U.S. patent application number 15/344281 was filed with the patent office on 2017-03-16 for monoclonal antibodies to interleukin 35 and methods of use thereof to inhibit regulatory t cell function.
The applicant listed for this patent is St. Jude Children's Research Hospital. Invention is credited to Lauren Collison, Dario Vignali, Kate Vignali, Creg Workman.
Application Number | 20170073410 15/344281 |
Document ID | / |
Family ID | 39167363 |
Filed Date | 2017-03-16 |
United States Patent
Application |
20170073410 |
Kind Code |
A1 |
Vignali; Dario ; et
al. |
March 16, 2017 |
MONOCLONAL ANTIBODIES TO INTERLEUKIN 35 AND METHODS OF USE THEREOF
TO INHIBIT REGULATORY T CELL FUNCTION
Abstract
Methods for regulating T cell function in a subject,
particularly regulatory T cell activity are provided. Methods of
the invention include administering to a subject a therapeutically
effective amount of an Interleukin 35-specific binding agent, such
as an antibody or small molecule inhibitor. The invention further
provides methods for enhancing the immunogenicity of a vaccine or
overcoming a suppressed immune response to a vaccine in a subject,
including administering to the subject a therapeutically effective
amount of an IL35-specific binding agent and administering to the
subject a vaccine. In one embodiment the vaccine is a cancer
vaccine.
Inventors: |
Vignali; Dario; (Germantown,
TN) ; Workman; Creg; (Memphis, TN) ; Collison;
Lauren; (Memphis, TN) ; Vignali; Kate;
(Germantown, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
St. Jude Children's Research Hospital |
Memphis |
TN |
US |
|
|
Family ID: |
39167363 |
Appl. No.: |
15/344281 |
Filed: |
November 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14796494 |
Jul 10, 2015 |
9518113 |
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15344281 |
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14187751 |
Feb 24, 2014 |
9217135 |
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14796494 |
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12441166 |
Aug 7, 2009 |
8784807 |
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PCT/US07/79310 |
Sep 24, 2007 |
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14187751 |
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60846434 |
Sep 22, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/0011 20130101;
C07K 16/244 20130101; C07K 2317/21 20130101; A61P 35/00 20180101;
C07K 2317/35 20130101; C12N 5/0636 20130101; A61K 39/00114
20180801; C07K 2317/622 20130101; A61K 39/3955 20130101; C07K
2317/76 20130101; C07K 2317/24 20130101; A61K 2039/505 20130101;
A61K 39/39533 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; A61K 39/00 20060101 A61K039/00; A61K 39/395 20060101
A61K039/395 |
Claims
1. A pharmaceutical composition comprising an excipient and a
monoclonal antibody that specifically binds IL35.
2. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody specifically binds to the EBI3 subunit of
IL35.
3. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody specifically binds to the p35 subunit of
IL35.
4. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody does not specifically bind to IL12 and/or
IL27.
5. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody specifically binds to the EBI3 subunit of IL35
and specifically blocks IL35 regulatory T-cell activity.
6. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody specifically binds to the p35 subunit of IL35
and specifically blocks IL35 regulatory T-cell activity.
7. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody specifically interferes with IL35
formation.
8. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody is present in the composition in an amount
effective to inhibit IL35 immunoregulatory activity.
9. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody is present in the composition in an amount
effective to reduce and/or block of one or more of the suppressive
effects mediated by regulatory T-cells.
10. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody is present in the composition in an amount
effective to inhibit the activation and/or proliferation of
regulatory T-cells relative to the activation and/or proliferation
of effector T-cells.
11. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody is present in the composition in an amount
effective to prevent or overcome induction of tumor antigen
tolerance by regulatory T-cells in a subject
12. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody is monovalent, bivalent, or a single chain
antibody.
13. The pharmaceutical composition of claim 1, wherein the
monoclonal antibody is present in the composition in an amount from
about 0.1 to about 200 mg/kg body weight in single or divided
doses.
14. A method of inhibiting a regulatory T-cell function in a
subject, comprising administering to a subject in need of
inhibiting a regulatory T-cell function a therapeutically effective
amount of the pharmaceutical composition of claim 1, wherein the
therapeutically effective amount of said antibody inhibits a
regulatory T-cell function in said subject.
15. A method of inhibiting a regulatory T cell function in a
subject, comprising administering to the subject a therapeutically
effective amount of a specific binding agent, wherein said specific
binding agent binds to IL35.
16. A method of inhibiting a regulatory T cell function in a
subject, comprising administering to the subject a therapeutically
effective amount of a specific binding agent, wherein said specific
binding agent binds to Interleukin 35 (IL35).
17. The method of claim 16, wherein said specific binding agent
comprises an anti-IL35 antibody that specifically binds to
IL35.
18. The method of claim 16, wherein said specific binding agent
comprises a small molecule inhibitor that specifically binds to
IL35.
19. The method of claim 18, wherein said small molecule inhibitor
is a chemical compound.
20. A method of treating a subject having a cancer with a cancer
vaccine, comprising: (a) administering to the subject a
therapeutically effective amount of a specific binding agent,
wherein said specific binding agent binds to Interleukin 35 (IL35);
and (b) administering to the subject a cancer vaccine, wherein said
specific binding agent enhances the efficacy of said cancer
vaccine.
21. The method of claim 20, wherein said specific binding agent
comprises an anti-IL35 antibody that specifically binds to IL35.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
14/796,494, filed Jul. 10, 2015, which is a divisional of U.S. Ser.
No. 14/187,751, now U.S. Pat. No. 9,217,135, filed Feb. 24, 2014,
which is a continuation of U.S. Ser. No. 12/441,166, now U.S. Pat.
No. 8,784,807, filed Aug. 7, 2009, which was a national stage
filing under 35 U.S.C. 371 of PCT/US07/079310, filed Sep. 24, 2007,
which International Application was published by the International
Bureau in English on Mar. 27, 2008, and which claims the benefit of
U.S. Application 60/846,434, filed Sep. 22, 2006, each of which are
hereby incorporated herein in its entirety by reference, for all
purposes.
RECOGNITION OF RESEARCH FUNDING
[0002] This invention was supported by funds received from the
American Lebanese Syrian Associated Charities (ALSAC).
FIELD OF THE INVENTION
[0003] The present invention relates to methods for regulating T
cell function in a subject, particularly regulatory T cell
activity.
BACKGROUND OF THE INVENTION
[0004] The Epstein-Barr virus-induced gene 3 (EBI3; IL27b) product
is a novel soluble hematopoietin component related to the p40
subunit (IL12b) of Interleukin 12 (IL12). EBI3 is widely expressed
in cells and accumulates in the endoplasmic reticulum and
associates with the molecular chaperone calnexin. Besides promoting
Th1 cytokine production, EBI3 plays a critical regulatory role in
the induction of Th2-type immune responses and the development of
Th2-mediated tissue inflammation in vivo, which may be mediated
through the control of invariant natural killer (NK) T cell
function.
[0005] Interleukin 12 was identified and purified from the cell
culture media of Epstein-Barr virus (EBV)-transformed B
lymphoblastoid cell lines. Interleukin 12 is a 70 kDa heterodimeric
cytokine composed of two disulfide-linked glycoproteins, p40 and
p35 (IL12a). Interleukin 12 is primarily produced by macrophages
and other antigen-presenting cells. Interleukin 12 has pleiotropic
effects in the development of Th1 responses in NK and T
lymphocytes, including induction of interferon (INF)-.gamma.
production, proliferation, and enhancement of cytotoxic activity,
and inhibits Th2 responses.
[0006] Multiple, complex and interconnecting mechanisms control
discrimination between self and non-self, including the thymic
deletion of autoreactive T cells and the induction of anergy in
peripheral T cells. In addition to these passive mechanisms, active
suppression of autoreactive responder T cells is mediated by
regulatory or suppressor T cells. Regulatory T (T.sub.R) cells are
powerful inhibitors of T cell activation both in vivo and in vitro.
Regulatory T cells inhibit autoimmunity and inflammation, maintain
immunologic tolerance, and are involved in the induction of tumor
antigen tolerance (for reviews, see, Shevach, E. M., Nat. Rev.
Immunol. 2:389-400, 2002; Sakaguchi, S., Ann. Rev. Immunol.
22:531-562, 2004; and Mapara and Sykes, J. Clin. Oncology
22:1136-51, 2004).
[0007] A major factor limiting immune recognition of cancer cells
is the fact that tumors arise from a subject's own tissue and
therefore express mainly self antigens to which the subject's T
cells have been tolerized, either centrally (i.e., in the thymus)
or peripherally. This situation is manifested as tolerance of T
cells that display a high avidity for the normal self antigens
expressed by the tumor, leaving only functional T cells with low
avidity. This problem is exemplified by p53. Because of its high
level of expression in certain malignancies, wild-type p53 is a
potential target antigen for immunotherapy in a broad spectrum of
neoplastic diseases. However, because of low-level expression in
normal tissues, T cell tolerance by clonal deletion of high-avidity
T cells in the thymus is an obstacle to generating an effective
immune response following vaccination with a wild-type p53 antigen
(Theobald et al., J. Exp. Med. 185:833-41, 1997). Nevertheless, it
is possible to detect and clonally expand T cells specific for
tumor-associated antigens (TAA) from tumor-bearing subjects.
However, even if TAA-specific cells are present at detectable
levels in tumor-bearing subjects, they are often incompetent to
reject the tumor (Lee et al., Nat. Med. 5:677-85, 1999).
[0008] A number of vaccination approaches are currently being
evaluated in clinical trials in efforts to induce host immune
responses against a variety of solid tumors (e.g., colon cancer,
prostate cancer, melanoma, and renal cell carcinoma). These
strategies are all based on the observation that tumors are often
poor antigen presenting cells. The lack of costimulatory molecules
on their surface and the failure to produce stimulatory cytokines
may make them poorly immunogenic and sometimes even tolerogenic.
The approaches investigated include the use of gene-modified tumor
cells (Soiffer et al., Proc. Natl. Acad. Sci. USA 95:13141-46,
1998), the use of professional antigen presenting cells (e.g.,
dendritic cells) or dendritic cells fused to tumor cells (Gong et
al., Blood 99:2512-17, 2002; Gong et al., Nat. Med. 3:558-61,
1997), and DNA transfer using naked DNA or viral vectors.
[0009] Vaccination with dendritic cells has led to systemic T cell
responses in treated subjects. However, clinical responses have
been less striking, although some patients showed significant
antitumor responses, including some complete responses (Nestle et
al., Nat. Med. 4:328-32, 1998; Tjoa et al., Prostate 40:125-29,
1999; Murphy et al., Prostate 39:54-59, 1999). Therefore, there
remains a need for the development of effective therapies for
enhancing antitumor immunity.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to methods for inhibiting
a regulatory T cell function in a subject. In one embodiment,
methods of the invention include administering to the subject a
therapeutically effective amount of an Interleukin 35 (IL35;
previously designated Interleukin 34, IL34)-specific binding agent.
Interleukin 35-specific binding agents include antibodies, such as
monoclonal antibodies, or fragments thereof, modified polypeptides
designed to interfere with IL35 formation or activity, or small
molecule inhibitors, such as chemical compounds.
[0011] A method for treating a subject having a cancer with a
cancer vaccine is also provided. The method includes (i)
administering to the subject a therapeutically effective amount of
an IL35-specific binding agent and (ii) administering to the
subject a cancer vaccine, where the IL35-specific binding agent
enhances the efficacy of the cancer vaccine. In specific,
non-limiting examples, the IL35-specific binding agent includes an
antibody, such as a monoclonal antibody, or fragments thereof, or a
small molecule inhibitor, such as a chemical compound. In one
embodiment, administration of the therapeutically effective amount
of the IL35-specific binding agent and administration of the cancer
vaccine is sequential, in any order. Alternatively, administration
of the therapeutically effective amount of the IL35-specific
binding agent and administration of the cancer vaccine is
simultaneous.
[0012] Methods for enhancing the immunogenicity of a vaccine or
overcoming a suppressed immune response to a vaccine in a subject
are further provided. These methods include (i) administering to
the subject a therapeutically effective amount of an IL35-specific
binding agent and (ii) administering to the subject a vaccine,
where the IL35-specific binding agent enhances the immunogenicity
of the vaccine or overcomes the suppressed immune response to the
vaccine. In specific, non-limiting examples, the IL35-specific
binding agent includes an antibody, such as a monoclonal antibody,
or fragments thereof, or a small molecule inhibitor, such as a
chemical compound. In one embodiment, administration of the
therapeutically effective amount of the IL35-specific binding agent
and administration of the vaccine is sequential, in any order.
Alternatively, administration of the therapeutically effective
amount of the IL35-specific binding agent and administration of the
vaccine is simultaneous.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 illustrates that EBI3 and p35 (IL12a) are highly
expressed by T.sub.R cells. Real-time RT-PCR analysis of
IL12-related genes was performed on T cells sorted from C57BL/6
mice. Data presented as relative mRNA expression.
[0014] FIG. 2A illustrates T.sub.R-restricted expression of EBI3
and IL12a. Effector T (T.sub.E) or T.sub.R cells from the spleens
and lymph nodes of C57BL/6, Foxp3.sup.gfp or EBI3.sup.-1- mice were
purified by FACS as indicated. For FIG. 2A RNA was extracted and
cDNA generated. Quantitative real-time PCR analysis was performed
using .beta.-actin as an endogenous control. Relative mRNA
expression was determined by the comparative CT method (* p=0.008,
** p=0.06). Data represent the mean.+-.SEM of 4 (FIG. 2A)
independent experiments.
[0015] FIG. 2B illustrates T.sub.R-restricted expression of EBI3
and IL12a. Effector T (T.sub.E) or T.sub.R cells from the spleens
and lymph nodes of C57BL/6, Foxp3.sup.gfp or EBI3.sup.-/- mice were
purified by FACS as indicated. For FIG. 2B RNA was extracted and
cDNA generated. Quantitative real-time PCR analysis was performed
using .beta.-actin as an endogenous control. Relative mRNA
expression was determined by the comparative CT method (* p=0.008,
** p=0.06). Data represent the mean.+-.SEM of 2 (FIG. 2B)
independent experiments.
[0016] FIG. 2C illustrates T.sub.R-restricted expression of EBI3
and IL12a. Effector T (T.sub.E) or T.sub.R cells from the spleens
and lymph nodes of C57BL/6, Foxp3.sup.gfp or EBI3.sup.-/- mice were
purified by FACS as indicated. For FIG. 2C sorted T.sub.E and
T.sub.R cells (3.times.10.sup.6 cells/lane) were cultured for 36
hours in the absence of stimuli. Cells were lysed and supernatant
collected for overnight IP with an anti-IL12a (p35) mAb, eluted
proteins resolved on an SDS-PAGE gel and blotted with anti-EBI3
mAb. Two exposure times are shown in FIG. 2C. Data are
representative of 2 independent experiments.
[0017] FIG. 2D illustrates T.sub.R-restricted expression of EBI3
and IL12a. Effector T (T.sub.E) or T.sub.R cells from the spleens
and lymph nodes of C57BL/6, Foxp3.sup.gfp or EBI3.sup.-/- mice were
purified by FACS as indicated. FIG. 2D shows relative mRNA
expression was determined from purified T.sub.E or T.sub.R cells
under indicated conditions; unstimulated, stimulated for 48 hours
with anti-CD3/CD28 or activated in culture containing both T.sub.E
and T.sub.R cells. Data shown in FIG. 2D represent the mean.+-.SEM
of 2 independent experiments (* p=0.008).
[0018] FIG. 2E illustrates T.sub.R-restricted expression of EBI3
and IL12a. Effector T (T.sub.E) or T.sub.R cells from the spleens
and lymph nodes of C57BL/6, Foxp3.sup.gfp or EBI3.sup.-/- mice were
purified by FACS as indicated. For FIG. 2E 6.5 (TCR
transgenic-hemagglutinin specific) CD4.sup.+ T.sub.E cells were
purified by MACS and activated with anti-CD3/CD28 for 2 days. T
cells were retrovirally transduced with vector alone or Foxp3.
After resting, qPCR was performed as described herein. Data shown
in FIG. 2E represent the mean.+-.SEM of 2 independent experiments
(* p=0.002, ** p=0.02).
[0019] FIG. 3A demonstrates that EBI3 and p35 (IL12a) are required
for optimal T.sub.R cell function. For FIG. 3A splenic T.sub.E
cells (2.5.times.10.sup.4) were incubated with irradiated
splenocytes as antigen-presenting cells (2.5.times.10.sup.4) and
T.sub.R cells as indicated in the presence of anti-CD3 mAb (2C11)
for 60 hours, pulsed with [.sup.3H]thymidine for 8 hours and cell
proliferation measured.
[0020] FIG. 3B demonstrates that EBI3 and p35 (IL12a) are required
for optimal T.sub.R cell function. For FIG. 3B T.sub.E and T.sub.R
cells were sorted from spleens and lymph nodes of wild-type (WT),
EBI3.sup.-/- and IL12a.sup.-/- mice. Sorted T.sub.R cells were
mixed at different ratios with antigen-presenting cells, naive
wild-type T.sub.E cells (2.5.times.10.sup.4 cells/well) and 5 .mu.M
anti-CD3. Cells were cultured for 72 hours and pulsed with
[.sup.3H]-thymidine (1 .mu.Ci/well) for the last 8 hours of
culture. Data in FIG. 3B represent mean.+-.SEM of 5 (4 for
IL12a.sup.-/- T.sub.R) independent experiments (* p=0.0002, **
p=0.008).
[0021] FIG. 3C demonstrates that EBI3 and p35 (IL12a) are required
for optimal T.sub.R cell function. For FIG. 3C wild-type T.sub.E
cells (2.times.10.sup.6) alone or with WT, EBI3.sup.-/- or
IL12a.sup.-/- T.sub.R cells (5.times.10.sup.5) were injected
intravenously into RAG1.sup.-/- mice. Seven days post-transfer the
mice were sacrificed and splenic T cell numbers determined by flow
cytometry. Data in FIG. 3C represent mean.+-.SEM of 3 independent
experiments with 8-12 mice per group (* p=0.002, ** p=0.02).
[0022] FIG. 4A illustrates that EBI3.sup.-/- T.sub.R cells fail to
treat inflammatory bowel disease (IBD). RAG1.sup.-/- mice received
CD4.sup.+CD25.sup.-CD45RB.sup.hi T.sub.E cells via the tail vein.
After 3-4 weeks, mice developed clinical symptoms of IBD and were
given a second transfer of wild-type or EBI3.sup.-/- T.sub.R cells.
FIG. 4A shows percent weight change following T.sub.R cell
transfer.
[0023] FIG. 4B illustrates that EBI3.sup.-/- T.sub.R cells fail to
treat inflammatory bowel disease (IBD). RAG1.sup.-/- mice received
CD4.sup.+CD25.sup.-CD45RB.sup.hi T.sub.E cells via the tail vein.
After 3-4 weeks, mice developed clinical symptoms of IBD and were
given a second transfer of wild-type or EBI3.sup.-/- T.sub.R cells.
FIG. 4B shows colonic histology scores of experimental mice. Data
in both panels represent mean.+-.SEM of 8-11 mice per group from 4
independent experiments (* p=0.02, ** p=0.05).
[0024] FIG. 5A demonstrates that ectopic expression of IL35 and
recombinant IL35 suppress T.sub.E cell proliferation. For FIG. 5A
naive splenic T cells were activated for 48 hours with anti-CD3 mAb
prior to transduction with EBI3, p35 (IL12a), EBI3+p35 (IL35), or
pMIG (vector control). Following transduction, cells were expanded
for 6 days, rested for 2 days and sorted for equal expression of
the constructs. The T cells were then tested for their ability (at
indicated cell numbers) to suppress proliferation of T.sub.E cells
activated with irradiated splenocytes as antigen-presenting cells
(2.5.times.10.sup.4) and T.sub.R cells as indicated in the presence
of anti-CD3 mAb. Effector T cells were allowed to proliferate for
60 hours, then were pulsed with [.sup.3H]thymidine for 8 hours and
cell proliferation measured.
[0025] FIG. 5B demonstrates that ectopic expression of IL35 and
recombinant IL35 suppress T.sub.E cell proliferation. For FIG. 5B
6.5 (TCR transgenic-HA specific) CD4.sup.+ T.sub.E cells were
purified by MACS and activated with anti-CD3/CD28 for 2 days. T
cells were retrovirally transduced, sorted and titrated into an in
vitro T.sub.R assay with antigen-presenting cells, 10 .mu.g/ml HA
110-120 peptide and naive 6.5 CD4.sup.+CD25.sup.- T.sub.E cells.
Data in FIG. 5B represent mean.+-.SEM of 3 independent
experiments.
[0026] FIG. 5C demonstrates that ectopic expression of IL35 and
recombinant IL35 suppress T.sub.E cell proliferation. For FIG. 5C
HEK293T cells were transiently transfected with empty GFP encoding
vector or vectors containing "native" or "single chain" IL35. Cells
were sorted for equivalent GFP expression and cultured for 36 hours
to facilitate protein secretion. Dialyzed, filtered supernatant
from cells was mixed at indicated ratios with anti-CD3/CD28 coated
sulfate latex beads and T.sub.E cells in a proliferation assay.
Data in FIG. 5C represent mean.+-.SEM of 4 independent
experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Compositions and methods for modulating T cell function in a
subject are provided. The compositions comprise antagonists that
are specific for IL35, or the IL35 subunits EBI3 and p35 (IL12a),
but do not recognize other cytokines or cytokine combinations
(e.g., an IL35-specific binding agent). In particular, the
antagonists of the invention do not recognize or bind IL12, IL27,
and the like. By "specific binding agent" is intended an agent that
binds substantially only to a defined target. Thus an IL35-specific
binding agent binds substantially only to a subunit (i.e., EBI3 or
p35) of the heterodimeric glycoprotein or to the heterodimer
itself, or inhibits IL35 activity. Likewise, an IL35 receptor
(IL35R)-specific binding agent binds substantially only the IL35
receptor. As IL35 shares subunits with IL12 (p35) and IL27 (EBI3),
an IL35-specific binding agent that binds substantially only to
IL35 but not to IL12 or IL27 is preferred. Specific binding agents
include, but are not limited to, antibodies, proteins that are
designed to interfere with IL35 binding, formation or activity,
proteins that compete with binding of a subunit (i.e., EBI3 or p35)
to its complement subunit, proteins that bind IL35, and small
molecules. A binding agent specifically binds if it binds only to
EBI3, p35, or IL35, or fragments and closely related variants that
share at least 80%, at least 90%, at least 95% or greater sequence
identity to EBI3, p35, or IL35.
[0028] For purposes of the present invention, percent sequence
identity is determined using the Smith-Waterman homology search
algorithm using an affine gap search with a gap open penalty of 12
and a gap extension penalty of 2, BLOSUM matrix of 62. The
Smith-Waterman homology search algorithm is taught in Smith and
Waterman (Adv. Appl. Math. 2:482-489, 1981). A variant may, for
example, differ from the reference protein by as few as 1 to 15
amino acid residues, as few as 1 to 10 amino acid residues, such as
6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid
residue.
[0029] By "proteins that compete with binding of a subunit" is
intended a protein that is designed to compete with binding of a
subunit (i.e., EBI3 or p35) to its complement subunit. In this
manner, EBI3 and p35 modified proteins can be made that are capable
of binding to the complement subunit but that result in a defective
IL35 molecule. By "modified EBI3 or p35 protein" is intended an
amino acid sequence for EBI3 or p35 that has been modified by amino
acid substitutions, deletions, additions and the like. That is, the
resulting IL35 molecule does not retain the immunoregulatory
activity. In this manner, mutations can be introduced into the
EBI35 or p35 amino acid sequences and the resulting proteins tested
for their abilities to bind their complement subunit. Such modified
proteins can be made recombinantly, by proteolytic digestion, by
chemical synthesis, etc. Internal or terminal fragments of a
polypeptide can be generated by removing one or more nucleotides
from one end or both ends of a nucleic acid which encodes the
polypeptide. Mutations can be made in the corresponding nucleic
acid sequence encoding the EBI35 or p35 polypeptide and expression
of the mutagenized DNA produces modified polypeptide fragments or
proteins.
[0030] EBI3 and p35 are known in the art. The human EBI3 gene
encodes a protein of about 33 kDa. The protein shares about 27%
sequence identity to the p40 subunit of human IL12. Nucleic acid
and amino acid sequences for EBI3 are known. See, for example, SEQ
ID NOs:1 and 2 of WO97/13859 (human) and GenBank Accession Numbers
NM015766 and BC046112 (mouse). Nucleic acid and amino acid
sequences for p35 are also known in the art and include SEQ ID
NOs:3 and 4 of WO97/13859 (human) and GenBank Accession Numbers
NM_000882 and M86672 (mouse).
[0031] Interleukin 35 refers to any intramolecular complex or
single molecule comprising at least one EBI3 polypeptide component
and at least one p35 polypeptide component. Typically, in vivo,
EBI3 and p35 associate via non-covalent association. For purposes
of the present invention, the EBI3-p35 components may be associated
with one another either covalently or non-covalently for the
purpose of raising specific antibodies. In some examples, EBI3 and
p35 can be coexpressed as a fusion protein.
[0032] By "small molecule inhibitor" is intended a molecule of a
size comparable to those molecules generally used in
pharmaceuticals. The term excludes biological macromolecules (e.g.,
proteins, nucleic acids, etc.). Preferred small organic molecules
range in size up to about 5000 Da, more preferably up to 2000 Da,
and most preferably up to about 1000 Da. Small molecule inhibitors
can disrupt protein-protein interactions between a protein (both
membrane bound and soluble) and its receptor, such as between the
IL35 heterodimer and its receptor. The preparation of small
molecule inhibitors is well known in the art. For example, although
protein-protein interactions occur over a large surface area, X-ray
crystallography and site-directed mutagenesis can be used to map
the compact, centralized regions of protein-protein interfaces,
often termed "hot spots," that are crucial for the interaction.
[0033] Non-limiting examples of small molecule inhibitors include
chemical compounds, inorganic molecules, organic molecules, organic
molecules containing an inorganic component, molecules including a
radioactive atom, synthetic molecules, and peptidomimetics (e.g.,
short, peptide fragments that mimic the most common peptide motifs,
such as an .alpha.-helix or .beta.-sheet). As a specific binding
agent, small molecule inhibitors may be more permeable to cells,
less susceptible to degradation, and less apt to elicit an
undesired immune response than large molecules.
[0034] The present invention further provides methods for
inhibiting a regulatory T cell function in a subject. T.sub.R
cells, also known as suppressor T cells, downregulate immune
responses for both foreign and self antigens. Regulatory T cells
have immunoregulatory properties and are actively involved in
maintaining immune tolerance (i.e., in preventing autoimmunity),
but also control various immune reactions (Chatila, T. A., J.
Allergy Clin. Immunol. 116:949-59, 2005; Bluestone and Tang, Curr.
Opin. Immunol. 17:638-42, 2005; and Schwartz, R. H., Nat. Immunol.
6:327-30, 2005). One class of T.sub.R cells, CD4.sup.+CD25.sup.+
suppressor T cells, is characterized by the expression of CD4 and
CD25 (the Interleukin 2 receptor .alpha.-chain). These cells are
often referred to as "natural regulatory T cells" (Bluestone and
Abbas, Nat. Rev. Immunol. 3:253-57, 2003) or "innate regulatory T
cells" (Cortez et al., Transplantation 77:S12-15, 2004), and are
produced by the thymus as a functionally distinct subpopulation of
T cells. Their development critically depends on expression of the
forkhead transcription factor Foxp3 (Hori and Sakaguchi, Microbes
Infect. 6:745-51, 2004). CD4.sup.+Foxp3.sup.+ T.sub.R cells are
powerful inhibitors of T cell activation both in vivo and in
vitro.
[0035] Other classes of regulatory T cells with diverse phenotypes
and antigen specificities have been described (Maggi et al.,
Autoimmun. Rev. 4:579-586, 2005 and Levings and Roncarolo, Curr.
Topics Micro. Immunol. 293:303-26, 2005). For example, "adaptive
regulatory T cells," which are also referred to as "acquired
regulatory T cells," are a population of antigen-induced regulatory
T cells induced in the periphery after encounter with pathogens and
foreign antigens (Cortez et al., Transplantation 77:S12-15, 2004;
Mills and McGuirk, Seminars Immunol. 16:107-17, 2004; and Vigouroux
et al., Blood 104:26-33, 2004).
[0036] By "inhibiting a regulatory T cell function in a subject" is
intended reducing and/or blocking of one or more of the suppressive
effects mediated by T.sub.R cells. While not being bound by any
theory, it is believed that T.sub.R cells mediate their suppressive
effects through both cell contact-dependent mechanisms (involving
their T cell receptors and/or other cell surface-expressed
molecules), and cytokine-dependent mechanisms (including, e.g.,
IL10 and TGF-.beta.). In one embodiment, reducing and/or blocking
of one or more of the suppressive effects mediated by T.sub.R cells
is achieved by inhibiting the activation and/or proliferation of
T.sub.R cells. The inhibition of the activation and/or
proliferation of T.sub.R cells can be measured relative to a
control population of cells, such as responder or effector T cells.
For purposes of the invention, T.sub.R cell function is reduced at
least 30%, at least 50%, at least 60%, at least 70%, at least 80%,
or at least 90% as compared to control cells, such as responder T
cells.
[0037] As used herein, "responder T cells" or "effector T cells"
refers to a subpopulation of mature T cells that facilitate an
immune response through cell activation and/or the secretion of
cytokines. In one embodiment, the responder T cells are
CD4.sup.+CD25.sup.- T cells. In another embodiment, the responder T
cells are CD8.sup.+CD25.sup.- T cells. One example of a responder T
cell is a T lymphocyte that proliferates upon stimulation by an
antigen, such as a tumor antigen. Another example of a responder T
cell is a T lymphocyte whose responsiveness to stimulation can be
suppressed by T.sub.R cells.
Production of Anti-IL35 Antibodies
[0038] As noted herein, the invention includes antibodies
specifically reactive with IL35, EBI3 or p35. Antibodies, including
monoclonal antibodies (mAbs) can be made by standard protocols.
See, for example, Harlow and Lane, Using Antibodies: A Laboratory
Manual, CSHL, New York, 1999. Briefly, a mammal such as a mouse,
hamster or rabbit can be immunized with an immunogenic form of a
peptide. Techniques for conferring immunogenicity on a protein or
peptide include conjugation to carriers or other techniques, well
known in the art. In preferred embodiments, the subject antibodies
are immunospecific for antigenic determinants of EBI3, p35, or
IL35. See, SEQ ID NOs:1-4 of WO97/13859 for the human nucleic acid
and amino acid sequences for EBI3 and p35, respectively, and
GenBank Accession Numbers NM015766, BC046112, NM_000882, and M86672
for the mouse nucleic acid and amino acid sequences for EBI3 and
p35, respectively.
[0039] The antibodies of the invention include antibodies that
specifically bind IL35, EBI3 and p35. As discussed herein, these
antibodies are collectively referred to as "anti-IL35 antibodies".
Thus, by "anti-IL35 antibodies" is intended antibodies specific for
IL35, antibodies specific for EBI3 and antibodies specific for p35.
All of these antibodies are encompassed by the discussion herein.
The respective antibodies can be used alone or in combination in
the methods of the invention.
[0040] By "antibodies that specifically bind" is intended that the
antibodies will not substantially cross react with another
polypeptide. By "not substantially cross react" is intended that
the antibody or fragment has a binding affinity for a
non-homologous protein which is less than 10%, more preferably less
than 5%, and even more preferably less than 1%, of the binding
affinity for EBI3, p35, or IL35.
[0041] The anti-IL35 antibodies disclosed herein and for use in the
methods of the present invention can be produced using any antibody
production method known to those of skill in the art. Thus,
polyclonal sera may be prepared by conventional methods. In
general, a solution containing the IL35, EBI3 or p35 antigen is
first used to immunize a suitable animal, preferably a mouse, rat,
rabbit, or goat. Rabbits or goats are preferred for the preparation
of polyclonal sera due to the volume of serum obtainable, and the
availability of labeled anti-rabbit and anti-goat antibodies.
[0042] Polyclonal sera can be prepared in a transgenic animal,
preferably a mouse bearing human immunoglobulin loci. In a
preferred embodiment, Sf9 (Spodoptera frugiperda) cells expressing
IL35, EBI3 or p35 are used as the immunogen. Immunization can also
be performed by mixing or emulsifying the antigen-containing
solution in saline, preferably in an adjuvant such as Freund's
complete adjuvant, and injecting the mixture or emulsion
parenterally (generally subcutaneously or intramuscularly). A dose
of 50-200 .mu.g/injection is typically sufficient Immunization is
generally boosted 2-6 weeks later with one or more injections of
the protein in saline, preferably using Freund's incomplete
adjuvant. One may alternatively generate antibodies by in vitro
immunization using methods known in the art, which for the purposes
of this invention is considered equivalent to in vivo immunization.
Polyclonal antisera are obtained by bleeding the immunized animal
into a glass or plastic container, incubating the blood at
25.degree. C. for one hour, followed by incubating at 4.degree. C.
for 2-18 hours. The serum is recovered by centrifugation (e.g.,
1,000.times.g for 10 minutes). About 20-50 ml per bleed may be
obtained from rabbits.
[0043] Production of the Sf9 cells is disclosed in U.S. Pat. No.
6,004,552. Briefly, sequences encoding human IL35, EBI3 or p35 are
recombined into a baculovirus using transfer vectors. The plasmids
are co-transfected with wild-type baculovirus DNA into Sf9 cells.
Recombinant baculovirus-infected Sf9 cells are identified and
clonally purified. Recombinant baculovirus-infected Sf9 cells are
identified and clonally purified.
[0044] Preferably the antibody is monoclonal in nature. By
"monoclonal antibody" is intended an antibody obtained from a
population of substantially homogeneous antibodies, that is, the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. The term is not limited regarding the
species or source of the antibody. The term encompasses whole
immunoglobulins as well as fragments such as Fab, F(ab')2, Fv, and
others which retain the antigen binding function of the antibody.
Monoclonal antibodies are highly specific, being directed against a
single antigenic site on the target polypeptide. Furthermore, in
contrast to conventional (polyclonal) antibody preparations that
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler and Milstein (Nature
256:495-97, 1975), or may be made by recombinant DNA methods (see,
e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may
also be isolated from phage antibody libraries using the techniques
described in, for example, Clackson et al. (Nature 352:624-28,
1991), Marks et al. (J. Mol. Biol. 222:581-97, 1991) and U.S. Pat.
No. 5,514,548.
[0045] By "epitope" is intended the part of an antigenic molecule
to which an antibody is produced and to which the antibody will
bind. Epitopes can comprise linear amino acid residues (i.e.,
residues within the epitope are arranged sequentially one after
another in a linear fashion), nonlinear amino acid residues
(referred to herein as "nonlinear epitopes"-these epitopes are not
arranged sequentially), or both linear and nonlinear amino acid
residues.
[0046] As discussed herein, mAbs can be prepared using the method
of Kohler and Milstein, or a modification thereof. Typically, a
mouse is immunized with a solution containing an antigen
Immunization can be performed by mixing or emulsifying the
antigen-containing solution in saline, preferably in an adjuvant
such as Freund's complete adjuvant, and injecting the mixture or
emulsion parenterally. Any method of immunization known in the art
may be used to obtain the monoclonal antibodies of the invention.
After immunization of the animal, the spleen (and optionally,
several large lymph nodes) are removed and dissociated into single
cells. The spleen cells may be screened by applying a cell
suspension to a plate or well coated with the antigen of interest.
The B cells expressing membrane bound immunoglobulin specific for
the antigen bind to the plate and are not rinsed away. Resulting B
cells, or all dissociated spleen cells, are then induced to fuse
with myeloma cells to form hybridomas, and are cultured in a
selective medium. The resulting cells are plated by serial dilution
and are assayed for the production of antibodies that specifically
bind the antigen of interest (and that do not bind to unrelated
antigens). The selected mAb-secreting hybridomas are then cultured
either in vitro (e.g., in tissue culture bottles or hollow fiber
reactors), or in vivo (as ascites in mice).
[0047] Where the anti-IL35 antibodies of the invention are to be
prepared using recombinant DNA methods, the DNA encoding the
monoclonal antibodies is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of murine antibodies). The hybridoma cells described
herein serve as a preferred source of such DNA. Once isolated, the
DNA can be placed into expression vectors, which are then
transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, to obtain the
synthesis of monoclonal antibodies in the recombinant host cells.
Review articles on recombinant expression in bacteria of DNA
encoding an antibody includes Skerra, A. (Curr. Opinion in Immunol.
5:256-62, 1993) and Phickthun, A. (Immunol. Revs. 130:151-88,
1992). Alternatively, antibody can be produced in a cell line such
as a CHO cell line, as disclosed in U.S. Pat. Nos. 5,545,403;
5,545,405 and 5,998,144. Briefly the cell line is transfected with
vectors capable of expressing a light chain and a heavy chain,
respectively. By transfecting the two proteins on separate vectors,
chimeric antibodies can be produced. Another advantage is the
correct glycosylation of the antibody.
[0048] Additionally, the term "anti-IL35 antibody" as used herein
encompasses chimeric and humanized anti-IL35 antibodies. By
"chimeric" antibodies is intended antibodies that are most
preferably derived using recombinant deoxyribonucleic acid
techniques and which comprise both human (including immunologically
"related" species, e.g., chimpanzee) and non-human components.
Thus, the constant region of the chimeric antibody is most
preferably substantially identical to the constant region of a
natural human antibody; the variable region of the chimeric
antibody is most preferably derived from a non-human source and has
the desired antigenic specificity to the IL35 antigen. The
non-human source can be any vertebrate source that can be used to
generate antibodies to a human IL35 antigen or material comprising
a human IL35 antigen. Such non-human sources include, but are not
limited to, rodents (e.g., rabbit, rat, mouse, etc.; see, e.g.,
U.S. Pat. No. 4,816,567) and non-human primates (e.g., Old World
Monkeys, Apes, etc.; see, e.g., U.S. Pat. Nos. 5,750,105 and
5,756,096). As used herein, the phrase "immunologically active"
when used in reference to chimeric/humanized anti-IL35 antibodies
means chimeric/humanized antibodies that bind human IL35.
[0049] By "humanized" is intended forms of anti-IL35 antibodies
that contain minimal sequence derived from non-human immunoglobulin
sequences. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
hypervariable region (also known as complementarity determining
region or CDR) of the recipient are replaced by residues from a
hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit, or nonhuman primate having the desired
specificity, affinity, and capacity. The phrase "complementarity
determining region" refers to amino acid sequences which together
define the binding affinity and specificity of the natural Fv
region of a native immunoglobulin binding site. See, for example,
Chothia et al. (J. Mol. Biol. 196:901-17, 1987) and Kabat et al.
(U. S. Dept. of Health and Human Services, NIH Publication No.
91-3242, 1991). The phrase "constant region" refers to the portion
of the antibody molecule that confers effector functions.
[0050] Humanization can be essentially performed following the
methods described by Jones et al. (Nature 321:522-25, 1986),
Riechmann et al. (Nature 332:323-27, 1988) and Verhoeyen et al.
(Science 239:1534-36, 1988), by substituting rodent or mutant
rodent CDRs or CDR sequences for the corresponding sequences of a
human antibody. See also U.S. Pat. Nos. 5,225,539; 5,585,089;
5,693,761; 5,693,762; and 5,859,205. In some instances, residues
within the framework regions of one or more variable regions of the
human immunoglobulin are replaced by corresponding non-human
residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761;
5,693,762; and 6,180,370). Furthermore, humanized antibodies may
comprise residues that are not found in the recipient antibody or
in the donor antibody. These modifications are made to further
refine antibody performance (e.g., to obtain desired affinity). In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
framework regions are those of a human immunoglobulin sequence. The
humanized antibody optionally also will comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin. Accordingly, such "humanized" antibodies may
include antibodies wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species.
[0051] Also encompassed by the term "anti-IL35 antibodies" are
xenogeneic or modified anti-IL35 antibodies produced in a non-human
mammalian host, more particularly a transgenic mouse, characterized
by inactivated endogenous immunoglobulin loci. In such transgenic
animals, competent endogenous genes for the expression of light and
heavy subunits of host immunoglobulins are rendered non-functional
and substituted with the analogous human immunoglobulin loci. These
transgenic animals produce human antibodies in the substantial
absence of light or heavy host immunoglobulin subunits. See, for
example, U.S. Pat. Nos. 5,877,397 and 5,939,598. Preferably, fully
human antibodies to IL35 can be obtained by immunizing transgenic
mice. One such mouse is disclosed in U.S. Pat. Nos. 6,075,181;
6,091,001; and 6,114,598.
[0052] Fragments of the anti-IL35 antibodies are suitable for use
in the methods of the invention so long as they retain the desired
affinity of the full-length antibody. Thus, a fragment of an
anti-IL35 antibody will retain the ability to bind to IL35, EBI3 or
p35. Such fragments are characterized by properties similar to the
corresponding full-length anti-IL35 antibody; that is, the
fragments will specifically bind IL35, EBI3 or p35. Such fragments
are referred to herein as "antigen-binding" fragments.
[0053] Suitable antigen-binding fragments of an antibody comprise a
portion of a full-length antibody, generally the antigen-binding or
variable region thereof. Examples of antibody fragments include,
but are not limited to, Fab, F(ab').sub.2, and Fv fragments and
single-chain antibody molecules. By "Fab" is intended a monovalent
antigen-binding fragment of an immunoglobulin that is composed of
the light chain and part of the heavy chain. By F(ab').sub.2 is
intended a bivalent antigen-binding fragment of an immunoglobulin
that contains both light chains and part of both heavy chains. By
"single-chain Fv" or "sFv" antibody fragments is intended fragments
comprising the V.sub.H and V.sub.L domains of an antibody, wherein
these domains are present in a single polypeptide chain. See, for
example, U.S. Pat. Nos. 4,946,778; 5,260,203; 5,455,030; and
5,856,456. Generally, the Fv polypeptide further comprises a
polypeptide linker between the V.sub.H and V.sub.L domains that
enables the sFv to form the desired structure for antigen binding.
For a review of sFv see Pluckthun, A. (1994) in The Pharmacology of
Monoclonal Antibodies, Vol. 113, ed. Rosenburg and Moore
(Springer-Verlag, New York), pp. 269-315.
[0054] Antibodies or antibody fragments can be isolated from
antibody phage libraries generated using the techniques described
in, for example, McCafferty et al. (Nature 348:552-54, 1990) and
U.S. Pat. No. 5,514,548. Clackson et al. (Nature 352:624-28, 1991)
and Marks et al. (J. Mol. Biol. 222:581-97, 1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology 10:779-83, 1992), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nucleic. Acids Res.
21:2265-66, 1993). Thus, these techniques are viable alternatives
to traditional monoclonal antibody hybridoma techniques for
isolation of monoclonal antibodies.
[0055] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., J. Biochem. Biophys. Methods 24:107-17, 1992 and Brennan et
al., Science 229:81-3, 1985). However, these fragments can now be
produced directly by recombinant host cells. For example, the
antibody fragments can be isolated from the antibody phage
libraries discussed above. Alternatively, Fab fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab').sub.2 fragments (Carter et al., Bio/Technology 10:163-67,
1992). According to another approach, F(ab').sub.2 fragments can be
isolated directly from recombinant host cell culture. Other
techniques for the production of antibody fragments will be
apparent to the skilled practitioner.
[0056] A representative assay to detect anti-IL35 antibodies
specific to the IL35, EBI3 or p35-antigenic epitopes identified
herein is a "competitive binding assay." Competitive binding assays
are serological assays in which unknowns are detected and
quantitated by their ability to inhibit the binding of a labeled
known ligand to its specific antibody. Antibodies employed in such
immunoassays may be labeled or unlabeled. Unlabeled antibodies may
be employed in agglutination; labeled antibodies may be employed in
a wide variety of assays, employing a wide variety of labels.
Detection of the formation of an antibody-antigen complex between
an anti-IL35 antibody and an epitope of interest can be facilitated
by attaching a detectable substance to the antibody. Suitable
detection means include the use of labels such as radionuclides,
enzymes, coenzymes, fluorescers, chemiluminescers, chromogens,
enzyme substrates or co-factors, enzyme inhibitors, prosthetic
group complexes, free radicals, particles, dyes, and the like. Such
labeled reagents may be used in a variety of well-known assays,
such as radioimmunoassays, enzyme immunoassays, e.g., ELISA,
fluorescent immunoassays, and the like. See, for example, U.S. Pat.
Nos. 3,766,162; 3,791,932; 3,817,837; and 4,233,402.
Small Molecule Screening
[0057] The likelihood of an assay identifying an agent that acts as
an IL35 small molecule inhibitor is increased when the number and
types of test agents used in the screening system is increased.
Recently, attention has focused on the use of combinatorial
chemical libraries to assist in the generation of new small
molecule inhibitor leads. A combinatorial chemical library is a
collection of diverse chemical compounds generated by either
chemical synthesis or biological synthesis by combining a number of
chemical "building blocks." For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks (see, e.g., Gallop et al., 37:1233-50, 1994).
[0058] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, for example, peptide
libraries (see, e.g., U.S. Pat. No. 5,010,175). Peptide synthesis
is by no means the only approach envisioned and intended for use
with the present invention. Other chemistries for generating
chemical diversity libraries can also be used. Such chemistries
include: peptoids (see, e.g., WO 91/19735), encoded peptides (see,
e.g., WO 93/20242), random bio-oligomers (see, e.g., WO 92/00091),
benzodiazepines (see, e.g., U.S. Pat. No. 5,288,514), diversomers
such as hydantoins, benzodiazepines and dipeptides (see, e.g.,
Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-13, 1993),
vinylogous polypeptides (see, e.g., Hagihara et al., J. Amer. Chem.
Soc. 114:6568-70, 1992), nonpeptidal peptidomimetics with a
.beta.-D-Glucose scaffolding (see, e.g., Hirschmann et al., J.
Amer. Chem. Soc. 114:9217-18, 1992), analogous organic syntheses of
small compound libraries (see, e.g., Chen et al., J. Amer. Chem.
Soc. 116:2661-62, 1994), oligocarbamates (see, e.g., Cho et al.,
Science 261:1303-05, 1993), and peptidyl phosphonates (see, e.g.,
Campbell et al., J. Org. Chem. 59:658-60, 1994). In addition, a
number of combinatorial libraries are commercially available, as is
well known to one of skill in the art.
[0059] High throughput techniques are used when screening any of
the various libraries described herein. As is well known to one of
skill in the art, a number of high throughput screening systems are
commercially available (e.g., Zymark Corp., Hopkinton, Mass.; Air
Technical Industries, Mentor, OH; Beckman Instruments, Inc.,
Fullerton, Calif.; and Precision Systems, Inc., Natick, Mass.).
These systems typically automate entire procedures including all
sample and reagent pipetting, liquid dispensing, timed incubations,
and final readings of the microplate in detector(s) appropriate for
the assay. These configurable systems provide high throughput and
rapid start up as well as a high degree of flexibility and
customization.
Methods of Therapy Using the Compositions of the Invention
[0060] As disclosed herein, methods of the invention are directed
to the use of specific binding agents for inhibiting T.sub.R cell
function. Thus, the compositions are useful for inhibiting T cell
function in a subject. EBI3 and p35 pair to form a novel cytokine,
IL35, with immunosuppressive activity. Interleukin 35 is secreted
by CD4.sup.+Foxp3.sup.+ T.sub.R cells, and may be secreted by other
cells (such as subpopulations of CD8.sup.+ T cells, .gamma..delta.
T cells and NK T cells that have regulatory function). It exhibits
immunoregulatory activity and is required for maximal T.sub.R cell
function. The ability to specifically inhibit IL35 can be used to
reduce or block regulatory T cell function. Inhibition may be by
antibodies, modified proteins or small molecules that specifically
block binding to its receptor or disrupt IL35 chain pairing. As
IL35 shares homology in some regions with IL12 and IL27, the
inhibitory molecules of the invention (i.e., IL35-specific
antagonists or IL35-specific binding agents) are designed to
recognize and interact with IL35 or its subunits but not IL12 or
IL27.
[0061] The compositions find use in boosting the efficacy of
vaccines. Since, T.sub.R cells are involved in the induction of
tumor antigen tolerance (Mapara and Sykes, J. Clin. Oncology
22:1136-51, 2004), the compositions are useful for increasing the
efficacy of anti-cancer vaccines. Reducing T.sub.R cell function
can also be beneficial for vaccines that are poorly immunogenic;
therefore, the compositions can be used with any vaccine including
vaccines for diphtheria, tetanus, pertussis, polio, measles, mumps,
rubella, hepatitis B, Haemophilus influenzae type b, varicella,
meningitis, human immunodeficiency virus, tuberculosis, Epstein
Barr virus, malaria, hepatitis E, dengue, rotavirus, herpes, human
papillomavirus, and cancers
[0062] In one embodiment, inhibition of a T.sub.R cell function in
a subject includes administering to the subject a therapeutically
effective amount of an IL35-specific binding agent. Administration
can begin whenever inhibition of a T.sub.R cell function in a
subject is desired, for example to prevent or overcome induction of
tumor antigen tolerance by T.sub.R cells in a subject.
[0063] As used herein, "a therapeutically effective amount" of an
IL35-specific binding agent is an amount which, when administered
to a subject, is sufficient to achieve a desired effect, such as
inhibiting a T.sub.R cell function, in a subject being treated with
that composition. For example, this can be the amount of an
IL35-specific binding agent useful in preventing or overcoming
induction of tumor antigen tolerance by T.sub.R cells in a subject,
or the amount required to enhance the efficacy of a vaccine (e.g.,
a cancer vaccine) in a subject. Ideally, a therapeutically
effective amount of an IL35-specific binding agent is an amount
sufficient to prevent or overcome induction of tumor antigen
tolerance by T.sub.R cells in a subject, or the amount required to
enhance the efficacy of a vaccine (e.g., a cancer vaccine) in a
subject, without causing a substantial cytotoxic effect in the
subject. The effective amount of an IL35-specific binding agent
useful for preventing or overcoming induction of tumor antigen
tolerance by T.sub.R cells in a subject and/or enhancing the
efficacy of a vaccine (e.g., a cancer vaccine) will depend on the
subject being treated, the severity of the affliction, and the
manner of administration of the IL35-specific binding agent.
[0064] In some embodiments a "therapeutically effective amount" or
"effective amount" (for non-topical administration, such as oral
administration, or intravenous or intraperitoneal injection) of a
pharmaceutical composition containing an IL35-specific binding
agent is from about 0.1 to about 200 mg/kg body weight in single or
divided doses; for example from about 1 to about 100 mg/kg, from
about 2 to about 50 mg/kg, from about 3 to about 25 mg/kg, or from
about 5 to about 10 mg/kg. Acceptable dosages of the IL35-specific
binding agent are, for example, dosages that achieve a target
tissue concentration similar to that which produces the desired
effect in vitro. Alternatively, therapeutically effective amounts
of an IL35-specific binding agent can be determined by animal
studies. When animal assays are used, a dosage is administered to
provide a target tissue concentration similar to that which has
been shown to be effective in the animal assays. It is recognized
that the method of treatment may comprise a single administration
of a therapeutically effective amount or multiple administrations
of a therapeutically effective amount of the IL35-specific binding
agents of the invention.
[0065] Any delivery system or treatment regimen that effectively
achieves the desired effect of inhibiting a T.sub.R cell function
can be used. Accordingly, pharmaceutical compositions including an
IL35-specific binding agent (such as an antibody and/or a small
molecule inhibitor) are also described herein. The IL35-specific
binding agent is present in the composition in a therapeutically
effective amount.
[0066] Formulations for pharmaceutical compositions are well known
in the art. For example, Remington's Pharmaceutical Sciences
(18.sup.th ed.; Mack Publishing Company, Eaton, Pa., 1990),
describes compositions and formulations suitable for pharmaceutical
delivery of one or more IL35-specific binding agents, such as one
or more anti-IL35 antibodies and/or small molecule inhibitors
combined with various pharmaceutically acceptable additives, as
well as a dispersion base or vehicle. Desired additives include,
but are not limited to, pH control agents, such as arginine, sodium
hydroxide, glycine, hydrochloric acid, citric acid, and the like.
In addition, local anesthetics (e.g., benzyl alcohol), isotonizing
agents (e.g., sodium chloride, mannitol, sorbitol), adsorption
inhibitors (e.g., Tween 80), solubility enhancing agents (e.g.,
cyclodextrins and derivatives thereof), stabilizers (e.g., serum
albumin), reducing agents (e.g., glutathione), and preservatives
(e.g., antimicrobials, and antioxidants) can be included.
[0067] Therapeutically effective amounts of an IL35-specific
binding agent, such as an antibody and/or a small molecule
inhibitor, for use in the present invention can be administered by
any route, including parenteral administration, for example,
intravenous, intraperitoneal, intramuscular, intraperitoneal,
intrasternal, or intraarticular injection, or infusion, or by
sublingual, oral, topical, intranasal, or transmucosal
administration, or by pulmonary inhalation. The pharmaceutical
compositions of the present invention can be administered at about
the same dose throughout a treatment period, in an escalating dose
regimen, or in a loading-dose regime (for example, in which the
loading dose is about two to five times the maintenance dose). In
some embodiments, the dose is varied during the course of a
treatment based on the condition of the subject being treated, the
apparent response to the therapy, and/or other factors as judged by
one of ordinary skill in the art. In some embodiments long-term
treatment with a disclosed pharmaceutical composition is
contemplated.
[0068] In a specific embodiment, it may be desirable to administer
a therapeutically effective amount of an IL35-specific binding
agent, such as an antibody and/or a small molecule inhibitor,
locally to an area in need of treatment (e.g., to an area of the
body where inhibiting a T.sub.R cell function is desired). This can
be achieved by, for example, local or regional infusion or
perfusion during surgery, topical application, injection, catheter,
suppository, or implant (for example, implants formed from porous,
non-porous, or gelatinous materials, including membranes, such as
sialastic membranes or fibers), and the like. In one embodiment,
administration can be by direct injection at the site (or former
site) of a cancer that is to be treated. In another embodiment, the
therapeutically effective amount of an IL35-specific binding agent
is delivered in a vesicle, such as liposomes (see, e.g., Langer,
Science 249:1527-33, 1990 and Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez Berestein and
Fidler (eds.), Liss, N.Y., pp. 353-65, 1989).
[0069] In yet another embodiment, the therapeutically effective
amount of an IL35-specific binding agent, such as an antibody
and/or a small molecule inhibitor, can be delivered in a controlled
release system. In one example, a pump can be used (see, e.g.,
Langer, Science 249:1527-33, 1990; Sefton, Crit. Rev. Biomed. Eng.
14:201-40, 1987; Buchwald et al., Surgery 88:507-16, 1980; Saudek
et al., N. Engl. J. Med. 321:574-79, 1989). In another example,
polymeric materials can be used (see, e.g., Levy et al., Science
228:190-92, 1985; During et al., Ann. Neurol. 25:351-56, 1989;
Howard et al., J. Neurosurg. 71:105-12, 1989). Other controlled
release systems, such as those discussed by Langer (Science
249:1527-33, 1990), can also be used.
[0070] Also provided by the present invention are methods for
enhancing the efficacy or immunogenicity of a vaccine in a subject,
or overcoming a suppressed immune response to a vaccine in a
subject, including (i) administering to the subject a
therapeutically effective amount of an IL35-specific binding agent
and (ii) administering to the subject a vaccine. In one embodiment,
the vaccine is a cancer vaccine. In a specific example, the method
further includes administering to the subject at least one
additional therapeutic agent, such as a cytokine, a glucocorticoid,
an anthracycline (e.g., doxorubicin or epirubicin), a
fluoroquinolone (e.g., ciprofloxacin), an antifolate (e.g.,
methotrexate), an antimetabolite (e.g., fluorouracil), a
topoisomerase inhibitor (e.g., camptothecin, irinotecan or
etoposide), an alkylating agent (e.g., cyclophosphamide,
ifosfamide, mitolactol, or melphalan), an antiandrogen (e.g.,
flutamide), an antiestrogen (e.g., tamoxifen), a platinum compound
(e.g., cisplatin), a vinca alkaloid (e.g., vinorelbine, vinblastine
or vindesine), or mitotic inhibitor (e.g., paclitaxel or
docetaxel). In some embodiments of the present invention, the
amount of the vaccine (and/or the additional therapeutic agent)
administered to the subject in the presence of the IL35-specific
binding agent is lower than when the vaccine (and/or the additional
therapeutic agent) is administered alone.
[0071] By "vaccine" is intended a composition useful for
stimulating a specific immune response (or immunogenic response) in
a subject. In some embodiments, the immunogenic response is
protective or provides protective immunity. For example, in the
case of a disease-causing organism the vaccine enables the subject
to better resist infection with or disease progression from the
organism against which the vaccine is directed. Alternatively, in
the case of a cancer, the vaccine strengthens the subject's natural
defenses against cancers that have already developed. These types
of vaccines may also prevent the further growth of existing
cancers, prevent the recurrence of treated cancers, and/or
eliminate cancer cells not killed by prior treatments. Without
being bound by theory, it is believed that an immunogenic response
arises from the generation of neutralizing antibodies, T helper
cells, or cytotoxic cells of the immune system, or all of the
above.
[0072] By "enhancing the efficacy" or "enhancing the
immunogenicity" with regard to a vaccine is intended improving an
outcome, for example, as measured by a change in a specific value,
such as an increase or a decrease in a particular parameter of an
activity of a vaccine associated with protective immunity. In one
embodiment, enhancement refers to at least a 25%, 50%, 100% or
greater than 100% increase in a particular parameter. In another
embodiment, enhancement refers to at least a 25%, 50%, 100% or
greater than 100% decrease in a particular parameter. In one
example, enhancement of the efficacy/immunogenicity of a vaccine
refers to an increase in the ability of the vaccine to inhibit or
treat disease progression, such as at least a 25%, 50%, 100%, or
greater than 100% increase in the effectiveness of the vaccine for
that purpose. In a further example, enhancement of the
efficacy/immunogenicity of a vaccine refers to an increase in the
ability of the vaccine to recruit the subject's natural defenses
against cancers that have already developed, such as at least a
25%, 50%, 100%, or greater than 100% increase in the effectiveness
of the vaccine for that purpose.
[0073] Similarly, by "overcoming a suppressed immune response" with
regard to a vaccine is intended improving an outcome, for example,
as measured by a change in a specific value, such as a return to a
formerly positive value in a particular parameter of an activity of
a vaccine associated with protective immunity. In one embodiment,
overcoming refers to at least a 25%, 50%, 100% or greater than 100%
increase in a particular parameter. In one example, overcoming a
suppressed immune response to a vaccine refers to a renewed ability
of the vaccine to inhibit or treat disease progression, such as at
least a 25%, 50%, 100%, or greater than 100% renewal in the
effectiveness of the vaccine for that purpose. In a further
example, overcoming a suppressed immune response to a vaccine
refers to a renewed ability of the vaccine to recruit the subject's
natural defenses against cancers that have already developed, such
as at least a 25%, 50%, 100%, or greater than 100% renewal in the
effectiveness of the vaccine for that purpose.
[0074] As disclosed herein, the present invention provides methods
for enhancing the efficacy or immunogenicity of a vaccine in a
subject, or overcoming a suppressed immune response to a vaccine in
a subject. Representative vaccines include, but are not limited to,
vaccines against diphtheria, tetanus, pertussis, polio, measles,
mumps, rubella, hepatitis B, Haemophilus influenzae type b,
varicella, meningitis, human immunodeficiency virus, tuberculosis,
Epstein Barr virus, malaria, hepatitis E, dengue, rotavirus,
herpes, human papillomavirus, and cancers.
[0075] Vaccines of interest include the two vaccines that have been
licensed by the U.S. Food and Drug Administration to prevent virus
infections that can lead to cancer: the hepatitis B vaccine, which
prevents infection with the hepatitis B virus, an infectious agent
associated with liver cancer (MMWR Morb. Mortal. Wkly. Rep.
46:107-09, 1997); and Gardasil.TM., which prevents infection with
the two types of human papillomavirus that together cause 70
percent of cervical cancer cases worldwide (Speck and Tyring, Skin
Therapy Lett. 11:1-3, 2006). Other treatment vaccines of interest
include therapeutic vaccines for the treatment of cervical cancer,
follicular B cell non-Hodgkin's lymphoma, kidney cancer, cutaneous
melanoma, ocular melanoma, prostate cancer, and multiple
myeloma.
[0076] The compositions of the invention can be coordinated with
treatment with other cancer therapies besides vaccines including
chemotherapy, anti-cancer antibody therapy, small molecule-based
cancer therapy, and vaccine/immunotherapy-based cancer therapy, and
combinations thereof. The compositions of the invention are
generally used prior to treatment with a vaccine; however, they can
be used either prior to, during, or after treatment of the subject
with the other cancer therapy or, in the case of multiple
combination therapies, either prior to, during, or after treatment
of the subject with the other cancer therapies.
[0077] As will be understood by one of skill in the art, the
methods disclosed herein for enhancing the efficacy or
immunogenicity of a cancer vaccine in a subject will be relevant
for various types of cancer vaccines, including, but not limited
to, antigen/adjuvant vaccines (i.e., one or more cancer cell
antigens combined with an adjuvant), whole cell tumor vaccines
(either autologous or allogenic), dendritic cell vaccines (i.e.,
isolated dendritic cells that are stimulated with the subject's own
cancer antigens and re-injected into the subject), and viral
vectors and DNA vaccines (which use the nucleic acid sequence of a
tumor antigen to produce a cancer antigen protein).
[0078] The immunosuppressive effects of T.sub.R cells (as well as
the inhibition those effects) can be evaluated using many methods
well known in the art. In one embodiment, a white blood cell count
(WBC) is used to determine the responsiveness of a subject's immune
system. A WBC measures the number of white blood cells in a
subject. Using methods well known in the art, the white blood cells
in a subject's blood sample are separated from other blood cells
and counted. Normal values of white blood cells are about 4,500 to
about 10,000 white blood cells/.mu.l. Lower numbers of white blood
cells can be indicative of a state of immunosuppression in the
subject. In another embodiment, immunosuppression in a subject can
be determined by way of a T lymphocyte count. T lymphocytes are
differentiated from other white blood cells using standard methods
in the art, such as, for example, immunofluorescence or
fluorescence activated cell sorting (FACS). Reduced numbers of T
cells, or a specific population of T cells (for example, CD8.sup.+
T cells) can be used as a measurement of immunosuppression. A
reduction in the number of T cells, or in a specific population of
T cells, compared to the number of T cells (or the number of cells
in the specific population) prior to a specific event can be used
to indicate that immunosuppression has been induced.
[0079] Methods for the isolation and quantitation of T.sub.R cells,
such as CD4.sup.+Foxp3.sup.+ T.sub.R cells, and other populations
of T cells (e.g., CD8.sup.+ cells), are well known in the art.
Typically, labeled antibodies specifically directed to one or more
cell surface markers are used to identify and quantify the T-cell
population. The antibodies can be conjugated to other compounds
including, but not limited to, enzymes, magnetic beads, colloidal
magnetic beads, haptens, fluorochromes, metal compounds,
radioactive compounds or drugs. The enzymes that can be conjugated
to the antibodies include, but are not limited to, alkaline
phosphatase, peroxidase, urease, and .crclbar.-galactosidase. The
fluorochromes that can be conjugated to the antibodies include, but
are not limited to, fluorescein isothiocyanate (FITC),
tetramethylrhodamine isothiocyanate, phycoerythrin (PE),
allophycocyanins, and Texas Red. For additional fluorochromes that
can be conjugated to antibodies see Haugland, R. P., Handbook of
Fluorescent Probes and Research Products, published by Molecular
Probes, 9.sup.th Edition (2002). The metal compounds that can be
conjugated to the antibodies include, but are not limited to,
ferritin, colloidal gold, and particularly, colloidal
superparamagnetic beads. The haptens that can be conjugated to the
antibodies include, but are not limited to, biotin, digoxigenin,
oxazalone, and nitrophenol. The radioactive compounds that can be
conjugated or incorporated into the antibodies are known to the
art, and include, but are not limited to, technetium 99
(.sup.99Tc), .sup.125I, and amino acids comprising any
radionuclides, including, but not limited to, .sup.14C, .sup.3H and
.sup.35S.
[0080] Fluorescence activated cell sorting can be used to sort
cells that are CD4.sup.+, CD25.sup.+, both CD4.sup.+ and
CD25.sup.+, or CD8.sup.+ by contacting the cells with an
appropriately labeled antibody. However, other techniques of
differing efficacy may be employed to purify and isolate desired
populations of cells. The separation techniques employed should
maximize the retention of viability of the fraction of the cells to
be collected. The particular technique employed will, of course,
depend upon the efficiency of separation, cytotoxicity of the
method, the ease and speed of separation, and what equipment and/or
technical skill is required.
[0081] Additional separation procedures may include magnetic
separation, using antibody-coated magnetic beads, affinity
chromatography, cytotoxic agents, either joined to a monoclonal
antibody or used in conjunction with complement, and "panning,"
which utilizes a monoclonal antibody attached to a solid matrix, or
another convenient technique. Antibodies attached to magnetic beads
and other solid matrices, such as agarose beads, polystyrene beads,
hollow fiber membranes and plastic Petri dishes, allow for direct
separation. Cells that are bound by the antibody can be removed
from the cell suspension by simply physically separating the solid
support from the cell suspension. The exact conditions and duration
of incubation of the cells with the solid phase-linked antibodies
will depend upon several factors specific to the system employed.
The selection of appropriate conditions, however, is well known in
the art.
[0082] Unbound cells then can be eluted or washed away with
physiologic buffer after sufficient time has been allowed for the
cells expressing a marker of interest (e.g., CD4 and/or CD25) to
bind to the solid-phase linked antibodies. The bound cells are then
separated from the solid phase by any appropriate method, depending
mainly upon the nature of the solid phase and the antibody
employed, and quantified using methods well known in the art. In
one example, bound cells separated from the solid phase are
quantified by FACS. Antibodies may be conjugated to biotin, which
then can be removed with avidin or streptavidin bound to a support,
or fluorochromes, which can be used with FACS to enable cell
separation and quantitation, as known in the art.
[0083] As used herein, the singular terms "a," "an," and "the"
include plural referents unless context clearly indicates
otherwise. Similarly, the word "or" is intended to include "and"
unless the context clearly indicates otherwise. It is further to be
understood that all base sizes or amino acid sizes, and all
molecular weight or molecular mass values, given for nucleic acids
or polypeptides are approximate, and are provided for description.
The subject matter of the present disclosure is further illustrated
by the following non-limiting examples.
EXPERIMENTAL
Example 1
Isolation of Interleukin 35 a Regulatory T Cell-Specific
Cytokine
[0084] An Affymetrix gene analysis was performed for the purpose of
identifying genes that are preferentially upregulated in or on
T.sub.R cells. This analysis identified EBI3 as one of those genes.
To verify the gene analysis data, quantitative real-time PCR (qPCR)
was used to measure EBI3 mRNA expression. PCR results confirmed the
upregulation of EBI3 expression in T.sub.R cells versus T.sub.E
cells (FIG. 1). Confirmation of T.sub.R-restricted expression of
EBI3 was obtained by additional qPCR analysis of peripheral
CD4.sup.+CD45RB.sup.lo CD25.sup.+ T.sub.R cells versus naive
CD4.sup.+CD45RB.sup.hiCD25.sup.- T.sub.E cells (the standard
phenotypic definition for T.sub.R and T.sub.E cells) purified from
C57BL/6 mice, and Foxp3.sup.+ T.sub.R cells versus Foxp3.sup.-
T.sub.E cells sorted from Foxp3.sup.gfp knockin mice (Fontenot et
al., Immunity 22:329-41, 2005), which express a GFP-Foxp3 chimeric
protein (FIG. 2A). Literature suggests that neither .alpha. or
.beta. chains will be secreted alone, but rather, need to pair
within the cell to be secreted. Interleukin 27 is a heterodimer of
EBI3 and p28, whereas p40 can pair with p19 to yield IL23, or with
p35 to yield IL12. Therefore, the expression of p40, EBI3, p35
(IL12a), p28, and p19 in T.sub.E cells and T.sub.R cells was
measured via qPCR to determine putative binding partners for EBI3
in T.sub.R cells. PCR results demonstrated that p35 was the only
IL12 family .alpha. chain expressed in T.sub.R cells (FIG. 2B). See
also, Devergne et al., Proc. Natl. Acad. Sci. USA 94:12041-46,
1997.
[0085] The expression of intracellular EBI3 was assessed by flow
cytometry in resting T.sub.R cells. Using three different
EBI3-specific mAbs, resting wild-type T.sub.R cells, but not
wild-type T.sub.E or EBI3.sup.-/- T.sub.R cells, were shown to
express intracellular EBI3. Finally, immunoblot analysis clearly
revealed the coimmunoprecipitation of EBI3 with IL12a in
supernatants from resting T.sub.R, but not T.sub.E cells or
EBI3.sup.-/- T.sub.R cells (FIG. 2C). Taken together, these data
demonstrate the preferential secretion of a novel EBI3/IL12a
heterodimeric cytokine by T.sub.R cells amongst CD4.sup.+ T cell
populations.
[0086] Given that T.sub.R cells require activation through their
TCR in order to exert their suppressive activity (Thornton et al.,
J. Exp. Med. 188:287-96, 1998; Thornton et al., J. Immunol.
164:183-90, 2000; Takahashi et al., Int. Immunol. 10:1969-80,
1998), an assessment of how EBI3 and IL12a mRNA levels were altered
following T.sub.R cell activation in the absence or presence of
T.sub.E cells was made. Both EBI3 and IL12a mRNA were significantly
reduced following anti-CD3 stimulation, but dramatically
upregulated (234- and 740-fold, respectively) in T.sub.R cells
recovered from an in vitro T.sub.R assay, and thus in the process
of active suppression (FIG. 2D). Indeed, the increase in IL12a mRNA
far exceeded that observed in activated macrophages. These data
demonstrate that a novel EBI3/IL12a heterodimeric cytokine is
produced by T.sub.R cells, which is potentiated during active
suppression of T.sub.E cells.
[0087] The discrete, differential expression of EBI3 in T.sub.R
versus T.sub.E cells suggests that its expression may be controlled
by transcriptional processes that regulate T.sub.R development and
function. Indeed, EBI3 expression was concordant with Foxp3, which
is required for T.sub.R development (Zheng et al., Nat. Immunol.
8:457-62, 2007). EBI3 mRNA was present in CD4.sup.+Foxp3.sup.+
thymocytes but essentially absent in CD4.sup.+CD8.sup.+ and
CD4.sup.+Foxp3.sup.- thymocytes. To determine if EBI3 is a
downstream target of Foxp3, purified T.sub.E cells were transduced
with retroviral vectors encoding Foxp3 plus GFP or GFP alone.
Foxp3-transduced T.sub.E cells exhibited considerably elevated EBI3
transcript levels compared with the GFP alone controls, while Foxp3
induced limited expression of IL12a mRNA (FIG. 2E). These data
provide a mechanistic basis for the restricted secretion of the
EBI3/IL12a heterodimer by T.sub.R cells, with EBI3 being a
downstream target of Foxp3.
Example 2
Interleukin 35 is Required for Optimal T.sub.R Cell Function
[0088] Neither EBI3.sup.-/- nor IL12a.sup.-/- mice have any overt
autoimmunity or inflammatory disease (Boirivant et al., J. Exp.
Med. 188:1929-39, 1998; Mattner et al., Eur. J. Immunol.
26:1553-59, 1996). Indeed, the percentage of T.sub.R cells in these
mice and their Foxp3 expression is comparable to wild-type mice.
This raises the possibility that the consequence of lacking a
negative regulatory EBI3/IL12a cytokine may be negated by the lack
of the proinflammatory cytokines IL27 and IL12 in the EBI3.sup.-/-
and IL12a.sup.-/- mice, respectively. Indeed, when challenged,
EBI3.sup.-/- mice are more susceptible to leishmaniasis (Zahn et
al., Eur. J. Immunol. 35:1106-12, 2005). Likewise, IL12a.sup.-/-,
distinct from IL12b.sup.-/- (p40) mice, are more susceptible to
Helicobacter-induced colitis (Kullberg et al., J. Exp. Med.
203:2485-94, 2006), Leishmania major infection (Mattner et al.,
Eur. J. Immunol. 26:1553-59, 1996), experimental autoimmune
encephalomyelitis (Gran et al., J. Immunol. 169:7104-10, 2002;
Becher et al., J. Clin. Invest. 110:493-97, 2002), and
collagen-induced arthritis (Murphy et al., J. Exp. Med.
198:1951-57, 2003).
[0089] To determine whether the loss of EBI3 or IL12a expression
would have functional implications for T.sub.R cells, T.sub.E cells
and T.sub.R cells were isolated from wild-type, EBI3.sup.-/- and
IL12a.sup.-/- mice (Boirivant et al., J. Exp. Med. 188:1929-39,
1998; Mattner et al., Eur. J. Immunol. 26:1553-59, 1996). An in
vitro T.sub.R cell assay was performed to determine whether T.sub.R
cells lacking EBI3 or IL12a could suppress T.sub.E cell
proliferation. Wild-type T.sub.R cells could suppress proliferation
of T.sub.E cells in a dose dependent manner. In contrast, both
EBI3.sup.-/- and IL12a.sup.-/- T.sub.R cells were less capable of
suppressing T.sub.E cell proliferation, showing that EBI3 and IL12a
are required for optimal T.sub.R cell function (FIGS. 3A &
3B).
[0090] To determine EBI3.sup.-/- and IL12a.sup.-/- T.sub.R cell
function in vivo, their ability to control the homeostatic
expansion of T.sub.E cells was evaluated. In vivo, T.sub.R cells
have been shown to control the homeostatic expansion of T.sub.E
cells in a lymphopenic, RAG1.sup.-/- environment (Annacker et al.,
Immunol. Rev. 182:5-17, 2001; Annacker et al., J. Immunol.
164:3573-80, 2000; Workman et al., J. Immunol. 174:688-95, 2004).
Therefore, to determine whether the expression of EBI3 and IL12a
influenced the ability of T.sub.R cells to control homeostatic
expansion, purified wild-type T.sub.E cells either alone, or in the
presence of wild-type, EBI3.sup.-/- or IL12a.sup.-/- T.sub.R cells,
were adoptively transferred into RAG1.sup.-/- mice. As RAG1.sup.-/-
mice lack T and B cells, expansion of adoptively transferred T
cells represent the only T cell population present in these mice.
Splenic T cell numbers were determined 7-10 days post-transfer. In
the presence of wild-type T.sub.R cells, T.sub.E cell expansion was
significantly reduced, while minimal reduction in wild-type T.sub.E
cell expansion was observed in the presence of either EBI3.sup.-/-
or IL12a.sup.-/- T.sub.R cells (FIG. 3C).
[0091] T.sub.R cells have also been shown to control colitis in
mice, resembling IBD, that is initiated experimentally by
transferring naive T cells into RAG1.sup.-/- recipients (Izcue et
al., Immunol. Rev. 212:256-71, 2006). In these experiments,
severity of disease is monitored clinically, by weight loss, and
histologically, utilizing a semi-quantitative grading scheme to
score pathology. Recovery from disease, marked by weight gain and
decreased histopathology, is observed only in mice that receive
purified T.sub.R cells approximately four weeks after the initial
T.sub.E cell transfer (Mottet et al., J. Immunol. 170:3939-43,
2003).
[0092] This recovery model of IBD was chosen to test the
functionality of EBI3.sup.-/- and IL12a.sup.-/- T.sub.R cells in
vivo. After wild-type T.sub.E-recipient RAG1.sup.-/- mice developed
clinical symptoms of IBD (approximately 4 weeks), they received
wild-type, EBI3.sup.-/- or IL12a.sup.-/- T.sub.R cells and were
monitored daily. Wild-type T.sub.R-recipient mice were noticeably
healthier within 5-7 days, had restored appetite, and resumed
weight gain (FIG. 4A). However, EBI3.sup.-/- and IL12a.sup.-/-
T.sub.R recipients continued to lose weight, with some mice dying
within the first 10 days post-T.sub.R cell transfer. After 4 weeks
(8 weeks after initial T.sub.E cell transfer), histological
analysis was performed to assess the extent of recovery. Severe IBD
pathology including loss of goblet cells and mucus secretion,
mucosal hyperplasia, extensive ulceration, marked transmural
lymphohistiocytic inflammation, extensive infiltration of CD3.sup.+
T cells, and effacement of the normal architecture by the
inflammatory infiltrate was observed in the non-T.sub.R recipients.
In wild-type T.sub.R recipients, there was substantial reduction of
the mean pathology score, significantly reduced inflammation,
reduced CD3.sup.+ T cell infiltration, and regeneration of goblet
cells, and mucus secretion. In contrast, EBI3.sup.-1 and
IL12a.sup.-/- T.sub.R recipients had only an approximately 50%
reduction in the pathology score, as defined by goblet cell
destruction, mucosal hyperplasia and cellular infiltration (FIG.
4B). Thus, the slightly improved histological score was
insufficient to mediate weight gain and recovery from colitis.
Similarly, EBI3.sup.-/- and IL12a.sup.-/- T.sub.R were unable to
reduce colitis and weight loss to the same extent as wild type
T.sub.R cells in a traditional co-transfer model of IBD. These
results demonstrate that EBI3 and IL12a are required by T.sub.R
cells for maximal regulatory activity in vitro and in vivo.
Example 3
Both EBI3 and IL12a are Required for the Generation of Interleukin
35
[0093] Several studies have shown that ectopic expression of Foxp3
or the regulatory protein LAG-3 can confer regulatory activity on
naive T.sub.E cells (Hori and Sakaguchi, Science 299:1057-61, 2003;
Fontenot et al., Nat. Immunol. 4:330-36, 2003; Huang et al.,
Immunity 21:503-13, 2004). As the qPCR data indicated that
EBI3+IL12a is a functional heterodimer important to T.sub.R cell
function, EBI3+IL12a was ectopically expressed to see if its
expression could confer regulatory activity to non-regulatory T
cells. Naive T.sub.E cells from hemagglutinin-specific clone 6.5
TCR transgenic mice were transduced with EBI3, IL12a, EBI3+IL12a,
or vector alone to assess the impact of expressing these proteins
on cellular function. With ectopic expression of EBI3+IL12a, but
not with either protein alone, transduced T.sub.E cells gained
T.sub.R cell function as measured by their ability to inhibit
proliferation of naive T cells (FIG. 5A). Recombinant IL35 derived
from 3T3 cells or 293T cells also inhibited T cell proliferation.
The observation that non-regulatory T cells gain regulatory
activity by the expression of EBI3+IL12a, but not independently,
demonstrates that both EBI3 and IL12a are required for the
generation of this regulatory cytokine.
[0094] Purified T.sub.E cells from the clone 6.5 TCR transgenic
mice were also transduced with retroviral vectors encoding the
expression of GFP alone, or GFP plus either Foxp3, EBI3, IL12a or
"native" IL35 (i.e., EBI3-2A-IL12a-stoichiometric, bicistronic
expression of EBI3 and IL12a in a single vector; Szymczak-Workman
et al. in Gene Transfer: Delivery and Expression, Friedmann and
Rossi (eds.), Cold Spring Harbor Laboratory Press, N.Y., pp.
137-47, 2006; Szymczak and Vignali, Exp. Opin. Biol. Ther.
5:627-38, 2005; Hoist et al., Nature Methods 3:191-97, 2006). T
cell transductants were sorted for GFP equivalency and co-cultured
with naive, wild-type T.sub.E cells in an antigen-driven
proliferation assay to determine if these proteins bestowed
regulatory potential. The results confirmed that T cells expressing
IL35, but not either chain alone, suppressed T.sub.E cell
proliferation in a titratable fashion, to a level that was
approximately two thirds of the regulatory activity observed with
the Foxp3-transduced T cells (FIG. 5B).
[0095] Given that IL35 is secreted by T.sub.R cells and forced
expression confers regulatory activity on an otherwise
non-regulatory T cell, an assessment was made as to whether
recombinant IL35 could directly inhibit T.sub.E cell proliferation.
HEK293T cells (human embryonic kidney) were transfected with
plasmids encoding expression of either "native" IL35
(EBI3-2A-IL12a) or "single chain" IL35 (i.e., EBI3 and IL12a
expressed as a single chain protein; Hisada et al., Cancer Res.
64:1152-56, 2004). Empty vector, EBI3 alone and IL12a alone
controls were also generated. Recombinant IL35 was then assessed to
determine if it could suppress the proliferation of T.sub.E cells
stimulated with anti-CD3/CD28-coated microbeads. Media containing
either form of recombinant IL35, but not any of the three controls,
potently suppressed T.sub.E cell proliferation in a titratable
fashion (FIG. 5C). Co-culture with irradiated HEK293T cell
transfectants gave identical results. These data demonstrate that
soluble, recombinant IL35 alone is sufficient to suppress T cell
proliferation.
[0096] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0097] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
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