent
IL-27RA+ DC(WT) cDCs (black) or DC(IL-27RA-KO) cDCs (red); dotted
line, isotype-matched control antibody. (FIG. 6B) Development of
EAE in DC(WT) and DC(IL-27RA-KO) mice, presented as clinical score
(left) and linear-regression curves (right; thinner lines indicate
95% confidence interval). (FIG. 6C) Frequency of IFN-.gamma.+,
IL-17+, IL-10+ and Foxp3+ cells among CNS-infiltrating CD4+ T
cells, analyzed by flow cytometry. (FIG. 6D) Recall response to
MOG(35-55) (MOG) by splenocytes isolated from DC(WT) and
DC(IL-27RA-KO) mice 21 d after EAE induction. (FIG. 6E) Frequency
of CD4+ CD44+ CD40L.sup.hi splenic IFN-.gamma.+, IL-17+,
IFN-.gamma.+ IL-17+ (DP), IL-10+ and Foxp3+ CD4+ T cells in DC(WT)
and DC(IL-27RA-KO) mice 21 d after EAE induction. Numbers in
quadrants or adjacent to outlined areas indicate percent cells in
each throughout. (FIG. 6F) Expression of Il27ra, Il6, Il12a, Il23a,
Il27, Ifnb1, Il10 and Tgfb1 mRNA in cDCs sorted from DC(WT) and
DC(IL-27RA-KO) mice 21 d after EAE induction, presented relative to
that of Gapdh. (FIG. 6G) Quantitative expression profiling of cDCs
isolated from DC(WT) and DC(IL-27RA-KO) mice 21 d after EAE
induction, presented relative to that of endogenous control genes.
(FIGS. 6H-6I) Proliferation (FIG. 6H) and cytokine secretion (FIG.
6I) of naive 2D2 CD4+ T cells labeled with the division-tracking
dye CFSE and stimulated with MOG(35-55) plus cDCs sorted from
DC(WT) and DC(IL-27RA-KO) mice 21 d after EAE induction. Numbers
above plots (FIG. 6H, left) indicate percent CFSE+ (proliferated)
cells; green line (FIG. 6H), unproliferated cells. *P<0.05,
**P<0.01 and ***P<0.001, compared with DC(WT) (Student's
t-test). Data are from one experiment representative of three
experiments with five or more mice per group (error bars (FIG.
6B-6I), s.e.m.).
[0035] FIGS. 7A-7D show transcriptional effects of IL-27 on cDCs.
(FIGS. 7A-7B) Ingenuity Pathway Analysis (IPA) of the
transcriptional effects of IL-27 in DCs identified significant
effects of IL-27 on NF-kB (FIG. 7A) and Toll-like Receptor (FIG.
7B) signaling pathways. In NF-kB and Toll-like Receptor Signaling
pathways, green shaded regions indicate down-regulation and red
shaded regions indicate up-regulation of genes. (FIG. 7C) Time
course of Ido1 and Ido2, Entpd1, Il27, Il10, Tnip3, Tnfaip3, Ramp3
and Esr1 expression measured by quantitative real-time PCR in cDCs
treated with IL-27 for 0, 2, 6, and 24 h. Results are shown
relative to the expression of mRNA encoding Gapdh. (FIG. 7D)
Computational model of the effects of IL-27 on DCs generated with
NetGenerator. Integrated interactions in splenic IL-27-treated cDCs
compared with untreated cDCs are shown. Black edges denote inferred
connections without prior knowledge, green edges present an
agreement, and grey dashed edges stand for prior knowledge not
reproduced in the inferred network. *P<0.05 and **P<0.01
(One-way ANOVA) compared with untreated cDCs (Time 0).
[0036] FIGS. 8A-8J show that CD39 is required for the inhibitory
effects of IL-27 on DCs. (FIGS. 8A-8B) Proliferation of naive CD4+
T cells stimulated with anti-CD3 plus wild-type cDCs treated with
ecLPS alone (-) or pretreated with IL-27 and treated with ecLPS
(+), in the presence of isotype-matched control antibody (IC) or
blocking antibody (Ab) to IL-27, IL-10, IFN-.gamma. or TGF-.beta.
(FIG. 8A) or in the presence (+) or absence (-) of 1-D-MT (FIG.
8B). (FIG. 8C) Proliferation of T cells stimulated with anti-CD3
plus DC(CD39-KO) cDCs treated with ecLPS alone or pretreated with
IL-27 and treated with ecLPS. (FIG. 8D) Quantitative PCR analysis
of Entpd1 mRNA (left) and flow cytometry of CD39 (right) in cDCs
sorted from naive DC(WT) and DC(IL-27RA-KO) mice; mRNA results are
relative to that of Gapdh. Numbers above bracketed lines (right)
indicate percent CD39+ cells; dotted line, isotype-matched control
antibody. (FIGS. 8E-8F) Immunoblot analysis (FIG. 8E) and flow
cytometry (FIG. 8F) of phosphorylated (p-) and total STAT1 and
STAT3 in splenic cDCs exposed for various times to IL-27 (20
ng/ml). (FIG. 8G) STAT1-binding site (green; IRF-1), STAT3-binding
sites (blue; SRE-1 and SRE-2) and STAT1-STAT3-binding site
(green-blue) in the Entpd1 promoter. (FIGS. 8H-8I)
Chromatin-immunoprecipitation analysis of the interaction of STAT3
(FIG. 8H) or STAT1 (FIG. 8I) with various binding sites of the
Entpd1 promoter as in g (above graphs) in cDCs left untreated
(None) or treated with IL-27 or ecLPS alone or sequentially. (FIG.
8J) Luciferase activity in HEK293 cells transfected with a CD39
luciferase reporter alone (Control) or together with a construct
encoding constitutively activated STAT1 (STAT1c) or STAT3 (STAT3c)
separately or together (STAT1c+STAT3c). *P<0.05 and **P<0.01
(one-way ANOVA). Data are representative of more than three
independent experiments with similar results (error bars (FIGS.
8A-8D, 8F, and 8H-8J), s.e.m.).
[0037] FIGS. 9A-9E show that ENTPD1 is required for the effects of
IL-27 on DCs. (FIG. 9A) PD-L1 expression in IL-27-treated cDC in
the presence or absence of ecLPS. Numbers adjacent to outlined
areas indicate percentage of CD11c PD-L1 positive cells. (FIG. 9B)
Naive CD4+ T cells were stimulated with anti-CD3 and ecLPS- or
ecLPS+IL-27-treated WT, Il27ra.sup.-, Il10.sup.-, CD274
(PD-L1).sup.- or Entpd1 (CD39)-deficient cDCs and proliferation was
analyzed. (FIG. 9C) Naive CD4+ T cells were stimulated with
anti-CD3 and ecLPS- or ecLPS+IL-27-treated Entpd1-deficient cDCs
and the differentiation of IFN.gamma.+, IL-17+, IL-10+ and Foxp3+ T
cells was analyzed by flow cytometry. (FIG. 9D) Entpd1 expression
in cDCs, Ly6C.sup.lo and Ly6C.sup.hi monocytes sorted from naive DC
(WT) and DC (CD39-KO) mice, analyzed by qPCR. (FIG. 9E) Frequency
(left panel) and absolute numbers of cDCs and pDCs in spleens from
DC (WT) and DC (CD39-KO) mice. *P<0.05; **P<0.01 (One-way
ANOVA). Data are representative of at least three independent
experiments
[0038] FIGS. 10A-10F show that IL-27-induced CD39 controls
extracellular ATP and activation of the NLRP3 inflammasome. (FIG.
10A) Extracellular ATP concentration in culture supernatants of
wild-type (WT), IL-27RA-deficient (Il27ra.sup.-/-) or
CD39-deficient (Entpd1.sup.-/-) cDCs treated with IL-27 or ecLPS
alone or sequentially. (FIG. 10B) Residual extracellular ATP in
culture supernatants of cDCs treated with ecLPS in the presence (+)
or absence (-) of LPS after incubation with 500 .mu.M exogenous
ATP. (FIG. 10C) Thin-layer chromatography assay of the enzymatic
activity of CD39 in cDCs as in FIG. 10A. (FIG. 10D) Quantification
of AMP band intensity, presented in arbitrary units (AU) relative
to that of ADP in CD39-deficient cDCs treated as in FIG. 10A. (FIG.
10E) Immunoblot analysis (left) and densitometry (right) of
caspase-1 and IL-1.beta. in cDCs as in FIG. 10A. (FIG. 10F)
Quantification of IL-1.beta. in culture supernatants of cDCs as in
FIG. 10A. *P<0.05, **P<0.01 and ***P<0.001 (one-way
ANOVA). Data are representative of two independent experiments with
similar results (error bars (FIG. 10A-10B, and 10D-10F),
s.e.m.).
[0039] FIGS. 11A-11H show that CD39 in DCs controls T cell
differentiation and EAE development. (FIG. 11A) Flow cytometry of
CD39 in splenic DC sorted from naive DC(WT) or DC(CD39-KO) mice.
Numbers above bracketed line (FIG. 11A) indicate percent CD39+
DC(WT) cDCs (black) or DC(CD39-KO) cDCs (red); dotted line,
isotype-matched control antibody. (FIG. 11B) Development of EAE in
DC(WT) and DC(CD39-KO) mice (presented as in FIG. 6B). (FIG. 11C)
Frequency of IFN-.gamma.+, IL-17+, IL-10+ and Foxp3+ cells among
CNS-infiltrating CD4+ T cells, analyzed by flow cytometry. (FIG.
11D) Recall response to MOG(35-55) in splenocytes isolated from
DC(WT) and DC(CD39-KO) mice 21 d after EAE induction. (FIG. 11E)
Frequency of CD4+ CD44+ CD40L.sup.hi splenic IFN-.gamma.+, IL-17+,
IFN-.gamma.+ IL-17+ (DP), IL-10+ and Foxp3+ CD4+ T cells in DC(WT)
and DC(CD39-KO) mice 21 d after EAE induction. (FIG. 11F)
Expression of Entpd1, Il6, Il12a, Il23a, Il27, Ifnb1, Il10 and
Tgfb1 in cDCs sorted from DC(WT) and DC(CD39-KO) mice 21 d after
EAE induction, presented relative to that of Gapdh. (FIGS. 11G-11H)
Proliferation (FIG. 11G) and cytokine secretion (FIG. 11H) of naive
CFSE-labeled 2D2 CD4+ T cells stimulated with MOG(35-55) plus cDCs
sorted from DC(WT) and DC(CD39-KO) mice 21 d after immunization
(results in g presented as in FIG. 6H). *P<0.05 and **P<0.01,
compared with DC(WT) (Student's t-test). Data are from one
experiment representative of three experiments with five or more
mice per group (error bars (FIGS. 11B-11H), s.e.m.).
[0040] FIGS. 12A-12F show that vaccination with IL-27 conditioned
DCs suppresses EAE. EAE was induced by immunization of naive SJL
mice with PLP (131-159), and DCs were administered i.v. 4 times,
once every 4 days, starting at day 20. (FIG. 12A) The course of EAE
is shown as the mean EAE score.+-.SEM (n=5 mice per group) for the
whole observation period (left panel), and also as the linear
regression curves of the disease for each group from day 20 until
the termination of the experiment. Arrows indicate DC vaccine
administration. (FIG. 12B-12D) EAE was induced by immunization of
naive B6 mice with MOG (35-55), and DCs were administered i.v. 4
times, once every 4 days, starting at day 10 after EAE induction.
(FIG. 12B & 12C) The course of EAE is shown as the mean EAE
score.+-.SEM (n=5 mice per group) for the whole observation period
(left panel), and also as the linear regression curves of the
disease for each group. Arrows indicate DC vaccine administration.
(FIG. 12D) Effects of therapeutic DC vaccination on B6 EAE. (FIGS.
12E & 12F) Recall proliferative and cytokine response to MOG
(35-55) in splenocytes taken from DCs-treated mice 21 days after
EAE induction. Data are representative of at least three
independent experiments. NS, not significant. *P<0.05,
**P<0.01 and **P<0.001 (One-way ANOVA) versus control
mice.
[0041] FIGS. 13A-13F show that vaccination with IL-27-conditioned
DCs suppresses EAE. (FIG. 13A) Course of EAE induced by no
treatment (None) or immunization of naive SJL mice with
PLP(131-151) alone (DC+PLP) or IL-17 alone (DC+IL-27) or both
(DC+IL-27+PLP), followed by intravenous administration of DCs
(downward arrows) four times once every 4 d starting at day 20,
presented as clinical scores for the entire observation period
(left) and as linear-regression curves from day 20 until the
termination of the experiment (right). (FIGS. 13B-13E) Recall
proliferative response (FIGS. 13B and 13D) and cytokine response
(FIGS. 13C and 13E) to PLP(131-151) (FIGS. 13B-13C) or PLP(178-191)
(FIGS. 13D-13E) in splenocytes obtained from DC-treated mice 55 d
after EAE induction as in FIG. 13A. (FIG. 13F) Heat map of the
antibody response to myelin antigens (right margin) on day 55 after
EAE induction as in FIG. 13A (assessed by antigen microarray); each
column represents the mean serum reactivity of immunoglobulin G
(IgG) to each treatment condition (key, below). *P<0.05,
**P<0.01 and ***P<0.001, versus untreated control mice
(one-way ANOVA). Data are representative of at least three
independent experiments (mean and s.e.m. of five mice per group in
FIG. 13A; error bars (FIGS. 13B-13E), s.e.m.)
[0042] FIGS. 14A-14C show that IL-27 acts on DCs to control Treg
and Teff differentiation via ENTPD1 (CD39) up-regulation. (FIG.
14A) eATP activates the NLRP3 inflammasome in DCs and promotes Teff
differentiation. (FIG. 14B) ENTPD1 (CD39) induced by IL-27 degrades
eATP, limits Teff differentiation and promotes Treg generation.
(FIG. 14C) Biotherapeutics of DC-targeting antibodies fused to
IL-27 or apyrase promote Treg generation and limit Teff
differentiation.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Embodiments of various aspects described herein are, in
part, based on the discovery that interleukin 27 (IL-27) acts on
dendritic cells (DCs) to expand regulatory T cells (Tregs) and/or
suppress T cell response (including, e.g., by limiting the
generation of effector cells of the Th1 and/or Th17 subsets of
helper T cells), which in turn inhibits development of an
autoimmune response. The inventors have also discovered that the
immunosuppressive effects of IL-27 on DCs are mediated at least in
part through induction of the immunoregulatory molecule
ectonucleotidase CD39 expression in DCs. Further, the inventors
have discovered that CD39 expressed by conventional DCs (cDCs)
reduced the extracellular concentration of ATP (eATP) and decreased
ATP-triggered activation of the NLRP3 inflammasome. The inventors
have also discovered that therapeutic vaccination with
IL-27-conditioned or IL-27-treated DCs can suppress established
relapsing-remitting experimental autoimmune encephalomyelitis
(EAE). Thus, not only can agents that modulate the activity and/or
expression/level of IL-27, IL-27RA, CD39 (or ectonucleotidase
CD39), and/or pro-inflammatory eATP be targeted to DCs for
treatment of immune-related diseases or disorders such as
autoimmune diseases, but IL-27-conditioned or IL-27-treated DCs can
also be administered to a subject for treatment of immune-related
diseases or disorders such as autoimmune diseases or disorders.
Accordingly, various aspects described herein provide for methods
for generating an immunosuppressive dendritic cell, as well as
methods and compositions for treating an immune-related disease or
disorder, including, e.g., autoimmune disease.
Immunosuppressive Dendritic Cells and Methods of Generating the
Same
[0044] One aspect provided herein relates to a method of generating
an immunosuppressive dendritic cell. The method comprises
contacting a dendritic cell with a composition comprising an
effective amount of an agent that stimulates or activates an
IL-27/ectonucleotidase CD39 axis signaling. The dendritic cell can
be obtained or derived from any source. For example, the dendritic
cell can be derived from a spleen, lymph node, blood, monocyte,
and/or hematopoietic progenitor cell.
[0045] DCs are antigen presenting cells (APC) that control the
activation and/or polarization of T cells into specific lineages.
The interplay between T cell lineages regulates the development of
an autoimmune disease or disorder, e.g., but not limited to
multiple sclerosis, autoimmune encephalomyelitis, and diabetes. DCs
express a functional IL-27 receptor (18); however, the
physiological relevance of IL-27 signaling in DCs and its effects
on the control of the T cell response and autoimmunity are unknown.
In accordance with various aspects described herein, an agent that
stimulates or activates an IL-27/CD39 axis signaling can act on DCs
to suppress the T cell response and autoimmunity.
[0046] As used herein and throughout the specification, the phrase
"agent that stimulates or activates an IL-27/ectonucleotidase CD39
axis signaling" or "IL-27/CD39 agonistic agent," as used
interchangeably herein, refers to an agent that induces
immunosuppression mediated by the IL-27/CD39 axis signaling as
defined earlier. The IL-27/CD39 axis suppresses proinflammatory
immune responses or induces immunosuppression, e.g., via limiting
generation of effector cells of the Th1 and Th17 subsets of helper
T cells. As noted above, the inventors have discovered that CD39
expressed by DCs decreases the extracellular concentration of ATP
(eATP) and thus reduces ATP-triggered activation of the NLRP3
inflammasome, as well as promoting Treg (regulatory T cell)
generation and/or limiting Teff (effector T cell)
differentiation.
[0047] In some embodiments, the IL-27/CD39 agonistic agent to be
contacted with a dendritic cell can be an IL-27 agonist. For
example, an IL-27 agonist can comprise a recombinant IL-27 protein
or peptide. In some embodiments, the IL-27/CD39 agonistic agent can
be a CD39 agonist. In some embodiments, the IL-27/CD39 agonistic
agent can be an ATP-degrading enzyme, including, e.g., apyrase.
[0048] An IL-27/CD39 agonistic agent can be present in any amount
sufficient to generate an immunosuppressive dendritic cell. For
example, the effective amount of an IL-27/CD39 agonistic agent can
be sufficient to upregulate the expression of CD39, phosphorylate
STAT3, and/or express one or more anti-inflammatory genes
(including, e.g., IDO1, IDO2, IL-10, IL-27, A20, TGF.beta.1, IL-10,
and/or IFN-.beta.) in DCs. For example, the effective amount of an
IL-27/CD39 agonistic agent can be sufficient to upregulate the
expression of CD39, phosphorylation of STAT3, and/or expression of
one or more anti-inflammatory genes (including, e.g., IDO1, IDO2,
IL-10, IL-27, A20, TGF.beta.1, IL-10, and/or IFN-.beta.) in DCs by
at least about 10% or more (including, e.g., at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 95%, at least about 97% or more, as compared to
DCs without the IL-27/CD39 agonistic agent. In some embodiments,
the effective amount of an IL-27/CD39 agonistic agent can be
sufficient to upregulate the expression of CD39, phosphorylation of
STAT3, and/or expression of one or more anti-inflammatory genes
(including, e.g., IDO1, IDO2, IL-10, IL-27, A20, TGF.beta.1, IL-10,
and/or IFN-.beta.) in DCs by at least about 1.1-fold or more
(including, e.g., at least about 1.5-fold, at least about 2-fold,
at least about 3-fold, at least about 4-fold, at least about
5-fold, at least about 6-fold, at least about 7-fold, at least
about 8-fold, at least about 9-fold, at least about 10-fold or
more, as compared to DCs without the IL-27/CD39 agonistic agent.
Methods for detecting and/or measuring these biological molecules
or cytokines are known in the art. For example, CD39 or
anti-inflammatory gene and/or protein expressions in DCs can be
analyzed by quantitative PCR, immunoassay, and/or FACS; while
phosphorylated STAT3 can be determined by FACS, immunoassay, and/or
western blot.
[0049] In some embodiments, the effective amount of the IL-27/CD39
agonistic agent can range from about 1 ng/mL to about 100 ng/mL. In
some embodiments, the effective amount of the IL-27/CD39 agonistic
agent can range from about 5 ng/mL to about 50 ng/mL, from about 10
ng/mL to about 40 ng/mL. In some embodiments, the effective amount
of the IL-27/CD39 agonistic agent can be about 10 ng/mL to about 30
ng/mL, or about 15 ng/mL to about 25 ng/mL.
[0050] In some embodiments, the effective amount of the IL-27/CD39
agonistic agent can be at least about 1 ng/mL, at least about 5
ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, at least
about 20 ng/mL, at least about 25 ng/mL, at least about 30 ng/mL,
at least about 40 ng/mL, at least about 50 ng/mL, at least about 60
ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, at least
about 90 ng/mL, or at least about 100 ng/mL.
[0051] In some embodiments, the effective amount of an IL-27
agonist can range from about 1 ng/mL to about 100 ng/mL. In some
embodiments, the effective amount of the IL-27 agonist can range
from about 5 ng/mL to about 50 ng/mL, from about 10 ng/mL to about
40 ng/mL. In some embodiments, the effective amount of the IL-27
agonist can be about 10 ng/mL to about 30 ng/mL, or about 15 ng/mL
to about 25 ng/mL. In some embodiments, the effective amount of an
IL-27 agonist can be at least about 1 ng/mL, at least about 5
ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, at least
about 20 ng/mL, at least about 25 ng/mL, at least about 30 ng/mL,
at least about 40 ng/mL, at least about 50 ng/mL, at least about 60
ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, at least
about 90 ng/mL, or at least about 100 ng/mL.
[0052] In some embodiments, the method can further comprise
contacting the dendritic cell with an autoimmune antigen. The
contact of dendritic cells with at least one or more autoimmune
antigens can occur prior to, concurrently with, or after the
contact of dendritic cells with a composition comprising an
IL-27/CD39 agonistic agent.
[0053] The term "antigen" as used herein means a substance,
molecule, or compound that stimulates an immune response. Although
usually a protein or polysaccharide, antigens may be any type of
molecule or microorganism (e.g., cells and/or virus), which can
include small molecules (haptens) that are optionally coupled to a
carrier-protein.
[0054] As used herein, an "immune response" being modulated refers
to a response by a cell of the immune system, such as a B cell, T
cell (CD4 or CD8), regulatory T cell, antigen-presenting cell,
dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil,
eosinophil, or neutrophil, to a stimulus. In some embodiments, the
response is specific for a particular antigen (an "antigen-specific
response"), and refers to a response by a CD4 T cell, CD8 T cell,
or B cell via their antigen-specific receptor. In some embodiments,
an immune response is a T cell response, such as a CD4+ response or
a CD8+response. Such responses by these cells can include, for
example, cytotoxicity, proliferation, cytokine or chemokine
production, trafficking, or phagocytosis, and can be dependent on
the nature of the immune cell undergoing the response.
[0055] As used herein, the term "autoimmune antigen" refers to any
self protein or self component that serves either as a target or
cause of an autoimmune disease. Examples of autoimmune antigens
include, but are not limited to, myelin basic protein (MBP);
proteolipid protein (PLP); myelin oligodendrocyte glycoprotein
(MOG), myelin-associated oligodendrocytic basic protein cardiac
myosin; outer surface protein (OSP); myelin associated glycoprotein
(MAG); neurofilaments; interferon omega; transglutaminase; aromatic
acid carboxylase; 17-hydroxylase; 21-hydroxylase, cardiolipin;
pyruvate dehydrogenase; .beta.2 glycoprotein I; phosphatidylserine;
apoH; Annexin A5; LKM-1; soluble liver antigen; carbonic anhydrase;
gpIIb-IIIa or lb-IX; type XVII collagen; tissue transglutaminase;
gliadin; GD1a; GQ1b; BP-1; BP-2; epidermal transglutaminase;
histidine-tRNA; signal recognition peptide; Mi-2; Jo1; Glutamic
acid decarboxylase, HSP60; HSP70; HSP90; IGRP; insulin;
carboxypeptidase H; insulinoma antigen-2; IA-2beta; ICA69; ZnT8;
chromogranin A; IAPP; scl70; topoisomerase; histones; Basement
Membrane Collagen Type IV; enolase; thyroid peroxidase;
thyroglobulin; complement component 3; voltage-gated calcium
channels; Q-type calcium channel, synaptogagmin, muscarinic
acetylcholine receptor M1; SMA; LKM-1; LKM-2; LKM-3; soluble liver
antigen; SLA; LP; major peripheral myelin protein P0;
myeloperoxidase; GQ1b; U1-RNP; Kir4.1; nicotinic acetylcholine
receptor; MuSK protein; hypocretin; orexin; keratin; AQP4;Yo; Hu;
glutamate receptor; Desmoglein 3; p62; sp100,; Ro; LA;
glycoproteins IIb-IIIa or Ib-IX; ADAMTS13; cardiolipin; .beta.2
glycoprotein I; HPA-1a; HPA-5b; IFN-gamma, IL-1, TNF-alpha; GMCSF,
portions thereof, and combinations thereof. Additional examples of
autoimmune antigens include, but are not limited to, peripheral
myelin proteins P0 and P2 (Guillain-Barre syndrome); acetylcholine
receptor (myasthenia gravis); cardiac myosin (rheumatic
fever/myocarditis); proteins of the beta cells in the Isles of
Langerhans-GAD (glutamic acid decarboxylase), insulin (Type I
autoimmune diabetes mellitus), the thyroid-stimulating hormone
receptor (Grave's disease), platelets (thrombocytopenic purpura),
neuromuscular junction (myasthenia gravis), red blood cells
(autoimmune hemolytic anemia and intracellular antigens
(spliceosomes, ribosomes, nucleic acid, etc in systemic lupus
erythematosus), portions thereof, and combinations thereof.
[0056] In some embodiments, the autoimmune antigen can encompass a
neuroantigen. As used herein, the term "neuroantigen" (NAg) refers
to a type of autoimmune antigen that is a nervous system protein
(central or peripheral) including an auto-reactive epitope. The
neuroantigen can be a myelin basic protein (MBP), a proteolipid
protein (PLP), myelin oligodendrocyte glycoprotein (MOG),
myelin-associated oligodendrocytic basic protein (MOG), or other
nervous system-derived proteins or a portion thereof and further
including those derived from any species, including, e.g., human,
rat and mouse.
[0057] The dendritic cell can be contacted with an amount of an
autoimmune antigen sufficient to establish tolerance to a specific
antigen. The term "tolerance" as used herein refers to a decreased
level of an immune response, a delay in the onset or progression of
an immune response and/or a reduced risk of the onset or
progression of an immune response. "Specific" immunological
tolerance occurs when immunological tolerance is preferentially
invoked against certain antigens in comparison with others.
"Active" immunological tolerance refers to a state in which the
tolerance effect(s) are the result of an ongoing biological
process: for example, down-regulation of specific effector cells by
suppressor cells. "Sustained tolerance" is tolerance that
measurably persists for an extended period of time.
[0058] In some embodiments, the autoimmune antigen to be contacted
with a dendritic cell can be in a concentration of about 0.01
.mu.g/mL to about 100 .mu.g/mL, about 0.1 .mu.g/mL to about 100
.mu.g/mL, about 1 .mu.g/mL to about 100 .mu.g/mL, about 5 .mu.g/mL
to about 90 .mu.g/mL, about 10 .mu.g/mL to about 80 .mu.g/mL, about
20 .mu.g/mL to about 70 .mu.g/mL, about 30 .mu.g/mL to about 60
.mu.g/mL. In some embodiments, the autoimmune antigen can have a
concentration of about 0.1 .mu.g/mL to about 10 .mu.g/mL.
[0059] The methods of generating an immunosuppressive dendritic
cell as described herein can be performed in a subject, ex vivo or
in vitro. Accordingly, in some embodiments, a dendritic cell can be
contacted ex vivo or in vitro with a composition comprising an
IL-27/CD39 agonistic agent. In alternative embodiments, a dendritic
cell can be contacted in vivo with a composition comprising an
IL-27/CD39 agonistic agent.
[0060] As used herein, the term "contacting" refers to any suitable
means for delivering, or exposing, an agent (e.g., an IL-27/CD39
agonistic agent and/or autoimmune antigen) to cells, e.g.,
dendritic cells. Exemplary delivery methods include, but are not
limited to, direct delivery to cell culture medium, delivery to a
cell culture, e.g., via perfusion, administration to a subject
(e.g., by injection, and/or implantation),or other delivery method
well known to one skilled in the art. In one embodiment, an
IL-27/CD39 agonistic agent and the optional autoimmune antigen can
be added to the cell culture medium in which the dendritic cells
are cultured. In another embodiment, an IL-27/CD39 agonistic agent
and optional autoimmune antigen can be coated on a solid support on
which the dendritic cells are cultured. In still another
embodiment, an IL-27/CD39 agonistic agent and optional autoimmune
antigen can be injected into a biocompatible gel or matrix (e.g.,
peptide gel, hydrogel) in which the dendritic cells are
encapsulated. In one embodiment, dendritic cells are contacted with
an IL-27/CD39 agonistic agent and optional autoimmune antigen added
to the cell culture medium. In another embodiment, an IL-27/CD39
agonistic agent and optional autoimmune antigen can be introduced
or targeted to dendritic cells in a subject. The term "conditioned"
or "treated" as used herein, with respect to exposing cells to an
agent, e.g., treatment of dendritic cells with an IL-27/CD39
agonistic agent and optional autoimmune antigen, is used herein
interchangeably with the term "contacting".
[0061] Dendritic cells can be contacted, treated or conditioned
with a composition comprising an IL-27/CD39 agonistic agent and
optional autoimmune antigen for any period of time, e.g., minutes,
hours, days, or weeks. In some embodiments, the dendritic cells can
be contacted with a composition comprising an IL-27/CD39 agonistic
agent and optional autoimmune antigen for at least about 5 minutes,
at least about 10 minutes, at least about 15 minutes, at least
about 30 minutes, at least about 1 hour, at least about 2 hours, at
least about 3 hours, at least about 4 hours, at least about 5
hours, at least about 6 hours, at least about 9 hours, at least
about 12 hours, at least about 18 hours, at least about 24 hours or
longer. In some embodiments, the dendritic cells can be contacted
with a composition comprising an IL-27/CD39 agonistic agent and
optional autoimmune antigen for at least about 1 day, at least
about 2 days, at least about 3 days, at least about 4 days, at
least about 5 days, at least about 6 days, at least about 7 days or
longer. In some embodiments, the dendritic cells can be in contact
with a composition comprising an IL-27/CD39 agonistic agent and
optional autoimmune antigen until they are ready for administration
to a subject in need thereof, e.g., diagnosed with an autoimmune
disease.
[0062] The dendritic cell can be obtained or derived from any
source. For example, the dendritic cell can be derived from a
spleen, lymph node, blood, monocyte, and/or hematopoietic
progenitor cell. In some embodiments, the dendritic cells comprise
conventional (myeloid) DCs. In some embodiments, the dendritic
cells comprise plasmacytoid DCs. Methods for isolation of dendritic
cells are known in the art. See, e.g., Current Protocols in
Immunology (1998) supplement 25: 3.7.1-3.7.15; Inaba et al. Curr
Protoc Immunol (2001) Chapter 3: Unit 3.7, and the Examples
described herein. Kits for isolation of dendritic cells are
commercially available (e.g., from STEMCELL.TM. Technologies,
and/or Life Technologies) and can be used to isolated dendritic
cells.
[0063] Immunosuppressive dendritic cells: In another aspect,
immunosuppressive dendritic cell produced by the methods described
herein involving an IL-27/CD39 agonistic agent are also provided
herein. The immunosuppressive dendritic cells generated by various
embodiments of the methods described herein are distinct and can be
identified from non-treated dendritic cells using methods known in
the art, including, but not limited to, FACS, western blot, qPCR,
and/or immunoassay. In some embodiments, the immunosuppressive
dendritic cells generated by the methods described herein can be
identified and isolated from non-treated dendritic cells by FACS
sorting based on expression of IL-27 and/or CD39, and/or
phosphorylation of STAT3, and/or expression of anti-inflammatory
genes, including, e.g., IDO1, IDO2, IL-10, IL-27, A20 and any other
anti-inflammatory genes discussed in the Examples herein.
[0064] In some embodiments, the immunosuppressive dendritic cells
described herein can comprise an increased expression of IL-27 by
at least about 10% or more (including, e.g., at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 95%, at least about 97%, or more, as compared
to dendritic cells not contacted with an IL-27/CD39 agonistic agent
(including, e.g., an IL-27 agonist). In some embodiments, the
immunosuppressive dendritic cells described herein can comprise an
increased expression of IL-27 by at least about 1.1-fold or more
(including, e.g., at least about 1.5-fold, at least about 2-fold,
at least about 3-fold, at least about 4-fold, at least about
5-fold, or more, as compared to dendritic cells not contacted with
an IL-27/CD39 agonistic agent (including, e.g., an IL-27
agonist).
[0065] In some embodiments, the immunosuppressive dendritic cells
described herein can comprise an increased expression of CD39 by at
least about 10% or more (including, e.g., at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 95%, at least about 97%, or more, as compared
to dendritic cells not contacted with an IL-27/CD39 agonistic agent
(including, e.g., an IL-27 agonist). In some embodiments, the
immunosuppressive dendritic cells described herein can comprise an
increased expression of CD39 by at least about 1.1-fold or more
(including, e.g., at least about 1.5-fold, at least about 2-fold,
at least about 3-fold, at least about 4-fold, at least about
5-fold, or more, as compared to dendritic cells not contacted with
a composition comprising an IL-27/CD39 agonistic agent (including,
e.g., an IL-27 agonist).
[0066] In some embodiments, the immunosuppressive dendritic cells
described herein can comprise an increased phosphorylation of STAT3
by at least about 10% or more (including, e.g., at least about 20%,
at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 95%, at least about 97%, or more, as
compared to dendritic cells not contacted with an IL-27/CD39
agonistic agent (including, e.g., an IL-27 agonist). In some
embodiments, the immunosuppressive dendritic cells described herein
can comprise an increased phosphorylation of STAT3 by at least
about 1.1-fold or more (including, e.g., at least about 1.5-fold,
at least about 2-fold, at least about 3-fold, at least about
4-fold, at least about 5-fold, or more, as compared to dendritic
cells not contacted with a composition comprising an IL-27/CD39
agonistic agent (including, e.g., an IL-27 agonist).
[0067] In some embodiments, the immunosuppressive dendritic cells
can comprise a reduced production of an effector polarizing
cytokine by at least about 10% or more (including, e.g., at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 97%, or up to
100%), as compared to dendritic cells not contacted with an
IL-27/CD39 agonistic agent (including, e.g., an IL-27 agonist).
Exemplary effector polarizing cytokines include, but are not
limited to IL-12 and/or IL-6.
[0068] In some embodiments, the immunosuppressive dendritic cells
can comprise an increased production of an anti-inflammatory
cytokine and/or expression of an anti-inflammatory gene by at least
about 10% or more (including, e.g., at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 95%, at least about 97%, or more), as compared to
dendritic cells not contacted with an IL-27/CD39 agonistic agent
(including, e.g., an IL-27 agonist). Exemplary anti-inflammatory
cytokine include, but are not limited to TGF.beta.1, IL-10,
IFN-.beta., or any combinations thereof. Exemplary
anti-inflammatory genes include, but are not limited to, IDO1,
IDO2, IL-10, IL-27, A20 and any other anti-inflammatory genes
discussed in the Examples herein.
[0069] In some embodiments, the immunosuppressive dendritic cells
can be tolerogenic. By the term "tolerogenic" as used herein, it is
meant that a response of immunological tolerance is induced by an
antigen or antigenic substance or an activity that results in the
induction of immunological tolerance toward an antigen or antigenic
substance.
[0070] It is also contemplated that immunoactive dendritic cells
can be produced by suppressing the IL-27/CD39 axis signaling. For
example, dendritic cells can be contacted with an agent that
suppresses one or more components of the IL-27/CD39 axis signaling.
In some embodiments, such an agent can be an IL-27 antagonist. In
some embodiments, such an agent can be a CD39 antagonist.
[0071] In some embodiments, dendritic cells can be obtained from a
subject and made to become immunosuppressive dendritic cells using
the methods described herein. The resultant, autologous
immunosuppressive cells can then be administered to the subject for
use in a therapeutic treatment, e.g., of an autoimmune disease.
Methods of Treating an Immune-Related Disease or Disorder, e.g.,
but Not Limited to Autoimmune Diseases or Disorders
[0072] As noted earlier, the inventors have discovered, among other
things that, IL-27 acts on DCs to expand Tregs, limit Teffs and
suppress autoimmune diseases, e.g., but not limited to type 1
diabetes, multiple sclerosis (MS) and encephalomyelitis in animal
models. The anti-inflammatory effects of IL-27 on DCs are mediated
at least in part by the up-regulation of the ectonucleotidase CD39
(encoded by ENTPD1) and the consequent decrease in the levels of
pro-inflammatory extracellular ATP (eATP). In some immune-related
diseases or disorders, e.g., but not limited to autoimmune
diseases, it can be desirable to suppress pro-inflammatory
responses, e.g., via Th1 and/or Th17 responses, or promote
anti-inflammatory resposnes, e.g., via expression and/or production
of anti-flammatory cytokines for a therapeutic effect. Accordingly,
these immune-related diseases or disorders can be treated by
inducing the IL-27/CD39 axis signaling in DCs to generate
immunosuppressive DCs.
[0073] In some aspects, provided herein are methods of treating an
autoimmune disease or disorder. "Autoimmune disease or disorder"
refers to a class of diseases or disorders in which a subject's own
antibodies react with host tissue or in which immune effector T
cells are autoreactive to endogenous self-peptides and cause
destruction of tissue. Thus an immune response is mounted against a
subject's own antigens, referred to as self-antigens. A
"self-antigen" as used herein refers to an antigen of a normal host
tissue. Normal host tissue does not include cancer cells.
[0074] Accordingly, in some embodiments, the autoimmune diseases to
be treated or prevented using the methods described herein,
include, but are not limited to: rheumatoid arthritis, Crohn's
disease, multiple sclerosis, systemic lupus erythematosus (SLE),
autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's
thyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigus
vulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, scleroderma with anti-collagen
antibodies, mixed connective tissue disease, polymyositis,
pernicious anemia, idiopathic Addison's disease,
autoimmune-associated infertility, glomerulonephritis (e.g.,
crescentic glomerulonephritis, proliferative glomerulonephritis),
bullous pemphigoid, Sjogren's syndrome, insulin resistance, and
autoimmune diabetes mellitus (type 1 diabetes mellitus;
insulin-dependent diabetes mellitus). Autoimmune disease has been
recognized also to encompass atherosclerosis and Alzheimer's
disease. In one embodiment of the aspects described herein, the
autoimmune disease is selected from the group consisting of
multiple sclerosis, type-I diabetes, Hashinoto's thyroiditis,
Crohn's disease, rheumatoid arthritis, systemic lupus
erythematosus, gastritis, autoimmune hepatitis, hemolytic anemia,
autoimmune hemophilia, autoimmune lymphoproliferative syndrome
(ALPS), autoimmune uveoretinitis, glomerulonephritis,
Guillain-Barre syndrome, psoriasis and myasthenia gravis.
[0075] The method of treatment comprises administering to a patient
in need thereof a dendritic cell (DC)-targeting composition
comprising (i) an agent that stimulates or activates
IL-27/ectonucleotidase CD39 axis signaling (or an IL-27/CD39
agonistic agent), and (ii) a DC-binding agent.
[0076] In some embodiments, the methods can be adapted to treat
type 1 diabetes.
[0077] In some embodiments, the methods can be adapted to treat
multiple sclerosis.
[0078] In some embodiments, the methods can be adapted to treat
encephalomyelitis.
[0079] DC-targeting compositions: A DC-targeting composition
comprises at least one or more (e.g., at least two, at least three
or more) IL-27/CD39 agonistic agents. In some embodiments, the
IL-27/CD39 agonistic agent(s) can be an IL-27 agonist. For example,
an IL-27 agonist can comprise a recombinant IL-27 protein or
peptide. In some embodiments, the IL-27/CD39 agonistic agent(s) can
be a CD39 agonist. In some embodiments, the IL-27/CD39 agonistic
agent(s) can be an ATP-degrading enzyme, including, e.g.,
apyrase.
[0080] An IL-27/CD39 agonistic agent can be administered to in any
amount sufficient to generate an immunosuppressive dendritic cell.
For example, the effective amount of an IL-27/CD39 agonistic agent
can be sufficient to upregulate the expression of CD39,
phosphorylation of STAT3, and/or expression of one or more
anti-inflammatory genes (including, e.g., IDO1, IDO2, IL-10, IL-27,
A20, TGF.beta.1, IL-10, and/or IFN-.beta.) in DCs, as compared to
DCs not contacted with the IL-27/CD39 agonistic agent.
[0081] In some embodiments, the effective amount of an IL-27/CD39
agonistic agent present in a DC-targeting composition can be
sufficient to upregulate expression of IL-27 in DCs by at least
about 10% or more (including, e.g., at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 95%, at least about 97%, or more, as compared to
dendritic cells not contacted with an IL-27/CD39 agonistic agent
(including, e.g., an IL-27 agonist). In some embodiments, the
effective amount of an IL-27/CD39 agonistic agent can be sufficient
to upregulate expression of IL-27 in DCs by at least about 1.1-fold
or more (including, e.g., at least about 1.5-fold, at least about
2-fold, at least about 3-fold, at least about 4-fold, at least
about 5-fold, or more, as compared to dendritic cells not contacted
with an IL-27/CD39 agonistic agent (including, e.g., an IL-27
agonist).
[0082] In some embodiments, the effective amount of an IL-27/CD39
agonistic agent can be sufficient to upregulate expression of CD39
in DCs by at least about 10% or more (including, e.g., at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 97%, or more,
as compared to dendritic cells not contacted with an IL-27/CD39
agonistic agent (including, e.g., an IL-27 agonist). In some
embodiments, the effective amount of an IL-27/CD39 agonistic agent
can be sufficient to upregulate expression of CD39 in DCs by at
least about 1.1-fold or more (including, e.g., at least about
1.5-fold, at least about 2-fold, at least about 3-fold, at least
about 4-fold, at least about 5-fold, or more, as compared to
dendritic cells not contacted with a composition comprising an
IL-27/CD39 agonistic agent (including, e.g., an IL-27 agonist).
[0083] In some embodiments, the effective amount of an IL-27/CD39
agonistic agent can be sufficient to upregulate phosphorylation of
STAT3 in DCs by at least about 10% or more (including, e.g., at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 95%, at least about 97%, or
more, as compared to dendritic cells not contacted with an
IL-27/CD39 agonistic agent (including, e.g., an IL-27 agonist). In
some embodiments, the effective amount of an IL-27/CD39 agonistic
agent can be sufficient to upregulate phosphorylation of STAT3 in
DCs by at least about 1.1-fold or more (including, e.g., at least
about 1.5-fold, at least about 2-fold, at least about 3-fold, at
least about 4-fold, at least about 5-fold, or more, as compared to
dendritic cells not contacted with a composition comprising an
IL-27/CD39 agonistic agent (including, e.g., an IL-27 agonist).
[0084] In some embodiments, the effective amount of an IL-27/CD39
agonistic agent can be sufficient to reduce DCs' production of an
effector polarizing cytokine by at least about 10% or more
(including, e.g., at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, at
least about 97%, or up to 100%), as compared to dendritic cells not
contacted with an IL-27/CD39 agonistic agent (including, e.g., an
IL-27 agonist). Exemplary effector polarizing cytokines include,
but are not limited to IL-12 and/or IL-6.
[0085] In some embodiments, the effective amount of an IL-27/CD39
agonistic agent can be sufficient to increase DCs' production of at
least one or more anti-inflammatory cytokine and/or expression of
one or more anti-inflammatory gene by at least about 10% or more
(including, e.g., at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, at
least about 97%, or more), as compared to dendritic cells not
contacted with an IL-27/CD39 agonistic agent (including, e.g., an
IL-27 agonist). Exemplary anti-inflammatory cytokine include, but
are not limited to TGF.beta.1, IL-10, IFN-.beta., or any
combinations thereof. Exemplary anti-inflammatory genes include,
but are not limited to, IDO1, IDO2, IL-10, IL-27, A20 and any other
anti-inflammatory genes discussed in the Examples herein.
[0086] In some embodiments, the effective amount of the IL-27/CD39
agonistic agent can range from about 1 ng/mL to about 100 ng/mL. In
some embodiments, the effective amount of the IL-27/CD39 agonistic
agent can range from about 5 ng/mL to about 50 ng/mL, from about 10
ng/mL to about 40 ng/mL. In some embodiments, the effective amount
of the IL-27/CD39 agonistic agent can be about 10 ng/mL to about 30
ng/mL, or about 15 ng/mL to about 25 ng/mL.
[0087] In some embodiments, the effective amount of the IL-27/CD39
agonistic agent can be at least about 1 ng/mL, at least about 5
ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, at least
about 20 ng/mL, at least about 25 ng/mL, at least about 30 ng/mL,
at least about 40 ng/mL, at least about 50 ng/mL, at least about 60
ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, at least
about 90 ng/mL, or at least about 100 ng/mL.
[0088] In some embodiments, the effective amount of an IL-27
agonist can range from about 1 ng/mL to about 100 ng/mL. In some
embodiments, the effective amount of the IL-27 agonist can range
from about 5 ng/mL to about 50 ng/mL, from about 10 ng/mL to about
40 ng/mL. In some embodiments, the effective amount of the IL-27
agonist can be about 10 ng/mL to about 30 ng/mL, or about 15 ng/mL
to about 25 ng/mL. In some embodiments, the effective amount of an
IL-27 agonist can be at least about 1 ng/mL, at least about 5
ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, at least
about 20 ng/mL, at least about 25 ng/mL, at least about 30 ng/mL,
at least about 40 ng/mL, at least about 50 ng/mL, at least about 60
ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, at least
about 90 ng/mL, or at least about 100 ng/mL.
[0089] The effective dosage of an IL-27/CD39 agonistic agent will
vary from composition to composition, patient to patient, and will
depend up on the physical and/or medical condition of a patient,
and/or the route of delivery. In some embodiments, the effective
amount of the IL-27/CD39 agonistic agent can vary with body weight
of a subject, e.g., ranging from about 1 ng/kg to about 200 mg/kg,
or from about 0.01 mg/kg to about 150 mg/kg, or from about 0.1
mg/kg to about 100 mg/kg, or from about 1 mg/kg to about 50 mg/kg.
The effective dosage can be administered to a subject in a single
dose or divided doses.
[0090] DC-binding agents: DC-targeting compositions comprising an
IL-27/CD39 agonistic agent described herein are adapted to
preferentially or specifically target DCs. Accordingly, the
DC-targeting compositions administered to a subject comprise one or
more DC-binding agents. As used herein, the term "DC-binding agent"
refers to any material, substance, agent or moiety which can
promote targeting of a composition comprising an IL-27/CD39
agonistic agent to dendritic cells in vivo and/or in vitro. The
DC-binding agent can be synthetic, semi-synthetic, or
naturally-occurring. Materials or substances which can serve as
DC-binding agents include, for example, proteins, including
antibodies, antibody fragments, hormones, hormone analogues,
glycoproteins, peptides, polypeptides, amino acids, sugars,
saccharides, including monosaccharides and polysaccharides,
carbohydrates, vitamins, steroids, steroid analogs, hormones,
cofactors, and genetic material, including nucleosides,
nucleotides, nucleotide acid constructs, peptide nucleic acids
(PNA), aptamers, and polynucleotides. Other DC-binding agents in
the present invention include cell adhesion molecules (CAM), among
which are, for example, cytokines, integrins, cadherins,
immunoglobulins and selectin. The DC-targeting compositions can
also encompass at least one or more precursor DC-binding agent. A
precursor to a DC-binding agent refers to any material or substance
which can be converted to a DC-binding agent. Such conversion can
involve, for example, anchoring a precursor to a DC-binding agent.
Exemplary targeting precursor moieties include maleimide groups,
disulfide groups, such as ortho-pyridyl disulfide, vinylsulfone
groups, and azide groups. The DC-binding agent(s) can be covalently
(e.g., cross-linked) or non-covalently linked to the DC-targeting
composition and/or IL-27/CD39 agonistic agent.
[0091] In some embodiments, a DC-binding agent is an agent or
moiety that specifically or preferentially targets or binds to DCs
such that a substantial amount of an IL-27/CD30 agonistic agent can
be delivered to DCs. In some embodiments, the DC-binding agent
specifically targets or binds DCs only. In some embodiments, the
DC-binding agent preferentially targets or binds DC and does not
target any T cells. As used herein, the term "preferentially"
refers to a greater amount of an IL-27/CD39 agonistic agent present
in a DC-targeting composition being delivered to dendritic cells
than any other cells. In some embodiments, at least 50% or more
(including, e.g., at least about 60%, at least about 70%, at least
about 80%, at least about 90%, at least about 95%, at least about
97%, or more, and up to 100%) of an IL-27/CD39 agonistic agent
present in a DC-targeting composition is delivered to dendritic
cells.
[0092] Any DC-binding agents known in the art can be used in the
DC-targeting compositions. Examples of DC-binding agents include,
but are not limited to agents that bind to a DC surface protein or
receptor. An exemplary DC-binding agent includes, but is not
limited to an antibody against Clec9A and/or DEC205. Additional
examples of DC-binding agents such as anti-DC receptor antibodies
and DC-binding peptides that can be used in the DC-targeting
compositions described herein include, but not limited to the ones
described in Flamar et al. Retrovirology (2009) 6 (Suppl 3): p286;
Sioud et al. FASEB J. (2013) 27: 3272-83; and Subramanya et al. J.
Virol (2010) 84: 2490-2501.
[0093] The term "antibody" as used herein generally refers to a
full length antibody or immunoglobulin, IgG, IgM, IgA, IgD or IgE
molecules, or a protein portion thereof that comprises only a
portion of an intact antibody, generally including an antigen
binding site of the intact antibody and thus retaining the ability
to bind a target, such as an epitope or antigen. Examples of
portions of antibodies or epitope-binding proteins encompassed by
the present definition include: (i) the Fab fragment, having VL,
CL, VH and CH1 domains; (ii) the Fab' fragment, which is a Fab
fragment having one or more cysteine residues at the C-terminus of
the CH1 domain; (iii) the Fd fragment having VH and CH1 domains;
(iv) the Fd' fragment having VH and CH1 domains and one or more
cysteine residues at the C terminus of the CH1 domain; (v) the Fv
fragment having the VL and VH domains of a single arm of an
antibody; (vi) the dAb fragment (Ward et al., 341 Nature 544
(1989)) which consists of a VH domain or a VL domain that binds
antigen; (vii) isolated CDR regions or isolated CDR regions
presented in a functional framework; (viii) F(ab')2 fragments which
are bivalent fragments including two Fab' fragments linked by a
disulphide bridge at the hinge region; (ix) single chain antibody
molecules (e.g., single chain Fv; scFv) (Bird et al., 242 Science
423 (1988); and Huston et al., 85 PNAS 5879 (1988)); (x)
"diabodies" with two antigen binding sites, comprising a heavy
chain variable domain (VH) connected to a light chain variable
domain (VL) in the same polypeptide chain (see, e.g., EP 404,097;
WO 93/11161; Hollinger et al., 90 PNAS 6444 (1993)); (xi) "linear
antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1)
which, together with complementary light chain polypeptides, form a
pair of antigen binding regions (Zapata et al., 8 Protein Eng. 1057
(1995); and U.S. Pat. No. 5,641,870).
[0094] "Antibodies" include antigen-binding portions of antibodies
such as epitope- or antigen-binding peptides, paratopes, functional
CDRs; recombinant antibodies; chimeric antibodies; tribodies;
midibodies; or antigen-binding derivatives, analogs, variants,
portions, or fragments thereof.
[0095] In some embodiments, a DC-binding agent can comprise a
single chain antibody against a dendritic cell surface protein or
receptor, including, but not limited to, Clec9A and/or DEC205.
[0096] DC-targeting fusion proteins: The DC-targeting composition
acting as an immunomodulator can be present in any appropriate
format to specifically or preferentially target or bind DCs. For
example, a DC-targeting composition can be a fusion protein
comprising a DC-binding agent described herein and at least one or
more IL-27/CD39 agonistic agents described herein. As used herein,
the term "fusion protein" refers to a fusion polypeptide comprising
a target polypeptide (e.g., an IL-27/CD39 agonistic agent) and at
least a second, heterologous fusion partner polypeptide. The fusion
partner can, for example, increase the in vivo stability of the
fusion polypeptide, modulate its biological activity or
localization, or facilitate purification of the fusion
polypeptide.
[0097] In some embodiments, the fusion partner can facilitate
targeting of the compositions described herein comprising at least
one or more (including, e.g., at least two or more) IL-27/CD39
agonistic agents to dendritic cells. In some embodiments, the
fusion partner can comprise or be a DC-binding agent described
herein.
[0098] Additional examples of heterologous fusion partner
polypeptides that can be further included to generate such fusion
polypeptides for use in the compositions and methods described
herein include, but are not limited to, polyhistidine (His or 6His
tag (SEQ ID NO: 1)), Glu-Glu tag, glutathione S transferase (GST),
thioredoxin, polypeptide A, polypeptide G, an immunoglobulin heavy
chain constant region (Fc), and maltose binding polypeptide (MBP),
which are particularly useful for isolation of the fusion
polypeptides by affinity chromatography. For the purpose of
affinity purification, relevant matrices for affinity
chromatography, such as glutathione-, amylase-, and nickel- or
cobalt-conjugated resins are used. In some embodiments, the fusion
polypeptides can have a protease cleavage site, such as for Factor
Xa or Thrombin, which allows the relevant protease to partially
digest the fusion polypeptides and thereby liberate the recombinant
polypeptides therefrom. The liberated polypeptides can then be
isolated from the fusion polypeptides by subsequent chromatographic
separation.
[0099] DC-targeting nanoparticles: In some embodiments, the
DC-targeting composition comprising at least one or more
(including, e.g., at least two, at least three or more) IL-27/CD39
agonistic agents can be formulated in the form of nanoparticle(s).
The IL-27/CD39 agonistic agent, DC-binding agent, and/or optional
autoimmune antigen can distribute on a surface of the
nanoparticle(s) or be encapsulated in the nanoparticle(s). In some
embodiments, the DC-binding agent can form on the surface of the
nanoparticle(s) while one or more IL-27/CD39 agonistic agents and
optional autoimmune antigen(s) can be encapsulated in the
nanoparticle(s), which can be released therefrom to DCs.
[0100] In some embodiments, the nanoparticle(s) can comprise on its
surface a biocompatible layer or material. As used herein, the term
"biocompatible layer or material" refers to any material or layer
that does not deteriorate appreciably and does not induce a
significant adverse effect, e.g., toxic reaction, over time when
placed adjacent to the biological tissue of a subject, or induce
blood clotting or coagulation when it comes in contact with blood.
Suitable biocompatible materials can include, but are not limited
to, polymers comprising an amino group (e.g., carbohydrate-based
amino-polymers, protein-based amino-polymers, or molecules
comprising at least one amino group), silk fibroin, derivatives and
copolymers of polyimides, polyvinyl alcohol, polyethyleneimine,
polyvinylamine, polyacrylates, polyamides, polyesters,
polycarbonates, polydimethylsiloxane, polyimide, polyethylene
terephthalate, polymethylmethacrylate, polyurethane,
polyvinylchloride, polystyrene, polysulfone, polycarbonate,
polymethylpentene, polypropylene, a polyvinylidine fluoride,
polysilicon, polytetrafluoroethylene, polysulfone, acrylonitrile
butadiene styrene, polyacrylonitrile, polybutadiene, poly(butylene
terephthalate), poly(ether sulfone), poly(ether ether ketones),
poly(ethylene glycol), styrene-acrylonitrile resin,
poly(trimethylene terephthalate), polyvinyl butyral,
polyvinylidenedifluoride, poly(vinyl pyrrolidone), polyethylene
glycol, and any combinations thereof. In some embodiments, the
nanoparticles can comprise on its surface a layer of polyethylene
glycol (PEG). In some embodiments, the biocompatible layer can be
selected to prolong the circulation time of the nanoparticles in a
subject. In some embodiments, the biocompatible layer can be
selected to induce antigen-specific immunity in a subject. In some
embodiments, the biocompatible layer can be selected to reduce or
minimize the exposure of the nanoparticle material to surrounding
tissue in a subject. In one embodiment, the nanoparticle(s) can
further comprise on its surface a PEG layer.
[0101] Generally, nanoparticles administered to a subject can be
made of any biocompatible material as described herein and/or inert
metals, e.g., but not limited to gold. As used herein, the term
"nanoparticle" refers to particle having a particle size of about
0.1 nm to about 1000 nm. Further, the nanoparticle can be of any
shape or form, e.g., spherical, rod, elliptical, cylindrical,
capsule, or disc. The term "nanoparticle" includes nanospheres,
nanorods, nanoshells, and nanoprisms and these nanoparticles can be
part of a nanonetwork. Without limitations, the nanoparticles used
herein can be any nanoparticle available in the art or available to
one of skill in the art. In some embodiments, the nanoparticle is
of size from about 10 nm to about 750 nm, from about 20 nm to about
500 nm, from about 25 nm to about 250 nm, or from about 50 nm to
about 150nm. In some embodiments, the nanoparticle is of size from
about 5 nm to about 75 nm, from about 10 nm to about 50 nm, from
about 15 nm to about 25 nm. The nanoparticles can be, e.g.,
monodisperse or polydisperse and the variation in diameter of the
particles of a given dispersion can vary. The nanoparticles can be
hollow or solid. In one embodiment, the nanoparticles are gold
nanoparticles.
[0102] In one embodiment, a DC-targeting composition described
herein comprises pegylated gold nanoparticles, wherein at least one
or more (including, e.g., at least two or more) IL-27/CD39
agonistic agents form on the surface of the gold nanoparticles. The
pegylated nanoparticles described in Yeste et al. PNAS (2012) 109:
1270-5 can be modified to deliver a composition comprising at least
one or more (including, e.g., at least two or more) IL-27/CD39
agonistic agents to dendritic cells.
[0103] In some embodiments, the DC-targeting composition described
herein can further comprise at least one or more (including, e.g.,
at least two, at least three or more) autoimmune antigens as
described herein. In some embodiments, the autoimmune antigen(s)
can be linked to a DC-targeting fusion protein described herein. In
some embodiments, the autoimmune antigen(s) can be coupled to a
DC-targeting nanoparticle described herein. The amount of an
autoimmune antigen present in the DC-targeting composition can be
sufficient to establish immune tolerance to a specific antigen in a
subject in need thereof. For example, the amount of an autoimmune
antigen in the DC-tar geting composition can range from about 0.01
.mu.g/mL to about 100 .mu.g/mL, about 0.1 .mu.g/mL to about 100
.mu.g/mL, about 1 .mu.g/mL to about 100 .mu.g/mL, about 5 .mu.g/mL
to about 90 .mu.g/mL, about 10 .mu.g/mL to about 80 .mu.g/mL, about
20 .mu.g/mL to about 70 .mu.g/mL, about 30 .mu.g/mL to about 60
.mu.g/mL. In some embodiments, the autoimmune antigen can have a
concentration of about 0.1 .mu.g/mL to about 10 .mu.g/mL.
[0104] In some embodiments, the amount of an autoimmune antigen in
the DC-targeting composition can range from about 0.01 .mu.g/kg to
about 1000 .mu.g/kg, or from about 0.1 .mu.g/kg to about 500
.mu.g/kg, or from about 0.5 .mu.g/kg to about 250 .mu.g/kg, or from
about 1 .mu.g/kg to about 100 .mu.g/kg. In some embodiments, the
amount of an autoimmune antigen in the DC-targeting composition can
range from about 1 mg/kg to about 500 mg/kg, or from about 5 mg/kg
to about 250 mg/kg, or from about 10 mg/kg to about 100 mg/kg, or
from about 20 mg/kg to about 50 mg/kg.
[0105] In another aspect, dendritic cells can be pre-treated with
one or more IL-27/CD39 agonistic agents as described herein
(including, e.g., IL-27 agonists) to generate immunosuppressive
dendritic cells, which can then be administered or implanted to a
subject in need thereof, e.g., a subject diagnosed with an
autoimmune disease or disorder. Accordingly, also provided herein
is a method of treating an autoimmune disease or disorder
comprising administering to or placing in a subject in need thereof
a composition comprising a population of immunosuppressive
dendritic cells that are generated by contacting dendritic cells
with at least one or more IL-27/CD39 agonistic agents (including,
e.g., IL-27 agonists).
[0106] In some embodiments, the population of immunosuppressive
dendritic cells comprise autologous dendritic cells. Thus, in some
embodiments, the method can further comprise obtaining dendritic
cells from a sample of a subject. For example, the sample can be a
tissue biopsy from a spleen or lymph node, or a blood sample. The
autologous dendritic cells can then be pre-treated ex vivo with at
least one or more IL-27/CD39 agonistic agents (including, e.g.,
IL-27 agonists), followed by implantation into the subject.
[0107] In some embodiments, the composition comprising
immunosuppressive dendritic cells can further comprise an
autoimmune antigen as described herein. The autoimmune antigen can
be administered prior to, concurrently with, or after the
administration or placement of the composition comprising
immunosuppressive dendritic cells at a target tissue or organ site
of a subject.
[0108] Vaccines: In some embodiments, the DC-targeting compositions
described herein and/or composition comprising immunosuppressive
dendritic cells described herein can be considered as therapeutic
vaccines for treatment of an autoimmune disease or disorder.
[0109] The term "vaccine" as used herein generally refers to an
immunological composition given to an animal to elicit an immune
response against an agent. As used herein, the term "vaccine"
includes both therapeutic and prophylactic vaccines. The term
"vaccine" is defined herein in its broad sense to mean a biological
agent used to produce active immunity. Vaccines generally employ
one of four categories of antigens: live microorganisms
administered via an unnatural route, live attenuated
microorganisms, killed microorganisms and fractions or even a
single antigen or product of a microorganism. In all situations,
the goal is to present antigens without giving the disease. In some
embodiments, the antigens comprise autoimmune antigens described
herein.
[0110] In some embodiments, the vaccine described herein can
further comprise an adjuvant. As used herein, the term "adjuvant"
refers to a substance distinct from target antigen that is capable
of enhancing or potentiating immune effector cell activation. Some
adjuvants are believed to enhance the immune response by slowly
releasing the antigen, while other adjuvants are strongly
immunogenic and are believed to function synergistically. Known
vaccine adjuvants include, but are not limited to, oil and water
emulsions (for example, complete Freund's adjuvant and incomplete
Freund's adjuvant, and adjuvants disclosed in US patent numbers
U.S. Pat. No. 7,371,395 to Merial Limited, which are herein
incorporated by reference in their entirety), Corynebacterium
parvum, Bacillus Calmette Guerin, aluminum hydroxide, glucan,
dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant,
certain synthetic polymers such as poly amino acids and co-polymers
of amino acids, saponin, "REGRESSIN" (Vetrepharm, Athens, Ga.),
"AVRIDINE"
(N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine),
paraffin oil, muramyl dipeptide and the like.
[0111] Accordingly, in another aspect, methods of treating an
autoimmune disease or disorder comprising vaccinating a subject
with a vaccine comprising a DC-targeting composition described
herein and/or a composition comprising a population of
immunosuppressive cells. The term "vaccinating" or "vaccination" is
well-understood in the art. For example, the term "vaccination" can
be understood to be a process that increases a subject's immune
reaction to antigen and therefore the ability to resist or overcome
infection. In the case of the present invention, vaccination or
immunization may decrease the recipient's immune response against
self antigens thereby decreasing the likelihood of an autoimmune
response.
[0112] It is contemplated that in other immune-related diseases or
disorder, including, e.g., cancer, it can be desirable to induce
proinflammatory responses, e.g., Th1/Th17 responses, at a target
site (e.g., a tumor) for a therapeutic effect. Accordingly, these
immune-related diseases or disorders, e.g., but not limited to
cancer, where upregulation of immune response is desirable, can be
treated by suppressing the IL-27/CD39 axis signaling. For example,
in some embodiments, a subject who is diagnosed with cancer can be
administered with a DC-targeting composition comprising a
DC-binding agent and an agent that suppresses IL-27/CD39 axis
signaling (also referred to as "IL-27/CD39 antagonistic
agent").
[0113] It is also contemplated that other inflammatory diseases or
disorders, including, e.g., allergy and asthma, where a dampening
Th2 response is desirable could be treated by downregulating or
suppressing the IL-27/CD39 axis signaling.
Agents that Stimulates or Activates the IL-27/CD39 Axis Signaling
("IL-27/CD39 Agonistic Agents") (I) IL-27 and Agonists Thereof
[0114] IL-27 is a cytokine structurally related to IL-12 and is
composed of a p28 subunit and the product of Epstein-Barr
virus-induced gene 3 (Ebi3) (8). IL-27 signals through a receptor
composed of the common IL-6 receptor chain gp130 (which is used by
several other members of the IL-6 and IL-12 families) and a unique
IL-27 receptor .alpha.-chain (IL-27RA) that is homologous to the
IL-12R.beta.2 chain of the IL-12 receptor (8). Previous report
shows that IL-27 can be produced by DCs in response to activation
via Toll-like receptors through a mechanism that involves the
autocrine effects of interferon-.beta. (IFN-.beta.) (9). However,
it is not known that IL-27 can act on DCs to suppress a T cell
response and autoimmunity.
[0115] On the basis of its structural homology to IL-12 and its
ability to trigger IFN-.gamma. production, IL-27 was initially
believed to be a proinflammatory cytokine (11). However, it was
subsequently reported that IL-27 suppresses TH1, TH2 and TH17
responses and limits CNS inflammation in several experimental
models (8). In the EAE model, the administration of IL-27 inhibits
disease development (12). Conversely, the lack of a functional
IL-27 receptor results in exacerbated TH17 responses and the
worsening of EAE (12, 13). It is previously reported that IL-27
acts directly on T cells to inhibit the development of pathogenic
TH17 cells and to promote the differentiation of IL-10-producing
type 1 Treg cells (Tr1 cells) (12-17). Thus, the arrest of EAE by
IL-27 is generally believed to reflect its direct effects on T
cells until the inventors discovered that IL-27 can act on
dendritic cells to suppress the T cell response and autoimmunity by
inducing expression of the immunoregulatory molecule CD39 as shown
herein in the Examples.
[0116] As used herein, the term "IL-27" generally refers to an
IL-27 polypeptide or an IL-27 polynucleotide that is similar or
identical to the sequence of a wild-type IL-27.
[0117] In some embodiments, the term "IL-27" refers to an IL-27
polypeptide having an amino acid sequence that is at least 70% or
more (including at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 97%, at least 99%, or 100%) identical
to that of a wild-type IL-27, and is capable of acting on DCs and
suppressing T cell response. In some embodiments, the IL-27
polypeptide can also increase expression and/or activity of CD39 to
mediate the immunosuppression.
[0118] In some embodiments, the term "IL-27" refers to an IL-27
polynucleotide having a nucleotide sequence that is at least 70% or
more (including at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 97%, at least 99%, or 100%) identical
to that of a wild-type IL-27 or a portion thereof, and encodes an
IL-27 polypeptide as described herein.
[0119] The wild-type IL-27 sequences of various species are
available on the world wide web from the NCBI, including human,
mouse, pig, rat, dog, and cattle. For example, the nucleotide
sequence encoding human IL-27 is available at NCBI under Accession
No. NM_145659 and its corresponding amino acid sequence is under
Accession No. NP_663634.
[0120] Where the term "IL-27" refers to an IL-27 polypeptide, the
term "IL-27 polypeptide" also encompasses a portion or fragment of
such an IL-27 polypeptide that retains at least about 70% or more
(including at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 99%, or 100%) of the activity
of the wild-type IL-27 polypeptide to act on DCs to suppress T cell
response. The term "IL-27 polypeptide" as used herein also
encompasses conservative substitution variants of an IL-27
polypeptide that retain at least about 70% or more (including at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 97%, at least 99%, or 100%) of the activity of the
wild-type IL-27 polypeptide to act on DCs to suppress T cell
response. Accordingly, an IL-27 polypeptide refers to any
immunosuppressive form of IL-27, including functional variants of
IL-27. For example, in some embodiments, an IL-27 polypeptide can
be a full-length IL-27. In some embodiments, an IL-27 polypeptide
refers to a functional domain or domains of IL-27 that acts on DCs
and induces immunosuppression and expression and/or activity of
CD39.
[0121] The amino acid identity between two polypeptides can be
determined, for example, by first aligning the two polypeptide
sequences using an alignment algorithm, such as BLAST.RTM. or by
other methods well-known in the art.
[0122] In a similar manner, the term "agonist" is used in the
broadest sense and includes any molecule that mimics a biological
activity of a native polypeptide disclosed herein. Suitable agonist
molecules specifically include agonistic antibodies or antibody
fragments, fragments or amino acid sequence variants of native
polypeptides, peptides, antisense oligonucleotides, small organic
molecules, recombinant proteins or peptides, etc. Methods for
identifying agonists of a polypeptide can comprise contacting a
polypeptide with a candidate agonist molecule and measuring a
detectable change in one or more biological activities normally
associated with the polypeptide.
[0123] As used herein, the term "IL-27 agonist" refers to an agent
that enhances or stimulates the normal functioning of IL-27, by
increasing transcription or translation of IL-27-encoding nucleic
acid, and/or by inhibiting or blocking activity of a molecule that
inhibits IL-27 expression or IL-27 activity, and/or by enhancing
normal IL-27 activity (including, but not limited to, enhancing the
stability of IL-27 or enhancing binding of IL-27 to one or more
target receptor such as IL-27RA). For example, the IL-27 agonist
can be selected from an antibody, an antigen-binding fragment, an
aptamer, an interfering RNA, a small molecule, a peptide, an
antisense molecule, and another binding polypeptide. In another
example, the IL-27 agonist can be a polynucleotide selected from an
aptamer, interfering RNA, or antisense molecule that interferes
with the transcription and/or translation of an IL-27-inhibitory
molecule. It will be understood by one of ordinary skill in the art
that in some instances, an IL-27 agonist can agonize one IL-27
activity without affecting another IL-27 activity. For example, a
desirable IL-27 agonist for use in certain of the methods herein is
an IL-27 agonist that agonizes IL-27 activity to act on DCs to
induce immunosuppression, e.g., without affecting or minimally
affecting any of the other IL-27 interactions.
[0124] In some embodiments, an IL-27 agonist is an agent that
directly or indirectly enhances or stimulates the IL-27-mediated
signaling in DCs to induce immunosuppression. Accordingly, an IL-27
agonist can target the IL-27 receptor or its corresponding ligand,
or any of IL-27's upstream molecules. Examples of IL-27 agonists
include, without limitations, IL-27 recombinant peptides or
proteins and/or IL-27 receptor agonists (e.g., IL-27RA agonists).
The IL-27 agonists can be a protein, a peptide, peptidomimetic, an
aptamer, a nucleic acid, an antibody, a small molecule, a vaccine,
a fusion protein, a recombinant molecule, or any combinations
thereof.
[0125] In some embodiments, an IL-27 agonist is a recombinant IL-27
protein (e.g., a recombinant human IL-27 protein). Recombinant
IL-27 proteins are commercially available (e.g., from Biolegend
(Cat. No. 589202); Affymetrix eBioscience (Cat. No. 34-8279-82);
and R&D Systems (Cat. No. 2526-IL-010/CF)).
[0126] IL-27 agonists can be obtained from known sources or
prepared using known techniques such as recombinant or synthetic
technology. The nucleic acid and protein sequences of IL-27 and its
receptors of different species (e.g., but not limited to, human,
mouse, rat, dog, chimpanzee) are known in the art, e.g., accessible
at world wide web from NCBI. Thus, one of skill in the art can
generate IL-27 agonists based on these sequences using
art-recognized molecular technologies such as cloning and
expression technologies. For example, a human IL-27 agonist (e.g.,
an IL-27 recombinant protein) can be generated based on the nucleic
acid sequence of human IL-27 accessible at NCBI under Accession No.
NM_145659 and/or its corresponding amino acid sequence under
Accession No. NP_663634, or fragments thereof.
(II) CD39 and Agonists Thereof
[0127] CD39 is a cell surface-located prototypic member of the
ecto-nucleoside triphosphate diphosphophydrolase (E-NTPDase)
family, and is also known as ENTPD1, ATPDase, or NTPDase-1. CD39 is
an ectonucleotidase that catalyzes the degradation of extracellular
ATP and ADP. Extracellular ATP triggers activation of the NLRP3
inflammasome, a process shown to control the differentiation of
encephalitogenic Th1 and Th17 cells during EAE.
[0128] As used herein, the term "CD39" generally refers to a CD39
polypeptide or a CD39 polynucleotide that is similar or identical
to the sequence of a wild-type CD39.
[0129] In some embodiments, the term "CD39" refers to a CD39
polypeptide having an amino acid sequence that is at least 70% or
more (including at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 97%, at least 99%, or 100%) identical
to that of a wild-type CD39, and is capable of catalyzing the
degradation of extracellular ATP (eATP).
[0130] In some embodiments, the term "CD39" refers to a CD39
polynucleotide having a nucleotide sequence that is at least 70% or
more (including at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 97%, at least 99%, or 100%) identical
to that of a wild-type CD39 or a portion thereof, and encodes a
CD39 polypeptide as described herein.
[0131] The wild-type CD39 sequences of various species are
available on the world wide web from the NCBI, including human,
mouse, pig, and rat. For example, the nucleotide sequence encoding
human CD39 is available at NCBI under Accession No. NM_001098175,
NM_001164178, NM_001164179, NM_001164181, NM_001164182,
NM_001164183, or NM_001776, and its corresponding amino acid
sequence is under Accession No. NP_001091645, NP_001157650,
NP_001157651, NP_001157653, NP_001157654, NP_001157655, or
NP_001767.
[0132] Where the term "CD39" refers to a CD39 polypeptide, the term
"CD39 polypeptide" also encompasses a portion or fragment of such a
CD39 polypeptide that retains at least about 70% or more (including
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at least 99%, or 100%) of the activity of the
wild-type CD39 polypeptide to catalyze the degradation of eATP. The
term "CD39 polypeptide" as used herein also encompasses
conservative substitution variants of a CD39 polypeptide that
retain at least about 70% or more (including at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 99%, or 100%) of the activity of the wild-type CD39
polypeptide to catalyze degradation of eATP. Accordingly, a CD39
polypeptide refers to any form of CD39 that can catalyze
degradation of eATP, including functional variants of CD39. For
example, in some embodiments, a CD39 polypeptide can be a
full-length CD39. In some embodiments, a CD39 polypeptide refers to
a functional domain or domains of CD39 that catalyze degradation of
eATP.
[0133] As used herein, the term "CD39 agonist" refers to an agent
that enhances or stimulates the normal functioning of CD39, by
increasing transcription or translation of CD39-encoding nucleic
acid, and/or by inhibiting or blocking activity of a molecule that
inhibits CD39 expression or CD39 activity, and/or by enhancing
normal CD39 activity (including, but not limited to, enhancing the
stability of CD39 or enhancing IL-27 signaling that induces CD39
expression. For example, the CD39 agonist can be selected from an
antibody, an antigen-binding fragment, an aptamer, an interfering
RNA, a small molecule, a peptide, an antisense molecule, and
another binding polypeptide. In another example, the CD39 agonist
can be a polynucleotide selected from an aptamer, interfering RNA,
or antisense molecule that interferes with the transcription and/or
translation of a CD39-inhibitory molecule. It will be understood by
one of ordinary skill in the art that in some instances, a CD39
agonist can agonize one CD39 activity without affecting another
CD39 activity. For example, a desirable CD39 agonist for use in
certain of the methods herein is a CD39 agonist that agonizes CD39
activity to catalyze degradation of eATP, e.g., without affecting
or minimally affecting any of the other CD39 interactions.
[0134] In some embodiments, a CD39 agonist is an agent that
directly or indirectly enhances or stimulates the CD39-mediated
degradation of eATP, which in turn down-regulate activation of the
NLRP3 inflammasome. Accordingly, a CD39 agonist can target the CD39
itself, CD39 ligands, or any of CD39's upstream molecules. Examples
of CD39 agonists include, without limitations, CD39 recombinant
peptides or proteins, and/or IL-27 agonists. The CD39 agonists can
be a protein, a peptide, peptidomimetic, an aptamer, a nucleic
acid, an antibody, a small molecule, a vaccine, a fusion protein, a
recombinant molecule, or any combinations thereof.
[0135] In some embodiments, a CD39 agonist is a recombinant CD39
protein (e.g., a recombinant human CD39 protein). Recombinant CD39
proteins are commercially available (e.g., from R&D Systems
(Cat. No. 4397-EN-010)).
[0136] CD39 agonists can be obtained from known sources or prepared
using known techniques such as recombinant or synthetic technology.
The nucleic acid and protein sequences of CD39 and its receptors of
different species (e.g., but not limited to, human, mouse, and rat)
are known in the art, e.g., accessible at world wide web from NCBI.
Thus, one of skill in the art can generate CD39 agonists based on
these sequences using art-recognized molecular technologies such as
cloning and expression technologies. For example, a human CD39
agonist (e.g., a CD39 recombinant protein) can be generated based
on the nucleic acid sequence of human CD39 accessible at NCBI under
Accession No. NM_001098175, NM_001164178, NM_001164179,
NM_001164181, NM_001164182, NM_001164183, or NM_001776, and its
corresponding amino acid sequence under Accession No. NP_001091645,
NP_001157650, NP_001157651, NP_001157653, NP_001157654,
NP_001157655, or NP_001767, or fragments thereof.
(III) ATP-Degrading Agents (e.g., Apyrase and Agonists Thereof)
[0137] ATP-degrading agents are molecules or compounds that can
degrade and/or catalyze the degradation/hydrolysis of eATP. In some
embodiments, an ATP-degrading agent comprises apyrase. Apyrase,
also known as ATP-diphosphatase, adenosine diphosphatase, ADPase,
ATP diphosphohydrolase is a calcium-activated plasma membrane-bound
enzyme that catalyzes the hydrolysis of ATP to yield AMP and
inorganic phosphate. Two isoenzymes are found in commercial
preparations from S. tuberosum.
[0138] As used herein, the term "apyrase" generally refers to an
apyrase polypeptide or an apyrase polynucleotide that is similar or
identical to the sequence of a wild-type apyrase.
[0139] In some embodiments, the term "apyrase" refers to an apyrase
polypeptide having an amino acid sequence that is at least 70% or
more (including at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 97%, at least 99%, or 100%) identical
to that of a wild-type apyrase, and is capable of catalyzing the
degradation/hydrolysis of extracellular ATP (eATP).
[0140] In some embodiments, the term "apyrase" refers to an apyrase
polynucleotide having a nucleotide sequence that is at least 70% or
more (including at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 97%, at least 99%, or 100%) identical
to that of a wild-type apyrase polynucleotide or a portion thereof,
and encodes an apyrase polypeptide as described herein.
[0141] The wild-type apyrase sequences of various species are
available on the world wide web from the NCBI, including human,
mouse, pig, rat, and potatoes. For example, the nucleotide sequence
encoding human apyrase is available at NCBI under Accession No.
NM_001159772, NM_001159773, or NM_138793, and its corresponding
amino acid sequence is under Accession No. NP_001153244,
NP_001153245, or NP_620148.
[0142] Where the term "apyrase" refers to an apyrase polypeptide,
the term "apyrase polypeptide" also encompasses a portion or
fragment of such an apyrase polypeptide that retains at least about
70% or more (including at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 97%, at least 99%, or 100%) of
the activity of the wild-type apyrase polypeptide to catalyze the
degradation/hydrolysis of eATP. The term "apyrase polypeptide" as
used herein also encompasses conservative substitution variants of
a apyrase polypeptide that retain at least about 70% or more
(including at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 99%, or 100%) of the activity
of the wild-type apyrase polypeptide to catalyze
degradation/hydrolysis of eATP. Accordingly, an apyrase polypeptide
refers to any form of apyrase that can catalyze
degradation/hydrolysis of eATP, including functional variants of
apyrase. For example, in some embodiments, an apyrase polypeptide
can be a full-length apyrase. In some embodiments, an apyrase
polypeptide refers to a functional domain or domains of apyrase
that catalyze degradation/hydrolysis of eATP.
[0143] As used herein, the term "apyrase agonist" refers to an
agent that enhances or stimulates the normal functioning of
apyrase, by increasing transcription or translation of
apyrase-encoding nucleic acid, and/or by inhibiting or blocking
activity of a molecule that inhibits apyrase expression or apyrase
activity, and/or by enhancing normal apyrase activity (including,
but not limited to, enhancing the stability of apyrase. For
example, the apyrase agonist can be selected from an antibody, an
antigen-binding fragment, an aptamer, an interfering RNA, a small
molecule, a peptide, an antisense molecule, and another binding
polypeptide. In another example, the apyrase agonist can be a
polynucleotide selected from an aptamer, interfering RNA, or
antisense molecule that interferes with the transcription and/or
translation of an apyrase-inhibitory molecule. It will be
understood by one of ordinary skill in the art that in some
instances, an apyrase agonist can agonize one apyrase activity
without affecting another apyrase activity. For example, a
desirable apyrase agonist for use in certain of the methods herein
is an apyrase agonist that agonizes apyrase activity to catalyze
degradation/hydrolysis of eATP, e.g., without affecting or
minimally affecting any of the other apyrase interactions.
[0144] In some embodiments, an apyrase agonist is an agent that
directly or indirectly enhances or stimulates the apyrase-mediated
degradation/hydrolysis of eATP, which in turn down-regulate
activation of the NLRP3 inflammasome. Accordingly, an apyrase
agonist can target the apyrase itself, or any of apyrase's upstream
molecules. Examples of apyrase agonists include, without
limitations, recombinant apyrase, CD39 agonists, and/or IL-27
agonists. The apyrase agonists can be a protein, a peptide,
peptidomimetic, an aptamer, a nucleic acid, an antibody, a small
molecule, a vaccine, a fusion protein, a recombinant molecule, or
any combinations thereof.
[0145] In some embodiments, an apyrase agonist is a recombinant
apyrase protein (e.g., a recombinant human apyrase protein).
Recombinant apyrase proteins are commercially available (e.g., from
Sigma-Aldrich (Cat. No. A6535) or New England Biolabs (Cat. No.
M0393). In some embodiments, apyrase from potatoes is used in the
methods and/or compositions described herein.
[0146] Apyrase agonists can be obtained from known sources or
prepared using known techniques such as recombinant or synthetic
technology. The nucleic acid and protein sequences of apyrase and
its receptors of different species (e.g., but not limited to,
human, mouse, pig, rat, and potatoes) are known in the art, e.g.,
accessible at world wide web from NCBI. Thus, one of skill in the
art can generate apyrase agonists based on these sequences using
art-recognized molecular technologies such as cloning and
expression technologies. For example, a human apyrase agonist
(e.g., a recombinant apyrase) can be generated based on the nucleic
acid sequence of human apyrase accessible at NCBI under Accession
No. NM_001159772, NM_001159773, or NM_138793, and its corresponding
amino acid sequence under Accession No. NP_001153244, NP_001153245,
or NP_620148, or fragments thereof. In some embodiments, an apyrase
agonist can be generated based on the nucleic acid sequence of
potato apyrase accessible at NCBI under XM_006349845 or its
corresponding amino acid sequence under Accession No.
XP_006349907.
Pharmaceutical Compositions
[0147] Pharmaceutical compositions for treatment of an
immune-related disease or disorder, including, e.g., autoimmune
diseases or disorders are also provided herein. More specifically,
the pharmaceutical composition comprises (i) a
pharmaceutically-acceptable excipient; and (ii) a DC-targeting
composition comprising at least one or more (including, e.g., at
least two or more) IL-27/CD39 agonistic agents, and/or an
immunosuppressive dendritic cell described herein.
[0148] In some embodiments, the pharmaceutical composition can
further comprise an agent for treatment of an autoimmune disease or
disorder. For example, the agent can comprise an agent that
increases an anti-inflammatory T cell response and/or an agent that
suppresses a proinflammatory T cell response.
[0149] The phrase "pharmaceutically acceptable" refers to those
compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable carrier" as used herein
means a pharmaceutically acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent, media, encapsulating material, manufacturing aid (e.g.,
lubricant, talc magnesium, calcium or zinc stearate, or steric
acid), or solvent encapsulating material, involved in maintaining
the stability, solubility, or activity of, an IL-27/CD39 agonistic
agent or an immunosuppressive dendritic cell described herein. Each
carrier must be "acceptable" in the sense of being compatible with
the other ingredients of the formulation and not injurious to the
patient. Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, methylcellulose, ethyl cellulose,
microcrystalline cellulose and cellulose acetate; (4) powdered
tragacanth; (5) malt; (6) gelatin; (7) excipients, such as cocoa
butter and suppository waxes; (8) oils, such as peanut oil,
cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil; (9) glycols, such as propylene glycol; (10) polyols,
such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG);
(11) esters, such as ethyl oleate and ethyl laurate; (12) agar;
(13) buffering agents, such as magnesium hydroxide and aluminum
hydroxide; (14) alginic acid; (15) pyrogen-free water; (16)
isotonic saline; (17) Ringer's solution; (19) pH buffered
solutions; (20) polyesters, polycarbonates and/or polyanhydrides;
(21) bulking agents, such as polypeptides and amino acids (22)
serum components, such as serum albumin, HDL and LDL; (23) C2-C12
alcohols, such as ethanol; and (24) other non-toxic compatible
substances employed in pharmaceutical formulations. Release agents,
coating agents, preservatives, and antioxidants can also be present
in the formulation. The terms such as "excipient", "carrier",
"pharmaceutically acceptable carrier" or the like are used
interchangeably herein.
[0150] The pharmaceutical compositions described herein can be
specially formulated for administration of a DC-targeting
composition or an immunosuppressive dendritic cell described herein
to a subject in solid, liquid or gel form, including those adapted
for the following: (1) parenteral administration, for example, by
subcutaneous, intramuscular, intravenous or epidural injection as,
for example, a sterile solution or suspension, or sustained-release
formulation; (2) topical application, for example, as a cream,
ointment, or a controlled-release patch or spray applied to the
skin; (3) intravaginally or intrarectally, for example, as a
pessary, cream or foam; (4) ocularly; (5) transdermally; (6)
transmucosally; or (79) nasally. Additionally, a bispecific or
multispecific polypeptide agent can be implanted into a patient or
injected using a drug delivery system. See, for example, Urquhart,
et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis,
ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum
Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No.
35 3,270,960.
[0151] Further embodiments of the formulations and modes of
administration of the pharmaceutical compositions described herein
that can be used in the methods described herein are illustrated
below.
[0152] Parenteral Dosage Forms. Parenteral dosage forms of a
DC-targeting composition comprising at least one or more
(including, e.g., at least two or more) IL-27/CD39 agonistic
agents, and/or an immunosuppressive dendritic cell described herein
can also be administered to a subject by various routes, including,
but not limited to, subcutaneous, intravenous (including bolus
injection), intramuscular, and intraarterial. Since administration
of parenteral dosage forms typically bypasses the patient's natural
defenses against contaminants, parenteral dosage forms are
preferably sterile or capable of being sterilized prior to
administration to a patient. Examples of parenteral dosage forms
include, but are not limited to, solutions ready for injection, dry
products ready to be dissolved or suspended in a pharmaceutically
acceptable vehicle for injection, suspensions ready for injection,
controlled-release parenteral dosage forms, and emulsions.
[0153] Suitable vehicles that can be used to provide parenteral
dosage forms of the disclosure are well known to those skilled in
the art. Examples include, without limitation: sterile water; water
for injection USP; saline solution; glucose solution; aqueous
vehicles such as but not limited to, sodium chloride injection,
Ringer's injection, dextrose Injection, dextrose and sodium
chloride injection, and lactated Ringer's injection; water-miscible
vehicles such as, but not limited to, ethyl alcohol, polyethylene
glycol, and propylene glycol; and non-aqueous vehicles such as, but
not limited to, corn oil, cottonseed oil, peanut oil, sesame oil,
ethyl oleate, isopropyl myristate, and benzyl benzoate.
[0154] Controlled and Delayed Release Dosage Forms. In some
embodiments of the methods described herein, a DC-targeting
composition comprising at least one or more (including, e.g., at
least two or more) IL-27/CD39 agonistic agents, and/or an
immunosuppressive dendritic cell described herein can be
administered to a subject by controlled- or delayed-release means.
Ideally, the use of an optimally designed controlled-release
preparation in medical treatment is characterized by a minimum of
drug substance being employed to cure or control the condition in a
minimum amount of time. Advantages of controlled-release
formulations include: 1) extended activity of the drug; 2) reduced
dosage frequency; 3) increased patient compliance; 4) usage of less
total drug; 5) reduction in local or systemic side effects; 6)
minimization of drug accumulation; 7) reduction in blood level
fluctuations; 8) improvement in efficacy of treatment; 9) reduction
of potentiation or loss of drug activity; and 10) improvement in
speed of control of diseases or conditions. (Kim, Cherng-ju,
Controlled Release Dosage Form Design, 2 (Technomic Publishing,
Lancaster, Pa.: 2000)). Controlled-release formulations can be used
to control a compound of formula (I)'s onset of action, duration of
action, plasma levels within the therapeutic window, and peak blood
levels. In particular, controlled- or extended-release dosage forms
or formulations can be used to ensure that the maximum
effectiveness of a compound of formula (I) is achieved while
minimizing potential adverse effects and safety concerns, which can
occur both from under-dosing a drug (i.e., going below the minimum
therapeutic levels) as well as exceeding the toxicity level for the
drug.
[0155] A variety of known controlled- or extended-release dosage
forms, formulations, and devices can be adapted for use with a
DC-targeting composition comprising at least one or more
(including, e.g., at least two or more) IL-27/CD39 agonistic
agents, and/or an immunosuppressive dendritic cell described
herein. Examples include, but are not limited to, those described
in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123;
4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543;
5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1, each of which is
incorporated herein by reference in their entireties. These dosage
forms can be used to provide slow or controlled-release of one or
more active ingredients using, for example, hydroxypropylmethyl
cellulose, other polymer matrices, gels, permeable membranes,
osmotic systems (such as OROS.RTM. (Alza Corporation, Mountain
View, Calif. USA)), multilayer coatings, microparticles, liposomes,
or microspheres or a combination thereof to provide the desired
release profile in varying proportions. Additionally, ion exchange
materials can be used to prepare immobilized, adsorbed salt forms
of the disclosed compounds and thus effect controlled delivery of
the drug. Examples of specific anion exchangers include, but are
not limited to, Duolite.RTM. A568 and Duolite.RTM. AP143 (Rohm
& Haas, Spring House, Pa. USA).
[0156] In some embodiments, a DC-targeting composition comprising
at least one or more (including, e.g., at least two or more)
IL-27/CD39 agonistic agents, and/or an immunosuppressive dendritic
cell described herein for use in the methods described herein is
administered to a subject by sustained release or in pulses. Pulse
therapy is not a form of discontinuous administration of the same
amount of a composition over time, but comprises administration of
the same dose of the composition at a reduced frequency or
administration of reduced doses. Sustained release or pulse
administrations are particularly preferred when the disorder occurs
continuously in the subject, for example where the subject has
continuous or chronic symptoms of a viral infection. Each pulse
dose can be reduced and the total amount of a DC-targeting
composition comprising at least one or more (including, e.g., at
least two or more) IL-27/CD39 agonistic agents, and/or an
immunosuppressive dendritic cell described herein administered over
the course of treatment to the patient is minimized.
[0157] The interval between pulses, when necessary, can be
determined by one of ordinary skill in the art. Often, the interval
between pulses can be calculated by administering another dose of
the composition when the composition or the active component of the
composition is no longer detectable in the subject prior to
delivery of the next pulse. Intervals can also be calculated from
the in vivo half-life of the composition. Intervals can be
calculated as greater than the in vivo half-life, or 2, 3, 4, 5 and
even 10 times greater the composition half-life. Various methods
and apparatus for pulsing compositions by infusion or other forms
of delivery to the patient are disclosed in U.S. Pat. Nos.
4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.
[0158] Embodiments of various aspects described herein can be
defined in any of the following numbered paragraphs: [0159] 1. A
method of generating an immunosuppressive dendritic cell comprising
contacting a dendritic cell with a composition comprising an
effective amount of an IL-27 agonist. [0160] 2. The method of
paragraph 1, wherein the effective amount of the IL-27 agonist
ranges from about 1 ng/mL to about 100 ng/mL. [0161] 3. The method
of paragraph 1 or 2, further comprising contacting the dendritic
cell with an autoimmune antigen. [0162] 4. The method of paragraph
3, wherein the autoimmune antigen has a concentration of about 1
.mu.g/mL to about 100 .mu.g/mL. [0163] 5. The method of paragraph 3
or 4, wherein the autoimmune antigen is selected from the group
consisting of myelin basic protein (MBP); proteolipid protein
(PLP); myelin oligodendrocyte glycoprotein (MOG), myelin-associated
oligodendrocytic basic protein cardiac myosin; outer surface
protein (OSP); myelin associated glycoprotein (MAG);
neurofilaments; interferon omega; transglutaminase; aromatic acid
carboxylase; 17-hydroxylase; 21-hydroxylase, cardiolipin; pyruvate
dehydrogenase; .beta.2 glycoprotein I; phosphatidylserine; apoH;
Annexin A5; LKM-1; soluble liver antigen; carbonic anhydrase;
gpIIb-IIIa or Ib-IX; type XVII collagen; tissue transglutaminase;
gliadin; GD1a; GQ1b; BP-1; BP-2; epidermal transglutaminase;
histidine-tRNA; signal recognition peptide; Mi-2; Jo1; Glutamic
acid decarboxylase, HSP60; HSP70; HSP90; IGRP; insulin;
carboxypeptidase H; insulinoma antigen-2; IA-2beta; ICA69; ZnT8;
chromogranin A; IAPP; scl70; topoisomerase; histones; Basement
Membrane Collagen Type IV; enolase; thyroid peroxidase;
thyroglobulin; complement component 3; voltage-gated calcium
channels; Q-type calcium channel, synaptogagmin, muscarinic
acetylcholine receptor M1; SMA; LKM-1; LKM-2; LKM-3; soluble liver
antigen; SLA; LP; major peripheral myelin protein P0;
myeloperoxidase; GQ1b; U1-RNP; Kir4.1; nicotinic acetylcholine
receptor; MuSK protein; hypocretin; orexin; keratin; AQP4; Yo; Hu;
glutamate receptor; Desmoglein 3; p62; sp100,; Ro; LA;
glycoproteins IIb-IIIa or Ib-IX; ADAMTS13; cardiolipin; .beta.2
glycoprotein I; HPA-1a; HPA-5b; IFN-gamma, IL-1, TNF-alpha; GMCSF,
portions thereof, and combinations thereof. [0164] 6. The method of
any of paragraphs 1-5, wherein the contacting is performed ex vivo
or in vitro. [0165] 7. The method of any of paragraphs 1-5, wherein
the contacting is performed in vivo. [0166] 8. The method of any of
paragraphs 1-7, wherein the immunosuppressive dendritic cell
comprises an increased expression of IL-27, as compared to a
dendritic cell not contacted with the IL-27 agonist. [0167] 9. The
method of any of paragraphs 1-8, wherein the immunosuppressive
dendritic cell comprises an increased expression of CD39, as
compared to a dendritic cell not contacted with the IL-27 agonist.
[0168] 10. The method of any of paragraphs 1-9, wherein the
immunosuppressive dendritic cell comprises a reduced production of
an effector polarizing cytokine and/or an increased production of
an anti-inflammatory cytokine, as compared to a dendritic cell not
contacted with the IL-27 agonist. [0169] 11. The method of
paragraph 10, wherein the effector polarizing cytokine comprises
IL-12 and/or IL-6. [0170] 12. The method of paragraph 10 or 11,
wherein the anti-inflammatory cytokine comprises TGF.beta.1, IL-10,
IFN-.beta., or any combinations thereof. [0171] 13. The method of
any of paragraphs 1-12, wherein the IL-27 agonist comprises a
recombinant IL-27 protein or peptide. [0172] 14. The method of any
of paragraphs 1-13, wherein the dendritic cell is derived from a
spleen, lymph node, blood, monocyte, and/or hematopoietic
progenitor cell. [0173] 15. An immunosuppressive dendritic cell
produced by the method of any of paragraphs 1-14. [0174] 16. A
method of treating an autoimmune disease or disorder comprising
administering to a patient in need thereof a dendritic cell
(DC)-targeting composition comprising (a) an agent that stimulates
IL-27/ectonucleotidase CD39 axis signaling, and (b) a DC-binding
agent. [0175] 17. The method of paragraph 16, wherein the agent
comprises an IL-27 agonist. [0176] 18. The method of paragraph 16
or 17, wherein the agent comprises a CD39 agonist. [0177] 19. The
method of any of paragraphs 16-18, wherein the agent comprises an
ATP degrading agonist. [0178] 20. The method of paragraph 19,
wherein the ATP degrading agonist comprises apyrase. [0179] 21. The
method of any of paragraphs 16-20, wherein the DC-binding agent
comprises an antibody against Clec9A and/or DEC205. [0180] 22. The
method of any of paragraphs 16-21, wherein the DC-targeting
composition further comprise an autoimmune antigen. [0181] 23. The
method of paragraph 22, wherein the autoimmune antigen is selected
from the group consisting of myelin basic protein (MBP);
proteolipid protein (PLP); myelin oligodendrocyte glycoprotein
(MOG), myelin-associated oligodendrocytic basic protein cardiac
myosin; outer surface protein (OSP); myelin associated glycoprotein
(MAG); neurofilaments; interferon omega; transglutaminase; aromatic
acid carboxylase; 17-hydroxylase; 21-hydroxylase, cardiolipin;
pyruvate dehydrogenase; .beta.2 glycoprotein I; phosphatidylserine;
apoH; Annexin A5; LKM-1; soluble liver antigen; carbonic anhydrase;
gpIIb-IIIa or Ib-IX; type XVII collagen; tissue transglutaminase;
gliadin; GD1a; GQ1b; BP-1; BP-2; epidermal transglutaminase;
histidine-tRNA; signal recognition peptide; Mi-2; Jo1; Glutamic
acid decarboxylase, HSP60; HSP70; HSP90; IGRP; insulin;
carboxypeptidase H; insulinoma antigen-2; IA-2beta; ICA69; ZnT8;
chromogranin A; IAPP; scl70; topoisomerase; histones; Basement
Membrane Collagen Type IV; enolase; thyroid peroxidase;
thyroglobulin; complement component 3; voltage-gated calcium
channels; Q-type calcium channel, synaptogagmin, muscarinic
acetylcholine receptor M1; SMA; LKM-1; LKM-2; LKM-3; soluble liver
antigen; SLA; LP; major peripheral myelin protein P0;
myeloperoxidase; GQ1b; U1-RNP; Kir4.1; nicotinic acetylcholine
receptor; MuSK protein; hypocretin; orexin; keratin; AQP4; Yo; Hu;
glutamate receptor; Desmoglein 3; p62; sp100; Ro; LA; glycoproteins
or Ib-IX; ADAMTS13; cardiolipin; .beta.2 glycoprotein I; HPA-1a;
HPA-5b; IFN-gamma, IL-1, TNF-alpha; GMCSF, portions thereof, and
combinations thereof. [0182] 24. The method of any of paragraphs
16-23, wherein the DC-binding agent and the optional autoimmune
antigen are fused to the agent that stimulates
IL-27/ectonucleotidase CD39 axis signaling. [0183] 25. The method
of any of paragraphs 16-23, wherein the DC-targeting composition
comprises a nanoparticle, the nanoparticle comprising the agent
that stimulates IL-27/ectonucleotidase CD39 axis signaling, the
DC-binding agent and the optional autoimmune antigen. [0184] 26.
The method of paragraph 25, wherein the agent and the optional
autoimmune antigen distribute on a surface of the nanoparticle.
[0185] 27. The method of paragraph 25 or 26, wherein the agent and
the optional autoimmune antigen are encapsulated in the
nanoparticle. [0186] 28. The method of any of paragraphs 25-27,
wherein the nanoparticle further comprises on its surface a PEG
layer. [0187] 29. The method of any of paragraphs 25-28, wherein
the nanoparticle is a gold nanoparticle. [0188] 30. The method of
any of paragraphs 25-29, wherein the autoimmune disease or disorder
is multiple sclerosis. [0189] 31. The method of any of paragraphs
25-30, wherein the autoimmune disease or disorder is
encephalomyelitis. [0190] 32. The method of any of paragraphs
25-31, wherein the autoimmune disease or disorder is type 1
diabetes. [0191] 33. A method of treating an autoimmune disease or
disorder comprising administering a composition comprising a
population of immunosuppressive dendritic cells of paragraph 15.
[0192] 34. The method of paragraph 33, wherein the population of
immunosuppressive dendritic cells are autologous dendritic cells.
[0193] 35. The method of paragraph 33 or 34, wherein the
composition further comprises an autoimmune antigen. [0194] 36. The
method of paragraph 35, wherein the autoimmune antigen is selected
from the group consisting of myelin basic protein (MBP);
proteolipid protein (PLP); myelin oligodendrocyte glycoprotein
(MOG), myelin-associated oligodendrocytic basic protein cardiac
myosin; outer surface protein (OSP); myelin associated glycoprotein
(MAG); neurofilaments; interferon omega; transglutaminase; aromatic
acid carboxylase; 17-hydroxylase; 21-hydroxylase, cardiolipin;
pyruvate dehydrogenase; .beta.2 glycoprotein I; phosphatidylserine;
apoH; Annexin A5; LKM-1; soluble liver antigen; carbonic anhydrase;
gpIIb-IIIa or Ib-IX; type XVII collagen; tissue transglutaminase;
gliadin; GD1a; GQ1b; BP-1; BP-2; epidermal transglutaminase;
histidine-tRNA; signal recognition peptide; Mi-2; Jo1; Glutamic
acid decarboxylase, HSP60; HSP70; HSP90; IGRP; insulin;
carboxypeptidase H; insulinoma antigen-2; IA-2beta; ICA69; ZnT8;
chromogranin A; IAPP; scl70; topoisomerase; histones; Basement
Membrane Collagen Type IV; enolase; thyroid peroxidase;
thyroglobulin; complement component 3; voltage-gated calcium
channels; Q-type calcium channel, synaptogagmin, muscarinic
acetylcholine receptor M1; SMA; LKM-1; LKM-2; LKM-3; soluble liver
antigen; SLA; LP; major peripheral myelin protein P0;
myeloperoxidase; GQ1b; U1-RNP; Kir4.1; nicotinic acetylcholine
receptor; MuSK protein; hypocretin; orexin; keratin; AQP4;Yo; Hu;
glutamate receptor; Desmoglein 3; p62; sp100,; Ro; LA;
glycoproteins IIb-IIIa or Ib-IX; ADAMTS13; cardiolipin; .beta.2
glycoprotein I; HPA-1a; HPA-5b; IFN-gamma, IL-1, TNF-alpha; GMCSF,
portions thereof, and combinations thereof.
Some Selected Definitions
[0195] For convenience, certain terms employed in the entire
application (including the specification, examples, and appended
claims) are collected here. Unless defined otherwise, all technical
and scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs.
[0196] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0197] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used to
described the present invention, in connection with numeric values
means.+-.5%.
[0198] As used herein and throughout the specification, the term
"antagonist" is used in the broadest sense, and includes any
molecule that partially or fully blocks, inhibits, or neutralizes a
biological activity of a native polypeptide disclosed herein. In a
similar manner, the term "agonist" is used in the broadest sense
and includes any molecule that mimics a biological activity of a
native polypeptide disclosed herein. Suitable agonist or antagonist
molecules specifically include agonist or antagonist antibodies or
antibody fragments, fragments or amino acid sequence variants of
native polypeptides, peptides, antisense oligonucleotides, small
organic molecules, recombinant proteins or peptides, etc. Methods
for identifying agonists or antagonists of a polypeptide can
comprise contacting a polypeptide with a candidate agonist or
antagonist molecule and measuring a detectable change in one or
more biological activities normally associated with the
polypeptide.
[0199] In one aspect, the present invention relates to the herein
described compositions, methods, and respective component(s)
thereof, as essential to the invention, yet open to the inclusion
of unspecified elements, essential or not ("comprising"). In some
embodiments, other elements to be included in the description of
the composition, method or respective component thereof are limited
to those that do not materially affect the basic and novel
characteristic(s) of the invention ("consisting essentially of").
This applies equally to steps within a described method as well as
compositions and components therein. In other embodiments, the
inventions, compositions, methods, and respective components
thereof, described herein are intended to be exclusive of any
element not deemed an essential element to the component,
composition or method ("consisting of").
[0200] All patents, patent applications, and publications
identified are expressly incorporated herein by reference for the
purpose of describing and disclosing, for example, the
methodologies described in such publications that might be used in
connection with the present invention. These publications are
provided solely for their disclosure prior to the filing date of
the present application. Nothing in this regard should be construed
as an admission that the inventors are not entitled to antedate
such disclosure by virtue of prior invention or for any other
reason. All statements as to the date or representation as to the
contents of these documents is based on the information available
to the applicants and does not constitute any admission as to the
correctness of the dates or contents of these.
[0201] As used herein, a "subject" can mean a human or an animal.
Examples of subjects include primates (e.g., humans, and monkeys).
Usually the animal is a vertebrate such as a primate, rodent,
domestic animal or game animal. Primates include chimpanzees,
cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
Rodents include mice, rats, woodchucks, ferrets, rabbits and
hamsters. Domestic and game animals include cows, horses, pigs,
deer, bison, buffalo, feline species, e.g., domestic cat, canine
species, e.g., dog, fox, wolf, and avian species, e.g., chicken,
emu, ostrich. A patient or a subject includes any subset of the
foregoing, e.g., all of the above, or includes one or more groups
or species such as humans, primates or rodents. In certain
embodiments of the aspects described herein, the subject is a
mammal, e.g., a primate, e.g., a human. The terms, "patient" and
"subject" are used interchangeably herein. A subject can be male or
female. The term "patient" and "subject" does not denote a
particular age. Thus, any mammalian subjects from adult to newborn
subjects, as well as fetuses, are intended to be covered.
[0202] In one embodiment, the subject or patient is a mammal. The
mammal can be a human, non-human primate, mouse, rat, dog, cat,
horse, or cow, but are not limited to these examples. In one
embodiment, the subject is a human being. In another embodiment,
the subject can be a domesticated animal and/or pet.
[0203] As used herein and throughout the specification, the terms
"administering," or "administration" refer to the placement of an
agent or composition (e.g., a DC-targeting composition described
herein that modulates the expression and/or activity of IL-27,
IL-27RA, and/or CD39 in DCs, and/or level of eATP, and/or a
composition comprising immunosuppressive dendritic cells described
herein) into a subject by a method or route which results in at
least partial localization of such agents at a desired site, such
as a target site, such that a desired effect(s) is produced.
[0204] In some embodiments of this aspect and other aspects
described herein, the DC-targeting compositions comprising an agent
for modulating the expression and/or activity of IL-27, IL-27RA,
and/or CD39 in DCs, and/or level of eATP, and/or a composition
comprising immunosuppressive dendritic cells can be administered to
a subject by any mode of administration that delivers the agent
systemically or to a desired surface, organ, or target, and can
include, but is not limited to injection, infusion, instillation,
and inhalation administration. To the extent that such agents can
be protected from inactivation in the gut, oral administration
forms are also contemplated. "Injection" includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intraventricular, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous,
subcuticular, intraarticular, sub capsular, subarachnoid,
intraspinal, intracerebro spinal, and intrasternal injection and
infusion. In some embodiments, the DC-targeting compositions
comprising an agent for modulating the expression and/or activity
of IL-27, IL-27RA, and/or CD39 in DCs, and/or level of eATP, and/or
a composition comprising immunosuppressive dendritic cells for use
in the methods described herein are administered by intravenous
infusion or injection. In some embodiments, a composition
comprising immunosuppressive dendritic cells described herein for
use in the methods described herein can be administered by
implantation, e.g., via a catheter.
[0205] The phrases "parenteral administration" and "administered
parenterally" as used herein, refer to modes of administration
other than enteral and topical administration, usually by
injection. The phrases "systemic administration," "administered
systemically", "peripheral administration" and "administered
peripherally" as used herein refer to the administration of a
DC-targeting composition comprising an agent for modulating the
expression and/or activity of IL-27, IL-27RA, and/or CD39 in DCs,
and/or level of eATP, and/or a composition comprising
immunosuppressive dendritic cells, other than directly into a
target site, tissue, or organ, such that it enters the subject's
circulatory system and, thus, is subject to metabolism and other
like processes.
[0206] As used herein, the term "peptidomimetic" refers to a
molecule capable of folding into a defined three-dimensional
structure similar to a natural peptide.
[0207] As used herein, the term "small molecules" refers to natural
or synthetic molecules including, but not limited to, peptides,
peptidomimetics, amino acids, amino acid analogs, polynucleotides,
polynucleotide analogs, aptamers, nucleotides, nucleotide analogs,
organic or inorganic compounds (i.e., including heteroorganic and
organometallic compounds) having a molecular weight less than about
10,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 5,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 1,000
grams per mole, organic or inorganic compounds having a molecular
weight less than about 500 grams per mole, and salts, esters, and
other pharmaceutically acceptable forms of such compounds.
EXAMPLES
[0208] The following examples illustrate some embodiments and
aspects of the invention. It will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be performed without altering the
spirit or scope of the invention, and such modifications and
variations are encompassed within the scope of the invention as
defined in the claims which follow. The following examples do not
in any way limit the invention.
Example 1
Comparison of Expression of IL-27 Receptor .alpha.-Chain (IL-27RA)
in Conventional Dendritic Cells (cDCs) and Plasmacytoid DCs
[0209] To determine the role of IL-27 signaling in DCs on the
regulation of autoimmunity, the expression of IL-27RA was first
analyzed in plasmacytoid DCs (pDCs;
F4/80.sup.-CD11b.sup.-CD11c.sup.lo B220.sup.+ MHC class II.sup.lo
Ly6c.sup.+) and cDCs (F4/80.sup.- CD11b.sup.+ CD11c.sup.+
B220.sup.- MHC class II.sup.+ Ly6c.sup.-) isolated from naive mice
by flow cytometry (FIG. 1). IL-27RA was expressed mainly in cDCs,
with only low or absent expression on pDCs (FIG. 2A). Similar
results were obtained for IL-27RA expression by quantitative PCR
and immunoblot analysis of sorted pDCs and cDCs (FIGS. 2B-2C).
Thus, there is higher IL-27RA expression in cDCs than in
plasmacytoid DCs. The expression pattern of IL-27RA indicated that
IL-27 controls the activity of cDCs.
Example 2
Effect of IL-27 on the Function of cDCs
[0210] After antigen uptake in the presence of DC-maturing stimuli,
DCs upregulate their expression of major histocompatibility complex
(MHC) class II and costimulatory molecules.sup.21. To determine the
effects of IL-27 on DC activation, splenic cDCs from naive mice
were pretreated with vehicle or IL-27 and their response to
activation with lipopolysaccharide from Escherichia coli (ecLPS)
were determined. Pretreatment of cDCs with IL-27 followed by
activation with ecLPS led to significantly lower expression of MHC
class II and the costimulatory molecules CD40, CD80 and CD86 than
that of cDCs activated with ecLPS without IL-27 pretreatment (FIGS.
3A-3I).
[0211] DCs control T cell differentiation via the secretion of
polarizing cytokines.sup.21. Pretreatment of splenic cDCs with
IL-27 followed by activation with ecLPS led to significantly lower
production of IL-12 and of IL-6 and IL-23 (which promote the
differentiation of TH1 and TH17 cells, respectively) than that of
cDCs activated with ecLPS without IL-27 pretreatment (FIG. 3B).
Pretreatment of cDCs with IL-27 followed by activation with ecLPS
also upregulated Il27 expression (FIG. 3C), which indicated a
positive feedback loop for IL-27 production. Indeed, in those
conditions increased production of IFN-.beta. was detected (FIG.
3B). Increased production of IFN-.beta. was previously reported to
act in an autocrine manner to trigger IL-27 production.sup.9.
Increased production of IL-10 in those conditions was detected; and
increased production of transforming growth factor-.beta.1
(TGF-.beta.1) were also detected only in response to treatment with
IL-27 (FIG. 3B). Together these data showed that IL-27 decreased
the production of cytokines that promote the differentiation of
effector TH1 and TH17 cells, while it enhanced the production of
anti-inflammatory cytokines by cDCs.
[0212] The effects of IL-27 on the expression of MHC class II,
costimulatory molecules and cytokines suggests that IL-27 affects
the ability of DCs to activate and polarize T cells into specific
subsets. Thus, cDCs were pretreated with IL-27 and they were
activated with ecLPS, then extensively washed and their ability to
activate naive 2D2 CD4+ T cells was assessed in the presence of
their cognate target antigen: an epitope of amino acids 35-55 of
myelin oligodendrocyte glycoprotein (MOG(35-55)). Pretreatment of
cDCs with IL-27 followed by activation with ecLPS led to a
significantly lower proliferative response of naive 2D2 T cells to
MOG(35-55) than that elicited by cDCs treated with ecLPS without
IL-27 pretreatment (FIG. 3D). Moreover, the cDCs pretreated with
IL-27 and activated with ecLPS had a decreased ability to induce
the production of IFN-.gamma. and IL-17 by T cells, as measured by
enzyme-linked immunosorbent assay and intracellular cytokine
staining (FIGS. 3E-3F). Conversely, pretreatment of cDCs with IL-27
before activation with ecLPS boosted their ability to promote the
differentiation of IL-10+ and Foxp3+ CD4+ T cells (FIGS. 3E-3F).
Similar effects for bone marrow-Derived DCs pretreated with IL-27
and activated with ecLPS were also observed (data not shown).
[0213] IL-27 is previously reported to act directly on T cells to
suppress their differentiation into effector T cells.sup.12, 15-17.
The inventors have found that cDCs pretreated IL-27 and activated
with ecLPS showed a diminished ability to trigger the production of
IFN-.gamma. and IL-17 by T cells in the presence of exogenously
added TH1- and TH17-polarizing cytokines than that of cDCs
activated with ecLPS without IL-27 pretreatment (FIG. 3G).
Conversely, pretreatment of cDCs with IL-27 increased IL-10
production and expression of the transcription factor Foxp3 in T
cells when Tr1-polarizing cytokines or cytokines that polarize
differentiation into Foxp3+ Treg cells were added to the coculture
(FIGS. 3G-3H), contemplating that IL-27 signaling in DCs modulated
T cell differentiation in vivo even in the context of inflammation
or other physiological conditions that generate a polarizing
cytokine milieu. Together these data showed that IL-27 signaling
controlled the antigen-presenting function of cDCs.
Example 3
Effect IL-27RA in DCs on Development of Experimental Autoimmune
Encephalomyelitis (EAE)
[0214] IL-27 has been previously reported to play a role in the
control of CNS inflammation during EAE.sup.12,13,15. A significant
worsening of EAE in IL-27RA-deficient (Il27ra-/-) mice,
characterized by an increase in the frequency of CNS-infiltrating
IFN-.gamma..sup.| and IL-17.sup.| CD4.sup.| T cells and a lower
frequency of IL-10+ CD4+ T cells were found (FIGS. 4A-4B).
Il27ra-/- mice also showed an increased recall response to
MOG(35-55) and an increased frequency of CD4.sup.+CD44.sup.+
CD40L.sup.hi IFN.gamma..sup.+, IL-17.sup.+ and IFN-.gamma..sup.+
IL-17.sup.+ CD4.sup.+ T cells in the lymph nodes and spleen,
concomitant with a decreased frequency of Foxp3.sup.+ and
IL-10.sup.+ CD4.sup.+ T cells (FIGS. 4C-4D).
[0215] The previously-reported effects of IL-27 on encephalitogenic
and Treg cells.sup.14,15,17 would indicate that the worsening of
EAE in the Il27ra.sup.-/- mice resulted from the lack of IL-27
signaling in T cells. It is not known whether IL-27 can act on
additional cells beyond T cells to limit the development of EAE.
The Il27ra.sup.-/- mice had non-cell-specific deletion of IL-27RA.
To determine the role of IL-27 signaling in DCs during EAE, cDCs
were isolated from wild-type and Il27ra-/- mice 21 d after disease
induction. It was found that cDCs from Il27ra-/- mice showed an
increased ability to activate naive 2D2 T cells in the presence of
MOG(35-55) (FIG. 4E), which indicated that defective IL-27
signaling in DCs contributed to the worsening of EAE in Il27ra-/-
mice.
[0216] In vivo, DCs are generally influenced by their interactions
with T cells and the cytokine milieu. Thus, the increased
antigen-presenting function of cDCs isolated from Il27ra-/- mice
might have reflected the exposure of the DCs to a more inflammatory
cytokine milieu and not direct effects of IL-27 on DCs. To
determine the role of IL-27 signaling in DCs during EAE, a
chimera-based approach was used to generate mice lacking IL-27RA
expression in DCs (FIG. 5A). For this, lethally irradiated
wild-type mice were reconstituted with bone marrow cells from mice
that express the diphtheria toxin receptor (DTR) under the control
of the promoter of the gene encoding CD11c (Itgax; called
`CD11c-DTR` mice herein). After reconstitution, these mice can be
depleted of CD11c.sup.+ DCs by the administration of diphtheria
toxin (DTx).sup.22 . DTx cannot be chronically administered to
CD11c-DTR mice because of adverse side effects; however, no adverse
effects are associated with the chronic administration of DTx to
chimeras generated by the reconstitution of wild-type mice with
bone marrow from CD11c-DTR mice (CD11c-DTR.fwdarw.WT).sup.4 . Thus,
2 months after reconstitution of wild-type mice with CD11c-DTR bone
marrow, those CD11c-DTR.fwdarw.WT chimeras of DTR+ DCs were
depleted by chronic administration of DTx and their DC compartment
was reconstituted with DC precursors from wild-type mice (to
generate `DC(WT)` mice) or from Il27ra-/- mice (to generate
`DC(IL-27RA-KO)` mice) (FIG. 5B). DTx was administered to DC(WT)
and DC(IL-27RA-KO) mice once every other day until the completion
of the experiment and detected no antibodies to DTx after 2 months
of DTx administration (FIG. 5C).
[0217] DC(IL-27RA-KO) mice had significantly lower IL-27RA
expression in cDCs but not in other antigen-presenting cell (APC)
populations than did DC (WT) mice (FIG. 6A and FIG. 5D). No
difference was detected between DC(WT) and DC(IL-27RA-KO) mice in
the frequency or absolute number of DCs (FIG. 5E). Treatment with
MOG(35-55) resulted in faster development of EAE in DC(IL-27RA-KO)
mice than in DC(WT) mice; DC(IL27RA-KO) mice also reached
significantly higher disease scores than did their DC(WT)
counterparts (FIG. 6B). The worsening of EAE in DC(IL-27RA-KO) mice
was associated with a greater frequency of TH1 and TH17 cells in
the CNS and significantly fewer IL-10+ T cells than that in DC(WT)
mice (FIG. 6C). Moreover, analysis of the CD4.sup.+ CD44.sup.+
CD40L.sup.hi splenic T cell compartment revealed a higher recall
proliferative response to MOG(35-55) and a higher frequency of
IFN-.gamma..sup.+ and IL-17.sup.+ CD4.sup.+ cells in DC(IL-27RA-KO)
mice than in DC(WT) mice, concomitant with a lower frequency of
Foxp3+ Treg cells and IL-10+ CD4+ cells in DC(IL-27RA-KO) mice than
in DC(WT) mice (FIGS. 6D-6E). Similar results were obtained when
EAE was induced by transferring MOG(35-55)-reactive TH1 or TH17
cells (FIGS. 5F-5H).
[0218] To study the effects of IL-27 signaling in DCs during EAE,
cDCs were isolated from DC(WT) and DC(IL-27RA-KO) mice 21 d after
EAE induction. cDCs from DC(IL-27RA-KO) mice had higher expression
of the proinflammatory cytokines IL-6, IL-12 and IL-23, concomitant
with lower expression of IL-10 and IL-27, than that of DC(WT) mice
(FIG. 6F). These results were confirmed by additional quantitative
profiling analyses, which detected a pronounced proinflammatory
transcription profile in cDCs isolated from DC(IL-27RA-KO) mice
during EAE (FIG. 6G). Moreover, cDCs from DC(IL-27RA-KO) mice
showed an greater ability to activate the proliferation of 2D2 CD4+
T cell and promoted the production of increased amounts of
IFN-.gamma. and IL-17 and decreased quantities of IL-10 and
TGF-.beta.1 than did their DC(WT) counterparts (FIGS. 6H-6I).
Together these data showed that IL-27 acted on cDCs in vivo to
limit the development of encephalitogenic T cells and EAE.
Example 4
Transcriptional Effects of IL-27 on DCs
[0219] To determine the mechanisms that mediate the effects of
IL-27 on DCs, the expression profiles of primary splenic cDCs
isolated from naive wild-type mice and treated in vitro with IL-27
were analyzed by microarray. Examination of the expression data by
Ingenuity pathway analysis identified significantly lower
expression of genes encoding proinflammatory molecules associated
with the NF-.kappa.B and Toll-like receptor signaling pathways in
IL-27-treated cDCs than in untreated cDCs (P=2.16.times.10.sup.-21
and P=4.55.times.10.sup.-27 , respectively; FIGS. 7A-7B).
Conversely, treatment with IL-27 led to a significant increase over
time in the expression of Tnip3 and Tnfaip3, which encode molecules
known to inhibit NF-.kappa.B activation23 (FIG. 7C). It was also
found that there was significant upregulation over time of Ido1,
Ido2, Il27, Il10 and Entpd1, which encode anti-inflammatory
molecules (FIG. 7C).
[0220] The NetGenerator algorithm was used to analyze the
transcriptional response of DCs to IL-27. This algorithm integrates
expression profiles in a model with prior `knowledge` of the genes
under investigation and their connections.sup.24. The resulting
model indicated that IL-27 controlled the expression of the
anti-inflammatory molecules described above in a manner dependent
on the transcription factors STAT1 and STAT3 (FIG. 7D). Together
these data showed that IL-27 limited the inflammatory response of
cDCs and triggered the expression of tolerogenic molecules.
Example 5
Identification of a Molecule/Ligand that Mediates the Inhibitory
Effects of IL-27 on DCs
[0221] The transcriptional profiling studies identified several
candidate molecules that probably mediate the effects of IL-27
signaling in cDCs on the activation of T cells. To identify the
mechanisms that mediate the effects of IL-27 on the
antigen-presenting function of cDCs, blocking antibodies to IL-27,
IL-10, IFN-.beta. or TGF-.beta., or the indoleamine 2,3-deoxygenase
(IDO)-specific inhibitor 1-methyl-dtryptophan (1-D-MT) was used.
Splenic cDCs were pre-treated with IL-27 and then treated with
ecLPS, extensively washed and used to activate naive CD4+ T cells
with antibody to CD3 (anti-CD3) in the presence of the blocking
antibodies to cytokines or 1-D-MT. The suppressive effect of
pretreating cDCs with IL-27 was not blocked by the antibodies or
1-D-MT (FIGS. 8A-8B), which indicated that neither IDO nor the
cytokines IL-27, IL-10, IFN-.beta. or TGF-.beta.mediated the
suppressive effects of cDCs pretreated with IL-27.
[0222] Expression of PD-L1 (CD274), the ligand for the T
cell-inhibitory receptor PD-1, is previously reported to be
upregulated following the treatment of mouse pDCs.sup.20 or human
monocyte-derived DCs.sup.19 with IL-27. No substantial upregulation
of PD-L1 expression was detected after treating cDC with IL-27
(FIG. 9A). Moreover, the suppressive effects of IL-27-treated cDCs
on the activation of T cells was also observed when we used PD-L1-
or IL-10-deficient cDCs (FIG. 9B). Together these data indicated
that the suppressive effects of IL-27 on the antigen-presenting
function of cDCs were independent of PD-L1 or IL-10.
[0223] CD39 has been previously linked to the suppressive activity
of mouse and human Treg cells.sup.25,26, but not to dendritic
cells. Entpd1 (which encodes CD39) was significantly upregulated in
DCs in response to treatment with IL-27 in vitro (P=0.01; FIG. 7C).
Hence, it was sought to determine the role of CD39 in the effects
of IL-27 on cDCs. Entpd1 deficiency abolished the suppressive
effects of the pretreatment of DCs with IL-27 on the activation and
polarization of T cells. Pretreatment of Entpd1-deficient cDCs with
IL-27 did not significantly decrease the proliferative response and
the production of IFN-.gamma. and IL-17, and had no significant
effect on IL-10 production and Foxp3 expression by 2D2 T cells
(P=0.2365; FIG. 8C and FIGS. 9B-9C). Thus, these data indicated
that CD39 mediated the effects of IL-27 signaling in cDCs on T cell
activation.
[0224] To further characterize the role of CD39 on the effects of
IL-27 in DCs, the regulation of CD39 expression by IL-27 was
evaluated. Freshly isolated cDCs from DC(IL-27RA-KO) mice showed
significantly lower expression of both Entpd1 mRNA and CD39
protein, reflected as a significant decrease in CD39.sup.+ DCs,
than did their DC(WT) counterparts (FIG. 8D). Thus, IL-27
controlled Entpd1 expression in cDCs in vitro and in vivo.
[0225] STAT1 and STAT3 mediate the response to IL-27 in T
cells.sup.17. Significant phosphorylation of STAT3 in cDCs 5 min
after treatment with IL-27 and a modest but significant increase in
the phosphorylation of STAT1 were detected (FIGS. 8E-8F).
Bioinformatics analysis identified a putative STAT1-binding element
(IRF-1), two STAT3-binding elements (SRE-1 and SRE-2) and a common
STAT1-STAT3-binding element upstream of the transcription start
site in the Entpd1 promoter (FIG. 8G).
Chromatin-immunoprecipitation (ChIP) assays detected binding of
STAT3 to SRE-1, SRE-2 and the STAT1-STAT3-binding element in the
Entpd1 promoter in response to IL-27 (FIG. 8H). Binding of STAT1 to
the Entpd1 promoter, however, was unresponsive to IL-27 and was
triggered by activation with ecLPS instead (FIG. 8I).
[0226] To determine the functional consequences of the interaction
of STAT1 and STAT3 with the Entpd1 promoter, reporter assays were
performed. Cotransfection of HEK293 human embryonic kidney cells
with a reporter construct containing the gene encoding firefly
luciferase under the control of the Entpd1 promoter together with a
construct encoding constitutively activated STAT1 or STAT3 led to
significantly more in luciferase activity than did transfection of
control cells with empty vector, but this effect was significantly
stronger in response to constitutively activated STAT3 (FIG. 8J).
These data indicated that IL-27 controlled CD39 expression in cDCs
via STAT3.
Example 6
CD39 Controls Activation of the NLRP3 Inflammasome
[0227] CD39 is an ectonucleotidase that catalyzes the degradation
of extracellular ATP and ADP.sup.28. Extracellular ATP triggers
activation of the NLRP3 inflammasome.sup.29, a process shown to
control the differentiation of encephalitogenic TH1 and TH17 cells
during EAE.sup.30. As presented herein, CD39 expression is required
for the effects of IL-27 signaling in cDCs on the activation and
differentiation of T cells (FIG. 8C and FIGS. 9A-9E). The effects
of IL-27 on the extracellular concentration of ATP and activation
of the NLRP3 inflammasome were thus evaluated. Pretreatment with
IL-27 and treatment with ecLPS led to a significant decrease the
extracellular concentration of ATP detected after activation of
wild-type cDCs with ecLPS (FIG. 10A). However, pretreatment with
IL-27 had no effect on the extracellular concentration of ATP
measured in the supernatants of IL-27RA- or CD39-deficient cDCs
treated with ecLPS (FIG. 10A). Moreover, deficiency in IL-27RA or
CD39 in cDCs led to a significantly higher extracellular
concentration of ATP detected after activation with LPS than that
of their wild-type counterparts (FIG. 10A).
[0228] To further explore the mechanisms linked to the reduced
amount of extracellular ATP detected in tissue culture supernatants
of wild-type cDCs treated with IL-27, exogenous ATP (500 .mu.M) was
added to cDCs treated with LPS, vehicle or IL-27 and the residual
extracellular ATP in the supernatants after 2 h of incubation was
quantified. Significantly lower concentrations of extracellular ATP
were found to remain in supernatants of wild-type cDCs treated with
ecLPS and IL-27 than in those of wild-type cDCs treated with ecLPS
(FIG. 10B). However, IL-27 did not affect the amount of residual
extracellular ATP in IL-27RA- or CD39-deficient cells (FIG. 10B),
which indicated that the lower abundance of ATP in supernatants of
wildtype cDCs treated with IL-27 resulted from its increased
catabolism by CD39. Further, treatment with IL-27 significantly
increased nucleoside triphosphate diphosphohydrolase activity in
wild-type cDCs but not in IL-27RA- or CD39-deficient cDCs (FIGS.
10C-10D). Moreover, less nucleoside triphosphate diphosphohydrolase
activity (manifested as greater residual amounts of ATP) was
detected in IL-27RA-deficient DCs than in wild-type cDCs (FIGS.
10C-10D), which indicated that the autocrine effects of IL-27
controlled the ability of cDCs to degrade extracellular ATP.
[0229] ATP activates the NLRP3 inflammasome in certain APCs such as
macrophages.sup.29. Activation of the NLRP3 inflammasome results in
the generation of active caspase 1, which leads to the maturation
and release of IL-18 and IL-1.beta..sup.31. It was found that
pretreatment with IL-27 significantly suppressed activation of the
NLRP3 inflammasome in wild-type cDCs activated with ecLPS, as shown
by a lower abundance of activated caspase-1 and mature IL-1.beta.
(FIG. 10E) and the secretion of significantly lower amounts of
IL-1.beta. into the culture medium (FIG. 10F). In accordance with
the findings presented herein on the extracellular concentrations
of ATP, no inhibitory effects of IL-27 on the activation of the
NLRP3 inflammasome and the release of IL-1.beta. in ecLPS-treated
IL-27RA- or CD39-deficient cDCs were detected (FIGS. 10E-10F).
Indeed, deficiency in IL-27RA or CD39 resulted in significantly
more release of IL-1.beta. than that of wild-type cDCs after
treatment with ecLPS (FIG. 10F). Together these data suggested that
without wishing to be bound by theory, the upregulation of CD39
expression triggered by IL-27 limited extracellular ATP
concentrations and, consequently, activation of the NLRP3
inflammasome in cDCs.
Example 7
Effect of CD39 in DCs on EAE Development
[0230] To determine the role of CD39 in DCs during EAE,
CD11c-DTR.fwdarw.WT chimeras were reconstituted with CD39-deficient
DC precursors to generate mice lacking Entpd1 expression in DCs
(DC(CD39-KO) mice). DC(WT) mice were used as controls.
Significantly lower expression of CD39 protein was found in cDCs
from DC(CD39-KO) mice than in cDCs from DC(WT) mice (FIG. 11A), but
this result was not obtained for other APC populations (FIG. 9D).
No difference was detected between DC(WT) and DC(CD39-KO) mice in
the frequency or absolute number of DCs (FIG. 9E). The induction of
EAE led to an earlier onset of EAE in DC(CD39-KO) mice than in
DC(WT) mice, and the EAE also reached significantly higher scores
in the former (FIG. 11B). The worsening of EAE in DC(CD39-KO) mice
was correlated with an increased frequency of TH1 and TH17 cells in
the CNS (FIG. 11C). In agreement with the worsening of EAE
associated with CD39 deficiency in DCs as shown herein, DC(CD39-KO)
splenic T cells had significantly enhanced proliferative recall
response to MOG(35-55), and DC(CD39-KO) mice had a significantly
greater frequency of CD4.sup.+ CD44.sup.+ CD40L.sup.hi splenic
IFN-.gamma.+ and IL-17+ CD4+ T cells (FIGS. 11D-11E). No difference
was observed between DC(WT) and DC(CD39-KO) mice in the frequency
of Foxp3+ Treg cells or IL-10+ CD4+ T cells (FIGS. 11D-11E).
[0231] Analysis of cDCs isolated 21 d after the induction of EAE
showed significantly lower CD39 expression, concomitant with higher
expression of mRNA encoding the proinflammatory cytokines IL-6,
IL-12 and IL-23, in DC(CD39-KO) mice than in DC(WT) cells (FIG.
11F). No significant difference was found between DC(WT) and
DC(CD39-KO) mice in the expression of mRNA encoding IL-10,
TGF-.beta., IFN-.beta. or IL-27 (FIG. 11F). In vitro, cDCs from
DC(CD39-KO) mice showed an increased ability to trigger the
proliferation of naive 2D2 CD4+ T cells in the presence of
MOG(35-55) and induce the production of IL-17 and IFN-.gamma., at
the expense of decreased secretion of IL-10 and TGF-.beta. (FIGS.
11G-11H). Thus, expression of CD39 in DCs limited the
encephalitogenic TH1 and TH17 T cell response and the development
of EAE.
Example 8
Vaccination with IL-27-Conditioned DCs Suppresses Autoimmune
Diseases
[0232] The tolerogenic effects of IL-27 signaling in T cells and
DCs provide therapeutic potential in disorders mediated by the
immune system. However, IL-27 is previously reported to act on T
cells, but not dendritic cells, to boost cytotoxic CD8+ T cell
responses.sup.11, which suggests that direct administration of
IL-27 could potentially have detrimental side effects in
immunologically mediated disorders. Vaccination with DCs can induce
immunity to tumors and pathogens.sup.32, but vaccination with
naturally-occurring tolerogenic DCs has been shown to induce
antigen-specific tolerance.sup.33. Thus, as shown herein the
tolerogenic effects of IL-27 signaling in DCs, and to avoid the
potential pathogenic effects of IL-27 administration, it was next
sought to determine the therapeutic effects of vaccination with
IL-27-conditioned DCs on CNS autoimmunity.
[0233] The effects of vaccination with IL-27-conditioned bone
marrow-derived DCs on the model of relapsing-remitting EAE induced
in SJL mice was assessed by immunization with a peptide of amino
acids 139-151 of proteolipid protein (PLP(139-151)). On day 20
after EAE induction, during the remission phase of the disease, the
mice were randomly allocated into four groups and treated as
follows: group 1, saline only; group 2, DCs loaded with
PLP(139-151); group 3, DCs treated with IL-27; group 4, DCs treated
with IL-27 and loaded with PLP(139-151). An additional control
group received DCs not loaded with peptide or pretreated with
cytokine. The treatment was repeated three additional times, once
every 4 d. DCs not loaded with peptide or pretreated with cytokine
had no significant effect on disease development (P=0.1487; FIG.
12A). However, the administration of IL-27-conditioned DCs loaded
with PLP(139-151) led to a significant reduction in EAE and a
significant reduction in the proliferative recall response
(measured as the production of IFN-.gamma. and IL-17) to
PLP(139-151), concomitant with increased production of IL-10 and
TGF-.beta., relative to results obtained for untreated mice (FIGS.
13A-13C).
[0234] In this model, the chronic phase of EAE is characterized by
spreading of the T cell response to the PLP epitope of amino acids
178-191 (PLP(178-191)).sup.7. Thus, the recall response to
PLP(178-191) in DC-vaccinated mice was analyzed. The administration
of IL-27-treated DCs loaded with PLP(139-151) led to significant
suppression of the proliferative response and production of
IFN-.gamma. and IL-17 triggered by PLP(178-191), concomitant with
increased production of IL-10 and TGF-.beta., relative to the
results obtained for untreated mice (FIGS. 13D-13E). Thus,
vaccination with IL-27-treated cDCs can also reduce epitope
spreading in this model of EAE.
[0235] To further investigate the effects of IL-27-conditioned DCs
on the immune response, myelin antigen arrays were used, which can
detect epitope spreading in EAE and MS.sup.34, 35. The microarrays
consisted of a collection of CNS-related autoantigens, including
tissue lysates, recombinant proteins, and peptide libraries
spanning the entire sequence of myelin proteins and lipids found in
the central and peripheral nervous system.sup.34. The therapeutic
effect of vaccination with DCs on EAE in SJL mice was accompanied
by a lower serum concentration of immunoglobulin G antibodies to 31
myelin antigens than that in untreated mice (FIG. 13F).
[0236] It was then sought to assess the mechanisms involved in the
suppression of EAE with IL-27-conditioned DCs; for this the C57BL/6
mouse model of EAE induced with MOG(35-55) was used. The
administration of IL-27-conditioned DCs was initiated 10 d after
the induction of EAE. Vaccination with IL-27-conditioned wild-type
DCs loaded with MOG(35-55) led to significantly diminished EAE
relative to that of untreated mice (FIGS. 12B-12F). Similar
suppression of EAE was observed after the administration of
MOG(35-55)-loaded, IL-27-conditioned DCs deficient in IL-10 or
PD-L1 (FIGS. 12B-12F), which indicated that these molecules did not
appear to have a role in regulating the encephalitogenic response
by IL-27 signaling in DCs. Conversely, MOG(35-55)-loaded,
IL-27-conditioned DCs deficient in IL-7RA or CD39 did not have a
significant effect on EAE (P=0.7979, P=05708 and P=0.5630,
respectively; FIGS. 12B-12F). Together these data showed that
vaccination with IL-27-treated DCs controlled the encephalitogenic
response and established relapsing-remitting EAE in a therapeutic
paradigm in an CD39-dependent manner.
Discussion for Examples 1-8
[0237] IL-27 is previously reported to limit tissue inflammation
and autoimmunity in various scenarios.sup.11. Deficiency in the
receptor for IL-27 has been previously reported to be linked to the
development of exacerbated inflammation in animal experimental
models, and polymorphisms in IL-27 have been previously reported to
be associated with human inflammatory disorders.sup.11. However, in
those previous reports, the immunoregulatory actions of IL-27 are
usually attributed to its direct effects on T cells, whereby it
arrests the development of TH17 cells and promotes the
differentiation of Tr1 cells through mechanisms that involve the
transcription factor AhR.sup.26,36. Thus, those previous reports
did not teach or suggest the immunosuppressive effects of IL-27 on
DCs. The present findings identified an important role for IL-27
signaling in DCs in the control of the T cell response and CNS
autoimmunity.
[0238] Presented herein shows that without wishing to be bound by
theory, CD39 was required for modulating the antigen presenting
function of cDCs by IL-27 in vitro and in vivo. However, IL-27 also
upregulated the expression of additional immunoregulatory
molecules, such as IL-27 itself, IFN-.beta., TGF-.beta. and IDO.
Although those molecules did not have a substantial role under the
experimental conditions shown herein, they might contribute to the
suppressive effects of IL-27 on DCs in alternative manners.
[0239] IL-10, known to activate STAT3 signaling, has also been
shown to induce a tolerogenic phenotype in DCs.sup.37. Conversely,
STAT3 deficiency restricted to DCs results in the spontaneous
development of inflammation.sup.38. Moreover, microglia and tissue
macrophages express CD39, and their function is also regulated by
cytokines that activate STAT3 signaling.sup.39,40. Thus, it is
contemplated that the induction of CD39 expression can constitute a
common immunoregulatory mechanism triggered by STAT3-activating
cytokines in cells of the innate immune system.
[0240] Without wishing to be bound by theory, CD39 mediates the
suppressive activity of mouse and human Treg cells, probably
through the generation of adenosine.sup.25, 26. The mechanisms by
which CD39 in APCs regulates adaptive immunity are less clear.
Extracellular ATP triggers such activation.sup.28. Augmented
contact hypersensitivity was previously observed in
Entpd1-deficient mice.sup.41. Conversely, Nlrp3 deficiency is
previously reported to result in decreased contact
hypersensitivity.sup.42. Follow-up studies have determined that
activation of the NLRP3 inflammasome in APCs controls TH1, TH2 and
TH17 responses.sup.43,44. Activation of NLRP3 inflammasome is
previously reported to be required for the differentiation of
encephalitogenic TH1 and TH17 cells and the development of
EAE.sup.30, probably as a result of its effects on the secretion of
IL-1.beta. and IL-18.
[0241] The present findings presented herein showed that the
induction of CD39 expression in DCs by IL-27 led to a significant
reduction in the extracellular concentration of ATP, concomitant
with a significant reduction in activation of the NLRP3
inflammasome and reduced differentiation of TH1 and TH17 cells.
These findings are compatible with a model in which, through the
upregulation of CD39 expression, IL-27 limits ATP-dependent
activation of the NLRP3 inflammasome in DCs and their ability to
promote the differentiation of pathogenic TH1 and TH17 cells.
Without wishing to be bound by theory, however, in addition to its
effects on CD39 expression, IL-27-triggered signaling can directly
interfere with the expression of components of the NLRP3
inflammasome and their activation in DCs.
[0242] The immunoregulatory properties of IL-27 can be used for the
treatment of human autoimmune diseases. However, IL-27 can boost
cytotoxic CD8+ T-cell responses.sup.11, which suggests that the
administration of IL-27 could potentially worsen disorders mediated
by the immune system. Hence, to evaluate the therapeutic potential
of IL-27 and avoid its potential deleterious side effects, the
effects of vaccination with IL-27-conditioned DCs were assessed.
Presented herein shows that vaccination with IL-27-conditioned DCs
suppressed the encephalitogenic TH1 and TH17 response and halted
established chronic EAE. Thus, vaccination with IL-27-conditioned
tolerogenic DCs can provide a new avenue for the treatment of
autoimmune diseases. Alternatively, nanoparticles have been
previously developed for delivery of antigens in vivo to induce
antigen-specific tolerance.sup.46. Accordingly, in some
embodiments, nanoparticles engineered to activate IL-27 signaling
and deliver myelin antigens, e.g., specifically to DCs, can induce
tolerogenic DCs in vivo and arrest pathogenic T cell responses.
That approach can exploit the immunoregulatory properties of IL-27
and overcome the limitations associated with the implementation of
cell-based therapies in a clinical setting. Presented herein shows
that IL-27 signaling in DCs limited the differentiation of
pathogenic TH1 and TH17 cells and the development of CNS
autoimmunity through a mechanism that was at least partially
dependent on CD39. This immunoregulatory pathway can provide new
therapeutic avenues for MS and other autoimmune disorders.
Exemplary Methods and Materials Used in Examples 1-8
[0243] Animals. IL-27RA- and CD39-deficient mice have been
previously described in Refs 25 and 36. SJL, 2D2, C57BL/6,
CD11c-Cre:DTR and IL-10 deficient mice were from the Jackson
Laboratories. For conditional ablation of DCs
(CD11c-DTR.fwdarw.WT), bone marrow chimeras were inoculated
intraperitoneally every second day for 2 weeks with 16 ng DTx per
gram body weight. For the generation of bone marrow chimeras,
recipient mice were lethally irradiated with a dose of 9.5 Gy and 1
d later were given intravenous injection of 5.times.10.sup.6 bone
marrow cells isolated from donor femora and tibiae. Recipients of
bone marrow were then allowed to rest for 8 weeks before use. Four
to five randomly assigned mice were used per experimental group per
experiment. Mice were kept in a conventional, pathogen-free
facility. All experiments were carried out in accordance with
guidelines of the Institutional Animal Care and Use Committee.
[0244] Isolation of splenic DCs and CNS infiltrates. Spleens were
incubated for 20 min with 2 mg/ml collagenase D and then were
mashed through a 70-.mu.m cell strainer. DCs were sorted as
previously described (in Ref. 36) into F4/80.sup.- CD11b.sup.-
CD11c.sup.lo B220.sup.+ MHCII.sup.lo Ly6C.sup.+pDCs and F4/80.sup.-
CD11b.sup.+ CD11c.sup.+ B220.sup.- MHCII.sup.+ Ly6C.sup.- cDCs
(eBioscience). cCs were cultured for 48 h with IL-27 (20 ng/ml) or
ecLPS (100 ng/ml; E. coli strain 0111:B4; Sigma). Parallel cultures
maintained without stimuli were used as controls. IL-27 was
prepared according to the manufacturer's protocol
(eBioscience).
[0245] CNS infiltrates were isolated as previously described in
Ref. 47. Mice were perfused with ice-cold PBS. The brain and spinal
cord were removed and incubated in PBS containing collagenase type
III (2 mg/ml; Worthington) and DNase (20 units/ml; Sigma-Aldrich).
Tissues were then homogenized and loaded on a 30%-37%-70% Percoll
gradient for enrichment of CNS infiltrates.
[0246] Flow cytometry staining and acquisition. The following
antibodies were used for flow cytometry: peridinin chlorophyll
protein-conjugated anti-LY6C (HK1.4) and allophycocyanin-conjugated
anti-CD11b (M1/70; both from BioLegend); phycoerythrin-conjugated
antibody to MHC class II (M5/114.15.2), fluorescein
isothiocyanate-conjugated anti-F4/80 (BM8) and Alexa Fluora
647-anti-CD39 (24DMS1; all from eBioscience);
allophycocyanin-indotricarbocyanine-conjugated anti-CD11c (HL3) and
phycoerythrin-conjugated anti-B220 (RA3-6B2; all from BD
Pharmingen); and fluorescein isothiocyanate-conjugated anti-IL-27RA
(263503; R&D Systems).
[0247] For analysis of intracellular Foxp3, cell preparations were
stained for cell surface markers with
allophycocyanin-indotricarbocyanine-conjugated anti-CD4 (RM4-5;
BioLegend), then were fixed and made permeable with
fixation-permeabilization buffers (eBioscience) and were stained
with peridinin chlorophyll protein-cyanine 5.5-conjugated
anti-Foxp3 (FJK-16s; eBioscience).
[0248] For intracellular cytokine staining, cells were stimulated
for 6 h with phorbol 12-myristate 13-acetate (50 ng/ml) and
ionomycin (500 ng/ml) in the presence of GolgiPlug (BD Pharmingen).
Then, cells were stained for surface molecules (antibodies
identified above), fixed and made permeable with a Cytofix/Cytoperm
Plus kit (BD Bioscience) and stained with the following antibodies:
phycoerythrin-indotricarbocyanine-conjugated anti-IFN-.gamma.
(XMG1.2; BioLegend), phycoerythrin-conjugated anti-IL-17 (eBio17B7;
eBioscience) and allophycocyanin-conjugated anti-IL-10 (JESS-16E3;
BD Pharmingen). Data were collected with a LSR II or FACSAria (BD
Biosciences), then were analyzed with FlowJo software
(Treestar).
[0249] Proliferation assays. For in vitro experiments, cDCs
pretreated with IL-27 (20 ng/ml) and activated for 48 h with ecLPS
(100 ng/ml; E coli strain 0111:B4; Sigma) were used (at a ratio of
1:10) to stimulate naive T cells from wild-type or 2D2 mice.
Polarizing conditions have been previously described in Ref. 48:
IL-12 (30 ng/ml) was used to generate TH1 cells, or IL-6 (30
ng/ml); TGF-.beta.1 (3 ng/ml) was used to generate TH17 cells; and
TGF-.beta.1 (5 ng/ml) and IL-27 (30 ng/ml) were used to generate
Treg cells and Tr1 cells, respectively. Mouse IL-6, IL-12, IL-23
and TGF-.beta.1 were all from R&D Systems For in vivo
experiments, splenic cells were obtained from wild type, IL27RA- or
CD39-deficient mice at day 21 after immunization with MOG(35-55)
and were restimulated in vitro for 3 d in the presence of
MOG(35-55). SJL mice were immunized with PLP(139-151), and at the
end of the experiment, splenic cells were collected and
restimulated in vitro for 3 din the presence of PLP(139-151) or
PLP(178-191). The cells were pulsed with [.sup.3H]thymidine (1
.mu.Ci/well) for the final 24 h of the incubation period. The
frequency of T cells producing IL-17 (eBio17B7; eBioscience),
IFN-.gamma. (XMG1.2; BioLegend) or IL-10 (JESS-16E3; BD Pharmingen)
and Foxp3.sup.+ T cells (FJK-16s; eBioscience) was assessed by flow
cytometry. For CFSE-based proliferation assay, 2D2 CD4+ T cells
were labeled with 1 .mu.M CFSE (carboxyfluorescein diacetate
succinimidyl ester; Molecular Probes) Data were acquired on an LSR
II (BD Biosciences) and were analyzed with FlowJo software
(TreeStar).
[0250] Measurement of cytokines. Secreted cytokines were measured
after 48 h by enzyme-linked immunosorbent assay as previously
described in Ref. 49.
[0251] Quantitative PCR analysis. RNA was extracted with RNAeasy
columns (Qiagen, USA), then cDNA was prepared according to the
manufacturer's instructions (Applied Biosystems) and was used as
template for real-time PCR All primers and probes were provided by
Applied Biosystems and were used on the ViiA 7 Real-Time PCR System
(Applied Biosystems). Expression was normalized to the expression
of the housekeeping gene Gapdh Primers-probe mixtures were as
follows (from Applied Biosystems; identifiers in parentheses): Il6
(Mm00446190_m1), Il10 (Mm0043614_m1), Il12a (Mm00434165_m1), Il23a
(Mm00518984_m1), Il27 (Mm00461162_m1), Il27ra (Mm00497259_m1),
Entpd1 (Mm00515447_m1), Tgfb1 (Mm01178820_m1), Ifnb1
(Mm00439552_s1), Ido1 (Mm0001218007_m1), Ido2 (Mm01218007_m1),
Tnip3 (Mm01181626_m1), Tnfaip3 (Mm00437121_m1), Ramp3
(Mm00840142_m1), Esr1 (Mm00433151_m1) and Gapdh
(Mm99999915_g1).
[0252] Gene-expression analysis. Transcriptomes were analyzed by
Affymetrix microarray MoGene1_0_st of samples obtained 0, 2 and 6 h
after stimulation with LPS or IL-27 and LPS. Data were normalized
with the robust multiarray average algorithm. Network activity was
determined with the NetGenerator algorithm and software of the R
project for statistical computing (version 2.1-3).sup.24 using as
input the change of gene expression (fold) over time points. The
network graph was produced with DOT plain text graph description
language through the open software collection Graphviz.
[0253] For nCounter analysis of gene expression, a `multiplexed`
target profiling of 146 inflammation- and immune system-related
transcripts was customized and used in accordance with the
manufacturer's protocol (Nanostring). This combination of genes and
their differences in expression in vivo in DCs allowed
investigation of immune system-related pathways during EAE with the
EXPANDER tool (`expression analyzer and displayer`).
[0254] Immunoblot analysis. For immunoblot analysis, cells were
lysed with radio-immunoprecipitation buffer supplemented with
protease inhibitor `cocktail` (Sigma). Total lysates of DCs (40
.mu.g) were resolved by electrophoresis through 4-12% Bis-Tris
Nupage gels (Invitrogen, USA) and were transferred onto PVDF
membranes (Millipore). The following primary antibodies were used:
anti-IL-27RA (MAB21091; R&D Systems); antibody to
phosphorylated STAT3 (9134), antibody to phosphorylated STAT1
(9167), anti-STAT3 (9132), anti-STAT1 (9172) and anti-GAPDH (2111;
all from Cell Signaling Technology); anti-caspase-1 (ab17820),
anti-IL-1.beta. (ab9722) and anti-.beta.-actin (ab20272; all from
Abcam). Immunoblot analysis was done as previously described in
Ref. 47 and blots were developed with SuperSignal West Femto
Maximum Sensitivity Substrate as suggested by the manufacturer
(Pierce).
[0255] Chromatin immunoprecipitation. DNA-protein complexes in
cells were crosslinked with 1% paraformaldehyde and lysed with 0.35
ml lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl, pH 8.1)
containing 1.times. protease inhibitor `cocktail` (Roche Molecular
Biochemicals). Chromatin was sheared by sonication and supernatants
collected after centrifugation were diluted in buffer (1% Triton
X-100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris-HCl, pH 8.1). 5 .mu.g
antibody was prebound for a minimum of 4 h to protein A and protein
G Dynal magnetic beads (Invitrogen) and samples were washed three
times with ice-cold PBS containing 5% BSA, and then were added to
the diluted chromatin, followed by immunoprecipitation overnight.
The magnetic bead-chromatin complexes were then washed three times
in radioimmunoprecipitation buffer (50 mM HEPES, pH 7.6, 1 mM EDTA,
0.7% Na deoxycholate, 1% NP-40 and 0.5 M LiCl), followed by two
washes with Tris-EDTA buffer. Immunoprecipitated chromatin was then
extracted with a solution of 1% SDS and 0.1 M NaHCO3 and was heated
at 65.degree. C. for at least 6 h for reversal of the
paraformaldehyde cross-linking. DNA fragments were purified with a
QIAquick DNA purification Kit (Qiagen) and were analyzed by SYBR
Green real-time PCR (Takara Bio). The following antibodies were
used for chromatin immunoprecipitation: anti-STAT3 (9132; Cell
Signaling Technology) and anti-STAT1 (9172; Cell Signaling
Technology). The following primer pairs were used: Stat3 (SRE-1),
forward, 5'-GCTGGGCTTTAGAGACTTGTGG GC-3' (SEQ ID NO: 2), and
reverse, 5'-ACCCATGCAAATGGTTTGGGCA-3' (SEQ ID NO: 3); Stat3
(SRE-2), forward, 5'-TGAGGGCCAGCCCACACTTCA3' (SEQ ID NO: 4), and
rev: 5'-GCTCACT GGGTACCTCTTGCCA-3' (SEQ ID NO: 5); Stat1 (IRF-1),
forward, 5'-GGAACAAAAATATA GAGAGAACTTGGGA-3' (SEQ ID NO: 6), and
reverse,5'-GTAGTTTGACCTAAGTGGACAT AGG-3' (SEQ ID NO: 7);
Stat1-Stat3, forward, 5'-AGGCTCTTGTATCCTTGCCACCTCT-3' (SEQ ID NO:
8), and reverse, 5'-TGATGGTGGAGTGCTGTGTGCTG -3' (SEQ ID NO: 9).
[0256] Transfection and luciferase assays. HEK293 cells were grown
in DMEM supplemented with 10% FBS and were transfected with FuGENE
HD transfection reagent and 2 .mu.g of each plasmids according the
manufacturer's instructions (Roche). Firefly and renilla luciferase
activity was analyzed 48 h after transfection with a Dual
Luciferase Assay kit (Promega).
[0257] Free ATP measurement. cDCs were cultured for 48 h with IL-27
or LPS as described above (Isolation of splenic DCs and CNS
infiltrates). Cells were then washed twice with phenol red-free
RPMI-1640 medium (Gibco) and were deprived of serum for 24 h.
Cell-free medium was then analyzed for endogenous ATP with ENTILEN
rLuciferase/Luciferin reagent (Promega). Bioluminescent activity
was measured with an Infinite 200 Proluminometer (Tecan).
[0258] Ectonucleotidase enzymatic activity analysis. Thin-layer
chromatography was done as previously described in Ref. 25 with
slight modifications. 1.times.10.sup.5 DCs were treated for 48 h
with IL-27 or LPS, then were incubated with 2 mCi/ml [.sup.14C]ADP
(GE Healthcare Life Sciences) in 10 mM Ca.sup.2| and 5 mM
Mg.sup.2|. Hydrolysis products of [.sup.14C]ADP were assessed by
TLC in 5-.mu.l aliquots collected at 1.5, 3 and 6 min and were
applied onto silica gel matrix plates (Sigma-Aldrich).
[.sup.14C]ADP and its radiolabeled derivatives were separated with
the appropriate solvent mixture as described previously in Refs. 25
and 50. Adenosine uptake and deamination was blocked with 10 .mu.M
dipyridamole. [.sup.14C]ADP, [.sup.14C]AMP and [.sup.14 C]ADO
incubated in PBS served as standards.
[0259] EAE induction and vaccination with DCs. EAE was induced by
subcutaneous immunization of mice with 150 .mu.g MOG(35-55) or 30
.mu.g PLP(139-151) (ANASPEC) as previously described in Ref. 49.
Adoptive transfer EAE was induced as previously described in Ref.
48 with some modifications. 2D2 mice were immunized with 150 .mu.g
MOG(35-55) in complete Freund's adjuvant, and draining lymph nodes
and spleens were collected 7 d after immunization and then were
cultured for 48 h with MOG(35-55) (20 .mu.g/ml) and carrier free
recombinant IL-12 (20 ng/ml; R&D Systems) or IL-23 (20 ng/ml;
R&D Systems). Subsequently, 5.times.10.sup.6 cells were
transferred intravenously into DC(WT) or DC(IL-27RA-KO) mice.
Clinical signs of EAE were assessed by investigators `blinded` to
treatment conditions, according to the following score: 0, no
disease; 1, loss of tail tone; 1.5, poor righting ability; 2,
hind-limb weakness; 3, hind-limb paralysis; 4, quadreparesis; and
5, moribund.
[0260] For the generation of bone marrow-derived DCs, bone marrow
cells isolated from the femurs of naive mice were cultured for 7 d
in the presence of the cytokine GM-CSF (20 ng/mL; Peprotech). On
day 7, cells were purified with CD11c+ magnetic beads (Miltenyi),
then were cultured for 18 h with IL-27 (20 ng/ml) and, for the
final 3 h before administration, were loaded with 20 .mu.g
MOG(35-55) or PLP(139-151) DCs (2.times.10.sup.6 cells per mouse)
were then extensively washed and administered intravenously four
times, once every 4 d All experiments were carried out in
accordance with guidelines prescribed by the Institutional Animal
Care and Use Committee.
[0261] Antigen microarray. Antigens were spotted onto Epoxy slides
(TeleChem) as describee . Nonspecific binding on microarrays was
blocked with 1% bovine serum albumin, followed by incubation with
test serum (1:100 dilution in blocking buffer). Arrays were then
washed and incubated with indocarbocyanine-conjugated goat antibody
to mouse immunoglobulin G (detection antibody; 115-166-072; Jackson
ImmunoResearch Labs). Antigen reactivity was defined by the mean
intensity of binding to the replicates of that antigen on the
microarray. Raw data were normalized and analyzed with GeneSpring
software (Silicon Genetics).
[0262] Statistical analysis. Prism software was used for
statistical analysis. Statistical analysis was done according to
the recommendations of Nature for reporting life sciences research.
For comparison of two groups, linear regression with 95% confidence
interval, and unpaired, two-tailed Student's t-test were used.
One-way ANOVA for paired data was used to determine the
significance of the time-response curves. P values of <0.05 were
considered statistically significant. For adjustment of the
significance value for multiple comparisons, a Bonferroni
correction was applied with a corrected significance value of
0.017.
Example 9
Tolerogenic Biotherapeutics Targeting the IL-27/Ectonucleotidase
Axis
[0263] Several Treg subsets enforce immune tolerance, of particular
importance are FoxP3+ Tregs and IL-10+ type 1 regulatory T cells
(Tr1 cells). Deficits in pancreatic FoxP3+ Tregs have been
described in T1D patients, and Teffs from T1D patients might be
resistant to regulation by FoxP3+ Tregs.sup.3. With regards to
IL-10+ Tr1 cells, IL-10 secretion by islet-specific T cells is
reduced in newly diagnosed T1D patients.sup.4. Conversely, Tr1
cells enforce tolerance in preclinical models of islet
transplantation.sup.s, and the arrest of diabetes by Teplizumab
(anti-CD3) is linked to IL-10 production by T cells.sup.6.
[0264] The re-establishment of immune tolerance can be beneficial
for the treatment of type 1 diabetes (T1D). Dendritic cells (DCs)
control the balance between effector and regulatory T cells (Teffs
and Tregs, respectively). As described in Examples 1-8, IL-27 acts
on DCs to expand Tregs, and thus can limit Teffs and suppress
disease in models of T1D and multiple sclerosis (MS) (FIGS.
14A-14B). Moreover, as presented earlier, the anti-inflammatory
effects of IL-27 on DCs were mediated by the up-regulation of the
ectonucleotidase CD39 (ENTPD1) and the consequent decrease in the
levels of pro-inflammatory extracellular ATP (eATP). Accordingly,
fusion proteins that incorporate DC-targeting antibodies and IL-27
or the ATP-degrading protein apyrase can be developed as therapy
for T1D (FIG. 14C). In some embodiments, these anti-inflammatory
biotherapeutics can be repurposed to treat a broad array of
immune-mediated diseases.
[0265] IL-27 induces Tr1 cell differentiation mediated by the aryl
hydrocarbon receptor .sup.7, 8. Thus, IL-27 and Tr1 cells play an
important role in suppressing pre-existing inflammation,
particularly because Teffs are resistant to the suppressive
activity of FoxP3+ Tregs in inflamed tissues.sup.9.
[0266] The IL-27 receptor (IL-27Ra) is expressed by T cells, and
also by innate immune cells such as DCs 10, 11. IL-27 polymorphisms
have been linked to T1D.sup.12. However, it is not known that IL-27
can act on DCs or induce an immunosuppressive effect on DCs.
Indeed, the inventors discovered that IL-27 acts on DCs to expand
Tregs, limit Teffs, and arrest the development of experimental T1D
and multiple sclerosis. These tolerogenic effects of IL-27 on DCs
were mediated by the up-regulation of the ectonucleotidase CD39 and
the consequent decrease in pro-inflammatory eATP (FIGS. 14A-14B).
The IL-27 also induced the expression of other tolerogenic
molecules in DCs, such as IDO 1.
[0267] While IL-27 can be administered to treat autoimmune
disorders, non-targeted systemic cytokine administration can be
associated with side effects that limit its clinical use.
Accordingly, in one aspect, a biotherapeutic comprising a
DC-targeting antibody fused to IL-27 or apyrase, an ATP-degrading
protein, can be used to target the IL-27/ectonucleotidase axis and
re-establish tolerance in T1D and other immune-mediated disorders
(FIG. 14C).
[0268] CD39-deficiency worsens diabetes in mice, and CD39 limits
renal injury and promotes .beta.-cell regeneration.sup.13-16. Thus,
in some embodiments, targeting the IL-27/ectonucleotidase axis can
activate additional tissue-protective mechanisms that may boost the
efficacy of a therapeutic in T1D and support its repurposing to
treat other inflammatory disorders (e.g. lupus nephritis).
[0269] Exemplary biotherapeutic drug candidate: In some
embodiments, a biotherapeutic can comprise a DC-targeting antibody
fused to IL-27 or an ATP-degrading protein (e.g., apyrase such as
potato (S. tuberosum) apyrase). In some embodiments, the
DC-targeting antibody can be a single chain antibody targeting
Clec9A, which is expressed by human DCs.sup.17. Alternatively, the
DC-targeting antibody can be a single chain antibody targeting
DEC205, although its expression is less specific, particularly in
humans.
[0270] While previous report shows that the .beta.-cell specific
delivery of NF-kB inhibitors suppresses experimental T1D.sup.18,
targeted delivery of anti-inflammatory IL-27 to .beta.-cells or the
kidney is not known. In some embodiments, it can be desirable to
conjugate apyrase or IL-27 to antibodies targeting .beta.-cells or
kidney, which can be used as a therapeutic approach for T1D and
nephritis. Similarly, the local delivery of apyrase to .beta.-cells
or the kidney might boost .beta.-cell regeneration and limit renal
injury. In some embodiments, the biotherapeutic described herein
can be used in combination with other immunotherapies in T1D (e.g.
IL-7Ra blocking antibodies.sup.19) to treat diabetic nephropathy or
promote .beta.-cell survival, and can alternatively be repurposed
for other clinical autoimmune indications (e.g. lupus
nephritis).
[0271] The biotherapeutics can be analyzed on murine T1D systems
and cells in vitro, and also on human cells from controls and T1D
patients. Further, IL-27Ra and CD39 expression can be analyzed in
T1D patients, to evaluate the relevance of the IL-27/CD39 pathway
in T1D and the utility of these molecules as biomarkers for patient
stratification and monitoring.
[0272] To study the effects of biotherapeutics described herein in
pre-clinical models of T1D, biotherapeutics, e.g., targeting
IL-27/ectonucleotidase pathway, are used to evaluate their effects
on murine T1D models. The targeting-specificity of the
biotherapeutics is analyzed using cells in vitro, and also upon in
vivo administration. The activity of the antibody-fused IL-27 or
apyrase on DCs in vitro is also measured to determine whether IL-27
or apyrase fusion to the antibody can affect their biological
activity. These in vivo and in vitro functional assays can analyze
the effects of the biotherapeutics on DC survival and maturation,
and T cell activation and polarization into Teffs and Tregs in vivo
and in vitro. IL-27- and CD39-deficient mice, as well as
recombinant IL-27 or apyrase, can be used as controls. In addition,
the effects of the biotherapeutics on murine models of T1D can be
assessed in preventive and therapeutic paradigms.
[0273] Dysregulated T-cell responses and IL-27 and CD39 (ENTPD1)
polymorphisms have been previously reported in T1D. To investigate
the effects of biotherapeutics described herein on T1D and control
samples, IL-27Ra, CD39 and the functional effects of targeting the
IL-27/ectonucleotidase axis with biotherapeutics are determined in
control and T1D samples. IL-27Ra and CD39 expression are first
analyzed in untreated or biotherapeutic-treated DCs and T cells
from controls and T1D samples. This can determine whether these
molecules can be used to stratify patients and monitor their
response to therapy.
[0274] To evaluate the efficacy of a biotherapeutic described
herein on treatment of T1D, newly diagnosed T1D patients within 6
weeks of diagnosis and positive for anti-GAD65, anti-ICA512, or
ICA. ENTPD1 polymorphisms are used in patient selection and treated
with either a placebo or a biotherapeutic described herein. Change
from baseline in mean C-peptide area under the curve, insulin use
and/or HbA1 levels can be measured at an indicated time point after
administration. To study the effects of the biotherapeutic on
IL-27/ectonucleotidase axis, CD39 expression is analyzed in blood
DCs, which is up-regulated by IL-27. As there is a transcriptional
signature of the response of DCs to IL-27, mRNA expression is also
determined. These parameters are then correlated with the
biodistribution and cell-targeting of the biotherapeutic in
patients. Further, the ability of blood DCs to activate T cells in
vitro is analyzed. In addition, T cells and DCs in PBMCs are
analyzed, e.g., by CyTOF, for the expression of surface and
intracellular markers (e.g. FoxP3, IL-10, IL-17, etc.);
insulin-specific T-cells are analyzed, e.g., with tetramers and by
ELISPOT.
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Sequence CWU 1
1
916PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 1His His His His His His1 5224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2gctgggcttt agagacttgt gggc 24322DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 3acccatgcaa atggtttggg ca
22421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4tgagggccag cccacacttc a 21522DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5gctcactggg tacctcttgc ca 22628DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6ggaacaaaaa tatagagaga
acttggga 28725DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 7gtagtttgac ctaagtggac atagg
25825DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8aggctcttgt atccttgcca cctct 25923DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9tgatggtgga gtgctgtgtg ctg 23
* * * * *