U.S. patent application number 14/151497 was filed with the patent office on 2014-07-10 for compositions and methods for the regulation of t regulatory cells using tl1a-ig fusion protein.
This patent application is currently assigned to University of Miami. The applicant listed for this patent is University of Miami. Invention is credited to Samia Q. Khan, Eckhard R. Podack, Taylor H. Schreiber.
Application Number | 20140193410 14/151497 |
Document ID | / |
Family ID | 51061114 |
Filed Date | 2014-07-10 |
United States Patent
Application |
20140193410 |
Kind Code |
A1 |
Podack; Eckhard R. ; et
al. |
July 10, 2014 |
Compositions and Methods for the Regulation of T Regulatory Cells
Using TL1A-Ig Fusion Protein
Abstract
Compositions comprising TL1A-Ig fusion proteins and methods of
their use, e.g., for the treatment of diseases and disorders
associated with antigen-specific immune responses, are described.
Also described are combination therapies that include the
administration of a TNFRSF25 agonist and an interleukin (e.g.,
IL-2) and/or an mTOR inhibitor (e.g., rapamycin).
Inventors: |
Podack; Eckhard R.; (Coconut
Grove, FL) ; Schreiber; Taylor H.; (Miami, FL)
; Khan; Samia Q.; (Miami, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Miami |
Miami |
FL |
US |
|
|
Assignee: |
University of Miami
Miami
FL
|
Family ID: |
51061114 |
Appl. No.: |
14/151497 |
Filed: |
January 9, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61750672 |
Jan 9, 2013 |
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61753634 |
Jan 17, 2013 |
|
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61842127 |
Jul 2, 2013 |
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61843558 |
Jul 8, 2013 |
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Current U.S.
Class: |
424/134.1 ;
435/252.33; 435/320.1; 435/69.6; 530/387.3; 536/23.4 |
Current CPC
Class: |
A61P 1/16 20180101; A61K
38/20 20130101; A61P 31/00 20180101; A61P 37/08 20180101; A61K
38/2013 20130101; A61K 31/436 20130101; A61K 39/39541 20130101;
A61P 7/00 20180101; C07K 14/52 20130101; A61K 31/7088 20130101;
A61K 39/3955 20130101; A61P 11/00 20180101; C07K 16/2878 20130101;
A61P 43/00 20180101; A61P 29/00 20180101; A61P 37/02 20180101; A61P
37/06 20180101; A61P 1/04 20180101; C07K 14/55 20130101; A61P 37/00
20180101; A61K 39/395 20130101; C07K 16/246 20130101; C07K 2319/30
20130101; A61P 1/12 20180101; A61P 17/00 20180101; A61P 21/00
20180101; A61K 39/39541 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/134.1 ;
536/23.4; 435/320.1; 435/69.6; 530/387.3; 435/252.33 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The research described in this application was supported in
part by a grant (NIH 5P01CA109094) from the National Institutes of
Health. Thus, the government has certain rights in the invention.
Claims
1. An isolated or recombinant nucleic acid comprising a
polynucleotide sequence which encodes a TL1A fusion protein, the
fusion protein comprising: (a) a first polypeptide comprising an
extracellular domain of a human TL1A polypeptide or a fragment
thereof that specifically binds to Tumor Necrosis Factor Receptor
Superfamily, Member 25 (TNFRSF25), and (b) a second polypeptide
comprising an immunoglobulin (Ig) polypeptide; or a complementary
polynucleotide sequence thereof.
2. The nucleic acid of claim 1, wherein said nucleic acid encodes a
monomeric fusion protein capable of forming a fusion protein
homomultimer, wherein the homomultimer is a dimer of trimers.
3. The nucleic acid of claim 1, wherein the nucleic acid encodes a
polypeptide that, when administered to a human in need thereof,
reduces the frequency of naive CD4 T cells in the human.
4. The nucleic acid of claim 1, wherein the second polypeptide
comprises one or more of a hinge region, a CH2 domain, and a CH3
domain of an IgG polypeptide.
5. The nucleic acid of claim 1, wherein the second polypeptide
comprises the amino acid sequence of SEQ ID NO: 14, or an amino
acid sequence that has at least 90% sequence identity to SEQ ID NO:
14.
6. The nucleic acid of claim 1, wherein the first polypeptide
comprises: (a) an amino acid sequence of SEQ ID NO: 12, or (b) an
amino acid sequence that has at least 90% sequence identity to SEQ
ID NO: 12.
7. The nucleic acid of claim 1, wherein the nucleic acid encodes a
fusion protein comprising SEQ ID NO: 12 and SEQ ID NO: 14.
8. The nucleic acid of claim 1, wherein the nucleic acid encodes a
fusion protein comprising SEQ ID NO: 16.
9. The nucleic acid of claim 1, wherein the nucleic acid further
comprises a nucleotide sequence that encodes a secretory or signal
peptide operably linked to the fusion protein.
10. A vector comprising the nucleic acid of claim 1.
11. An isolated or recombinant host cell comprising the vector of
claim 10.
12. A method of producing a TL1A fusion protein, the method
comprising: (a) introducing into a population of cells the nucleic
acid of claim 7, wherein the nucleic acid is operatively linked to
a regulatory sequence effective to produce the polypeptide encoded
by the nucleic acid; and (b) culturing the cells in a culture
medium to produce the polypeptide.
13. The method of claim 12, further comprising: (c) isolating the
polypeptide from the cells or culture medium.
14. A composition comprising a human TL1A-Ig fusion protein, the
fusion protein comprising (a) a first polypeptide comprising an
extracellular domain of a human TL1A polypeptide or a fragment
thereof that specifically binds to TNFRSF25; and (b) a second
polypeptide comprising an Ig polypeptide.
15. The composition of claim 14, wherein, when administered to a
human in need thereof, the composition reduces the frequency of
naive CD4 T cells in the human.
16. The composition of claim 14, wherein the first polypeptide
comprises: (a) an amino acid sequence of SEQ ID NO: 12, or (b) an
amino acid sequence that has at least 90% sequence identity to SEQ
ID NO: 12.
17. The composition of claim 14, wherein the fusion protein is a
homomultimer, and wherein the homomultimer is a dimer of
trimers.
18. The composition of claim 14, wherein the Ig polypeptide
comprises one or more of a hinge region, a CH2 domain, and a CH3
domain of an IgG polypeptide.
19. The composition claim 14, wherein the Ig polypeptide comprises:
(a) an amino acid sequence of SEQ ID NO: 14, or (b) an amino acid
sequence that has at least 90% sequence identity to SEQ ID NO:
14.
20. The composition of claim 14, wherein the fusion protein
comprises: (a) an amino acid sequence of SEQ ID NO: 16, or (b) an
amino acid sequence that has at least 90% sequence identity to SEQ
ID NO: 16.
21. The composition of claim 14, wherein the composition is
characterized by enhanced in vivo efficacy compared to the first
polypeptide when it is not coupled with an Ig polypeptide.
22. A method of modulating an antigen-specific immune response in a
human patient in need thereof, the method comprising: administering
to the patient the composition according to claim 14, wherein the
composition is administered in an amount that comprises a
therapeutically effective amount of the fusion protein.
23. The method of claim 22, wherein the patient in need thereof is
a patient selected from the group consisting of a patient
undergoing or about to undergo induction therapy in preparation for
a solid organ or stem cell transplant, a patient who is a solid
organ or stem cell transplant recipient and is undergoing or is
about to undergo maintenance therapy, a patient who is a solid
organ or stem cell transplant recipient, an allergic patient; a
patient who is receiving or about to receive a vaccine, and a
patient being treated or about to be treated with an immune
checkpoint inhibitor.
24. The method of claim 22, wherein the therapeutically effective
amount of the fusion protein is in a range selected from the group
consisting of 0.1-10 milligrams per kilogram of body weight per day
(mg/kg/day), 0.5-5 mg/kg/day, and 1-2 mg/kg/day.
25. A method of treating a disease or disorder associated with an
antigen-specific immune response, or treating one or more symptoms
of the disease or disorder, in a human patient in need thereof, the
method comprising: administering to the patient the composition of
claim 14, wherein the composition is administered in an amount that
comprises a therapeutically effective amount of the fusion
protein.
26. The method of claim 25, wherein the therapeutically effective
amount of the fusion protein is in a range selected from the group
consisting of 0.1-10 milligrams per kilogram of body weight per day
(mg/kg/day), 0.5-5 mg/kg/day, and 1-2 mg/kg/day.
27. The method of claim 25, wherein the disease or disorder is
selected from the group consisting of autoimmune disease or
disorder, transplant rejection, graft-versus-host disease,
inflammation, asthma, allergies, and chronic infection.
28. The method of claim 27, wherein the disease or disorder is
asthma.
29. The method of claim 22, wherein the composition is administered
to the patient in an amount that significantly increases
proliferation of Treg cells in the patient following the
administration.
30. The method of claim 22, wherein the composition is administered
to the patient in an amount that increases proliferation of Treg
cells by at least two-fold in the patient following the
administration.
31. The method of claim 22, comprising multiple administrations of
the composition to the patient.
32. The method of claim 22, wherein the disease or disorder is an
autoimmune disease selected from the group consisting of
inflammatory bowel disease and rheumatoid arthritis.
33. A method of reducing an adverse event associated with a therapy
involving the administration of a TNFRSF25 agonist, wherein the
method comprises administering to a patient in need thereof the
composition according to claim 14 in a physiologically acceptable
carrier.
34. The method of claim 33, wherein the adverse event is selected
from the group consisting of development of one or more symptoms of
inflammatory bowel disease, weight loss, rash, diarrhea, myalgias,
decreased platelet counts, elevated liver enzyme levels, and
death.
35. The method of claim 22, wherein the composition is formulated
as a pharmaceutical preparation.
36. The method of claim 25, wherein the composition is formulated
as a pharmaceutical preparation.
37. The method of claim 33, wherein the composition is formulated
as a pharmaceutical preparation.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
provisional application Ser. Nos. 61/750,672, filed Jan. 9, 2013;
61/753,634, filed Jan. 17, 2013; 61/842,127, filed Jul. 2, 2013;
and 61/843,558, filed Jul. 8, 2013; the entire contents of each are
incorporated by reference in their entireties.
TECHNICAL FIELD
[0003] This disclosure relates to the fields of molecular biology
and immunology.
BACKGROUND
[0004] Stimulation of tumor necrosis factor receptor superfamily,
member 25 (TNFRSF25) in vivo with its natural ligand TNFSF15 (also
known as TL1A), facilitates selective proliferation of Treg in mice
and suppression of immunopathology in allergic lung inflammation,
allogeneic heart transplantation and HSV-1 mediated ocular
inflammation. Progress in translating Treg therapy in humans has
been slow, however, and thus far limited to ex vivo cell culture
methodologies. Furthermore, such therapies must also be safe and
avoid dangerous side effects, such as susceptibilities to
inflammatory bowel disease (IBD). Prolonged stimulation with
certain TNFRSF25 agonists have been known to cause harmful side
effects in vivo, including, for example, increased inflammation in
mouse models of asthma, inflammatory bowel disease and arthritis.
Thus improved therapies that are safe and effective for Treg
therapy in humans are needed.
SUMMARY
[0005] As discussed above, safe and effective therapies for
treating autoimmune diseases and disorders, establishing tolerance
to allogeneic solid organ transplantation, and for modulating other
antigen-specific immune responses, particularly in human patients,
are needed in the art. The present disclosure provides these and
other related advantages.
[0006] In some embodiments, provided here is an isolated or
recombinant nucleic acid comprising a polynucleotide sequence which
encodes a fusion protein, the fusion protein comprising (a) a first
polypeptide comprising a polypeptide sequence that specifically
binds to Tumor Necrosis Factor Receptor Superfamily, Member 25
(TNFRSF25), and (b) a second polypeptide comprising an
immunoglobulin (Ig) polypeptide; or a complementary polynucleotide
sequence thereof. In some embodiments, the first polypeptide
comprises the extracellular domain of a human TL1A polypeptide or a
fragment thereof, wherein the fragment is capable of specifically
binding to TNFRSF25. In some embodiments, the nucleic acid encodes
a monomeric fusion protein capable of forming a fusion protein
homomultimer. In some embodiments, the homomultimer is a dimer of
trimers. In some embodiments, the nucleic acid encodes a
polypeptide that, when administered to a human in need thereof,
reduces the frequency of naive CD4 T cells in the human. In some
embodiments, the second polypeptide comprises one or more of a
hinge region, a CH2 domain, and a CH3 domain of an IgG polypeptide
(e.g., IgG1). In some embodiments, the second polypeptide comprises
two or more of a hinge region, a CH2 domain, and a CH3 domain of an
IgG polypeptide. In some embodiments, the second polypeptide
comprises a hinge region, a CH2 domain, and a CH3 domain of an IgG
polypeptide. In some embodiments, the second polypeptide comprises
the polypeptide sequence of SEQ ID NO: 14. In some embodiments, the
first polypeptide comprises a polypeptide sequence that has at
least 90% sequence identity to SEQ ID NO: 12. In some embodiments,
the first polypeptide comprises a polypeptide sequence that has at
least 95% sequence identity to SEQ ID NO: 12. In some embodiments,
the first polypeptide comprises a polypeptide sequence that has at
least 97%, 98%, or 99% sequence identity to SEQ ID NO: 12. In some
embodiments, the first polypeptide comprises the sequence of SEQ ID
NO: 12. In some embodiments, the first polypeptide consists of the
sequence of SEQ ID NO: 12. In some embodiments, the nucleic acid
encodes a fusion protein comprising SEQ ID NO: 12 and SEQ ID NO:
14. In some embodiments, the nucleic acid encodes a fusion protein
comprising SEQ ID NO: 16. In some embodiments, the nucleic acid
encodes a fusion protein consisting of SEQ ID NO: 16. In some
embodiments, the nucleic acid comprising a sequence that encodes a
fusion protein further comprises a nucleotide sequence that encodes
a signal peptide. In some embodiments, the nucleic acid further
comprises a nucleotide sequence that encodes a secretory and/or
signal peptide operably linked to the fusion protein. In some
embodiments, the fusion protein is secreted from the host cell as a
fusion protein homomultimer (e.g., a dimer of trimers).
[0007] Also provided herein is a vector comprising a nucleic acid
described above (e.g., an isolated or recombinant nucleic acid
comprising a polynucleotide sequence which encodes a fusion
protein, the fusion protein comprising (a) a first polypeptide
comprising a polypeptide sequence that specifically binds to
TNFRSF25, and (b) a second polypeptide comprising an Ig
polypeptide; or a complementary polynucleotide sequence thereof).
In some embodiments, the nucleic acid is operably linked to a
promoter. In some embodiments, the vector is an expression vector.
In some embodiments, the vector is a plasmid vector. In some
embodiments, also provided herein is an isolated or recombinant
host cell comprising the vector described above (e.g., a vector
comprising an isolated or recombinant nucleic acid comprising a
polynucleotide sequence which encodes a fusion protein, the fusion
protein comprising (a) a first polypeptide comprising a polypeptide
sequence that specifically binds to TNFRSF25, and (b) a second
polypeptide comprising an Ig polypeptide; or a complementary
polynucleotide sequence thereof). In some embodiments, also
provided herein is an isolated or recombinant host cell transfected
with any of the nucleic acids described above (e.g., an isolated or
recombinant nucleic acid comprising a polynucleotide sequence which
encodes a fusion protein, the fusion protein comprising (a) a first
polypeptide comprising a polypeptide sequence that specifically
binds to TNFRSF25, and (b) a second polypeptide comprising an Ig
polypeptide; or a complementary polynucleotide sequence thereof),
wherein the host cell is capable of expressing the fusion protein.
In some embodiments, the host cell is a eukaryotic cell.
[0008] Also provided herein is a method of producing a polypeptide,
comprising: (a) introducing into a population of cells a nucleic
acid described above (e.g., an isolated or recombinant nucleic acid
comprising a polynucleotide sequence which encodes a fusion
protein, the fusion protein comprising (a) a first polypeptide
comprising a polypeptide sequence that specifically binds to
TNFRSF25, and (b) a second polypeptide comprising an Ig
polypeptide; or a complementary polynucleotide sequence thereof),
wherein the nucleic acid is operatively linked to a regulatory
sequence effective to produce the polypeptide encoded by the
nucleic acid; and (b) culturing the cells in a culture medium to
produce the polypeptide. In some embodiments, the method further
comprises: (c) isolating the polypeptide from the cells or culture
medium. In some embodiments, the polypeptide is a fusion protein.
In some embodiments, the nucleic acid further comprises a third
nucleotide sequence that encodes a secretory or signal peptide
operably linked to the fusion protein. In some embodiments, the
fusion protein is secreted from the host cell as a fusion protein
homomultimer (e.g., dimer of trimers). In some embodiments, the
fusion protein homomultimer is recovered from the culture medium.
In some embodiments, the fusion protein is recovered from the
culture medium, host cell, or host cell periplasm. In some
embodiments, the fusion protein homomultimer comprises one or more
covalent disulfide bonds between a cysteine residue of the first
fusion protein and at least one cysteine residue of one or more
additional fusion proteins.
[0009] Also provided herein is a composition comprising a human
TL1A-Ig fusion protein, the fusion protein comprising (a) a first
polypeptide comprising a polypeptide that specifically binds to
TNFRSF25; and (b) a second polypeptide comprising an Ig
polypeptide. In some embodiments, the first polypeptide comprises
the extracellular domain of a human TL1A polypeptide or a fragment
thereof, wherein the fragment is capable of specifically binding to
TNFRSF25. In some embodiments, the composition, when administered
to a human in need thereof, reduces the frequency of naive CD4 T
cells in the human. In some embodiments, the first polypeptide
comprises a polypeptide sequence that has at least 90% sequence
identity to SEQ ID NO: 12. In some embodiments, the first
polypeptide comprises a sequence that has at least 95% sequence
identity to SEQ ID NO: 12. In some embodiments, the first
polypeptide comprises a sequence that has at least 98% sequence
identity to SEQ ID NO: 12. In some embodiments, the first
polypeptide comprises a sequence that has at least 99% or 100%
sequence identity to SEQ ID NO: 12. In some embodiments, the fusion
protein is a homomultimer. In some embodiments, the fusion protein
is a dimer of trimers. In some embodiments, the Ig polypeptide
comprises one or more of a hinge region, a CH2 domain, and a CH3
domain of an IgG polypeptide. In some embodiments, the Ig
polypeptide comprises two or more of a hinge region, a CH2 domain,
and a CH3 domain of an IgG polypeptide. In some embodiments, the Ig
polypeptide comprises a hinge region, a CH2 domain, and a CH3
domain of an IgG polypeptide. In some embodiments, the Ig
polypeptide comprises an amino acid sequence that has at least 90%
sequence identity to SEQ ID NO: 14. In some embodiments, the Ig
polypeptide comprises an amino acid sequence that has at least 95%
sequence identity to SEQ ID NO: 14. In some embodiments, the Ig
polypeptide comprises an amino acid sequence that has at least 97%
sequence identity to SEQ ID NO: 14. In some embodiments, the Ig
polypeptide comprises an amino acid sequence that has at least 99%
or 100% sequence identity to SEQ ID NO: 14. In some embodiments,
the fusion protein comprises an amino acid sequence that has at
least 90% sequence identity to SEQ ID NO: 16. In some embodiments,
the fusion protein comprises an amino acid sequence that has at
least 95% sequence identity to SEQ ID NO: 16. In some embodiments,
the fusion protein comprises an amino acid sequence that has at
least 97% sequence identity to SEQ ID NO: 16. In some embodiments,
the fusion protein comprises an amino acid sequence that has at
least 99% or 100% sequence identity to SEQ ID NO: 16. In some
embodiments, the composition is characterized by enhanced in vivo
efficacy compared to the first polypeptide when it is not coupled
with an Ig polypeptide.
[0010] Also provided herein is a method of modulating an
antigen-specific immune response in a human patient in need
thereof, the method comprising: administering to the patient a
composition described above (e.g., a composition comprising a human
TL1A-Ig fusion protein, the fusion protein comprising (a) a first
polypeptide comprising a polypeptide that specifically binds to
TNFRSF25; and (b) a second polypeptide comprising an Ig
polypeptide), wherein the composition is administered in an amount
that comprises a therapeutically effective amount of the fusion
protein. In some embodiments, the patient in need thereof is a
patient selected from the group consisting of a patient undergoing
or about to undergo induction therapy in preparation for a solid
organ or stem cell transplant, a patient who is a solid organ or
stem cell transplant recipient and is undergoing or is about to
undergo maintenance therapy, a patient who is a solid organ or stem
cell transplant recipient, an allergic patient; a patient who is
receiving or about to receive a vaccine, a patient being treated or
about to be treated with an immune checkpoint inhibitor (e.g.,
CTLA-4 or PD-1 inhibitor). In some embodiments, the therapeutically
effective amount of the fusion protein is in a range of 0.1-10
milligrams per kilogram of body weight per day (mg/kg/day). In some
embodiments, the therapeutically effective amount of the fusion
protein is in a range of 0.5-5 mg/kg/day. In some embodiments, the
therapeutically effective amount of the fusion protein is in a
range of 1-2 mg/kg/day. In some embodiments, the composition
reduces an antigen-specific immune response in the patient by at
least 20%. In some embodiments, the method comprises multiple
administrations of the composition to the patient. In some
embodiments, the above compositions further comprise an effective
amount of IL-2. In some embodiments, the effective amount of IL-2
is an amount that would induce suboptimal expansion of Treg cells
if administered alone to a subject. In some embodiments, the
effective amount of IL-2 is a dosage in the range of between 30,000
to 300,000 units per square meter per day. In some embodiments, the
effective amount of IL-2 is 30,000 units per square meter per day.
In some embodiments, the effective amount of IL-2 is 300,000 units
per square meter per day. In some embodiments, any of the above
compositions further comprise an effective amount of an mTOR
inhibitor. In some embodiments, the mTOR inhibitor is selected from
the group consisting of rapamycin (sirolimus), CI-779, everolimus
ABT-578, tacrolimus, AP-23675, BEZ-235, OSI-027, QLT-0447, ABI-009,
BC-210, salirasib, TAFA-93, deforolimus (AP-23573), temsirolimus,
2-(4-Amino-1-isopropyl-1H-pyrazolo
[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol (PP242), AP-23841,
32-deoxorapamycin, 16-pent-2-ynyloxy-32-deoxorapamycin,
16-pent-2-ynyloxy-32(S or R)-dihydro-rapamycin,
16-pent-2-ynyloxy-32(S or
R)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin
(CCI779), 40-epi-(tetrazolyl)-rapamycin (ABT578), biolimus-7,
biolimus-9, and AP23464.
[0011] Also provided herein is a method of treating a disease or
disorder associated with an antigen-specific immune response, or
treating one or more symptoms of the disease or disorder, in a
human patient in need thereof, the method comprising: administering
to the patient a composition described above (e.g., a composition
comprising a human TL1A-Ig fusion protein, the fusion protein
comprising (a) a first polypeptide comprising a polypeptide that
specifically binds to TNFRSF25; and (b) a second polypeptide
comprising an Ig polypeptide), wherein the composition is
administered in an amount that comprises a therapeutically
effective amount of the fusion protein. In some embodiments, the
therapeutically effective amount of the fusion protein is in a
range of 0.1-10 mg/kg/day. In some embodiments, the therapeutically
effective amount of the fusion protein is in a range of 0.5-5
mg/kg/day. In some embodiments, the therapeutically effective
amount of the fusion protein is in a range of 1-2 mg/kg/day. In
some embodiments, the disease or disorder is selected from the
group consisting of autoimmune disease or disorder, transplant
rejection, graft-versus-host disease, inflammation, asthma,
allergies, and chronic infection. In some embodiments, the disease
or disorder is asthma. In some embodiments, the method comprises
multiple administrations of the composition to the patient. In some
embodiments of the method of treating a disease or disorder
associated with an antigen-specific immune response, the
composition reduces an antigen-specific immune response in the
patient by at least 20%. In some embodiments, the method of
treating a disease or disorder associated with an antigen-specific
immune response further comprises administering to the patient an
effective amount of IL-2. In some embodiments, the effective amount
of IL-2 is an amount that would induce suboptimal, or fail to
induce, expansion of Treg cells if administered alone to the
patient. In some embodiments, the effective amount of IL-2 is a
dosage in the range of between 30,000 to 300,000 units per square
meter per day. In some embodiments, the effective amount of IL-2 is
30,000 units per square meter per day. In some embodiments, the
effective amount of IL-2 is 300,000 units per square meter per day.
In some embodiments, the method of treating a disease or disorder
associated with an antigen-specific immune response further
comprises administering to the patient an effective amount of an
mTOR inhibitor. In some embodiments, the mTOR inhibitor is selected
from the group consisting of rapamycin (sirolimus), CI-779,
everolimus ABT-578, tacrolimus, AP-23675, BEZ-235, OSI-027,
QLT-0447, ABI-009, BC-210, salirasib, TAFA-93, deforolimus
(AP-23573), temsirolimus,
2-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol
(PP242), AP-23841, 32-deoxorapamycin,
16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32(S or
R)-dihydro-rapamycin, 16-pent-2-ynyloxy-32(S or
R)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin
(CCI779), 40-epi-(tetrazolyl)-rapamycin (ABT578), biolimus-7,
biolimus-9, and AP23464.
[0012] Also provided herein is a method of reducing the severity
and/or frequency of an adverse event associated with a therapy that
comprises the administration of a TNFRSF25 agonist, wherein the
method of reducing the severity and/or frequency of the adverse
event comprises administering to a patient in need thereof any of
the compositions described above (e.g., a composition comprising a
human TL1A-Ig fusion protein, the fusion protein comprising (a) a
first polypeptide comprising a polypeptide that specifically binds
to TNFRSF25; and (b) a second polypeptide comprising an Ig
polypeptide, and/or an agonistic anti-TNFRSF25 antibody, and/or a
small molecule agonist of TNFRSF25, and/or an interleukin or analog
thereof (e.g., IL-2, IL-7, IL-15), and/or an mTOR inhibitor (e.g.,
rapamycin)) in a physiologically acceptable carrier. In some
embodiments, the adverse event is the development of one or more
symptoms of inflammatory bowel disease. In some embodiments, the
adverse event is development of inflammatory bowel disease. In some
embodiments, the adverse event is selected from the group
consisting of weight loss, rash, diarrhea, myalgias, decreased
platelet counts, elevated liver enzyme levels, and death. In some
embodiments, the composition is administered to the patient in an
amount that significantly increases proliferation of Treg cells in
the patient following the administration. In some embodiments, the
composition is administered to the patient in an amount that
increases proliferation of Treg cells by at least two-fold in the
patient following the administration. In some embodiments, the
method comprises multiple administrations of the composition to the
patient. In some embodiments, the disease or disorder is an
autoimmune disease. In some embodiments, the autoimmune disease is
selected from the group consisting of inflammatory bowel disease
and rheumatoid arthritis. In some embodiments, the composition is
formulated as a pharmaceutical preparation. In some embodiments,
the above methods for reducing the severity and/or frequency of an
adverse event further comprise administering to the patient an
effective amount of IL-2. In some embodiments, the effective amount
of IL-2 is an amount that would induce suboptimal, or fail to
induce, expansion of Treg cells if administered alone to the
patient. In some embodiments of the above methods of reducing the
severity and/or frequency of an adverse event, the effective amount
of IL-2 is a dosage in the range of between 30,000 to 300,000 units
per square meter per day. In some embodiments, the effective amount
of IL-2 is 30,000 units per square meter per day. In some
embodiments, the effective amount of IL-2 is 300,000 units per
square meter per day. In some embodiments, the above methods of
reducing the severity and/or frequency of an adverse event further
comprise administering to the patient an effective amount of an
mTOR inhibitor. In some embodiments, the mTOR inhibitor is selected
from the group consisting of rapamycin (sirolimus), CI-779,
everolimus ABT-578, tacrolimus, AP-23675, BEZ-235, OSI-027,
QLT-0447, ABI-009, BC-210, salirasib, TAFA-93, deforolimus
(AP-23573), temsirolimus,
2-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol
(PP242), AP-23841, 32-deoxorapamycin,
16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32(S or
R)-dihydro-rapamycin, 16-pent-2-ynyloxy-32(S or
R)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin
(CCI779), 40-epi-(tetrazolyl)-rapamycin (ABT578), biolimus-7,
biolimus-9, and AP23464.
[0013] Also provided herein is a pharmaceutical composition
comprising: (a) any of the compositions described above (e.g., a
composition comprising a human TL1A-Ig fusion protein, the fusion
protein comprising (a) a first polypeptide comprising a polypeptide
that specifically binds to TNFRSF25; and (b) a second polypeptide
comprising an Ig polypeptide, and/or an agonistic anti-TNFRSF25
antibody, and/or a small molecule agonist of TNFRSF25, and/or an
interleukin or analog thereof (e.g., IL-2, IL-7, IL-15), and/or an
mTOR inhibitor (e.g., rapamycin)); and (b) a pharmaceutically
acceptable carrier. In some embodiments, the pharmaceutical
composition further comprises an effective amount of IL-2. In some
embodiments, the effective amount of IL-2 is an amount that would
induce suboptimal, or fail to induce, expansion of Treg cells if
administered alone to the patient. In some embodiments, the
effective amount of IL-2 is a dosage in the range of between 30,000
to 300,000 units per square meter per day. In some embodiments, the
effective amount of IL-2 is 30,000 units per square meter per day.
In some embodiments, the effective amount of IL-2 is 300,000 units
per square meter per day.
[0014] Also provided herein are methods of modulating an
antigen-specific immune response, and/or for treating a disease or
disorder associated with an antigen-specific immune response,
and/or for treating one or more symptoms of the disease or
disorder, in a human patient in need thereof, the method
comprising: administering to the patient a combination therapy
comprising a TNFRSF25 agonist and an effective amount of
interleukin 2 and/or an mTOR inhibitor. In some embodiments of this
method, the TNFRSF25 agonist is a small molecule, an agonistic
anti-TNFRSF25 antibody, or a TL1A fusion protein, as described
herein. In some embodiments, the TL1A fusion protein is
administered in an amount in a range of 0.1-10 mg/kg/day, 0.5-5
mg/kg/day, or 1-2 mg/kg/day. In some embodiments, the effective
amount of IL-2 is an amount that would induce suboptimal, or fail
to induce, expansion of Treg cells if administered alone to the
patient. In some embodiments of the method, the effective amount of
IL-2 is a dosage in the range of between 30,000 to 300,000 units
per square meter per day. In some embodiments, the effective amount
of IL-2 is 30,000 units per square meter per day. In some
embodiments, the effective amount of IL-2 is 300,000 units per
square meter per day. In some embodiments, the TNFRSF25 agonist and
the effective amount of IL-2 are administered on the same day,
together or separately. In some embodiments, the TNFRSF25 agonist
and the effective amount of IL-2 are administered on different
days. In some embodiments of the method, the combination therapy
compriss a TNFRSF25 agonist and an effective amount of an mTOR
inhibitor. In some embodiments, the mTOR inhibitor is administered
in an amount in a range of 75 to 300 micrograms per kg body weight
per day. In some embodiments, the TNFRSF25 agonist and the mTOR
inhibitor are administered on the same day, together or separately.
In some embodiments, the TNFRSF25 agonist and the mTOR inhibitor
are administered on different days. In some embodiments of the
above methods comprising a combination therapy, the disease or
disorder is selected from the group consisting of autoimmune
disease or disorder, transplant rejection, graft-versus-host
disease, inflammation, asthma, allergies, and chronic infection. In
some embodiments of these methods, the mTOR inhibitor is selected
from the group consisting of rapamycin (sirolimus), CI-779,
everolimus ABT-578, tacrolimus, AP-23675, BEZ-235, OSI-027,
QLT-0447, ABI-009, BC-210, salirasib, TAFA-93, deforolimus
(AP-23573), temsirolimus,
2-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol
(PP242), AP-23841, 32-deoxorapamycin,
16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32(S or
R)-dihydro-rapamycin, 16-pent-2-ynyloxy-32(S or
R)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin
(CCI779), 40-epi-(tetrazolyl)-rapamycin (ABT578), biolimus-7,
biolimus-9, and AP23464.
[0015] The details of one or more embodiments of the present
disclosure are set forth in the accompanying drawings and the
description below. Other features, objects, and advantages of the
invention will be apparent from the description and drawings, and
from the claims.
[0016] Unless otherwise defined, 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 disclosure pertains. In
case of conflict, the present document, including definitions, will
control.
[0017] All publications, patent applications, patents, and other
references mentioned herein are each incorporated by reference in
their entirety. The materials, methods, and examples disclosed
herein are illustrative only and not intended to be limiting.
DESCRIPTION OF DRAWINGS
[0018] FIG. 1 is a schematic outline of the murine adoptive
transfer model.
[0019] FIG. 2 depicts a bar graph quantifying the proportion of
OT-II pTreg cells and FoxP3-RFP positive nTreg cells, expressed as
a percentage (%) FoxP3+ Ki67+ cells out of total CD4+ cells, in
mesenteric lymph node (mLN) (upper graph) and spleen cells (lower
graph). Data illustrate the mean.+-.S.E.M. with .gtoreq.3 mice per
group; * indicates a statistical significance of p<0.05.
[0020] FIG. 3 depicts line graphs quantifying the proportion of
FoxP3+Ki67+ mLN pTreg (upper panels) and in mLN tTregcells out of
total CD4+ cells in groups of mice that received 1% ovalbumnin
(ova) in drinking water continuously (left panels) or in groups of
mice that had 1% ova containing drinking water replaced with normal
water to `washout` ova for the indicated number of days (0-20)
(right panels). One group of mice in each graph corresponds to mice
that received isotype control IgG and the other group corresponds
to mice that received the 4C12 antibody. Data illustrate the
mean.+-.S.E.M. with .gtoreq.3 mice per group for the 0 and 10 day
time points and from 2 independent experiments with N.gtoreq.4 mice
per group for the 20 day time point; * indicates a statistical
significance of p<0.05.
[0021] FIG. 4 depicts line graphs quantifying the proportion of
splenic FoxP3+Ki67+pTreg (upper panels) and in splenic tTregcells
out of total CD4+ cells in groups of mice that received 1% ova in
drinking water continuously (left panels) or in groups of mice that
had 1% ova containing drinking water replaced with normal water to
`washout` ova for the indicated number of days (0-20) (right
panels). One group of mice in each graph corresponds to mice that
received isotype control IgG and the other group corresponds to
mice that received the 4C12 antibody. Data illustrate the
mean.+-.S.E.M. with .gtoreq.3 mice per group for the 0 and 10 day
time points and from 2 independent experiments with N.gtoreq.4 mice
per group for the 20 day time point; * indicates a statistical
significance of p<0.05.
[0022] FIG. 5 depicts a Western blot of purified hTL1A under
non-reduced and reduced conditions. Molecular weights are shown to
the left of the photograph.
[0023] FIG. 6 depicts a line graph quantifying the functional
activity (based on Rhodamine 110 counts) of human (h)TNFRSF25
transfected p815 cells treated with murine (ms) or human (hu)
TL1A-Ig fusion protein at the indicated dose (ng/ml).
[0024] FIGS. 7-9 depict bar graphs quantifying the % engraftment (%
of human CD45+ cells/total live lymphoid cells) (upper left graph),
frequency (%) of CD4+ cells (upper right graph), frequency (%) CD8+
cells (lower left graph), and frequency of human Treg cells (%
FoxP3+ out of total CD25+CD127-CD4+ cells) (lower right graph) in
the spleens (FIG. 7), mLN (FIG. 8), and small intestine (FIG. 9) of
NSG-hu mice 5 days after treatment with human (h)TL1A-Ig fusion
protein (100 .mu.g, intraperitoneally (i.p.)) or IgG control
(i.p.). In each graph, the light gray bars quantify the percentages
of Ki67-cells and the dark gray bars quantify the percentage of
Ki67+ cells. Data illustrate the mean.+-.S.E.M. with a total of 8
mice per group from 3 independent experiments.
[0025] FIG. 10 depicts a line graph quantifying the body weight
(kg) over time (experimental day) of rhesus macaques that were
administered rhesus macaque (rm) or human (h)TL1A-Ig fusion protein
at the indicated dose (mg/kg) on day 0.
[0026] FIG. 11 depicts line graphs quantifying the absolute number
of leukocytes (10.sup.3/.mu.l (upper panel) and total
polymorphonuclear (PMN) cells (10.sup.3/.mu.l (lower panel)
determined by peripheral blood CBC analysis on the indicated day of
study, following administering individual rhesus macaques rhTL1A-Ig
or hTL1A-Ig fusion protein at the indicated dose on day 0.
[0027] FIG. 12 depicts a line graph quantifying the mean
concentration (.mu.g/ml) of human TL1A-Ig fusion protein over time
in serum of rhesus macaques treated with the fusion protein.
[0028] FIG. 13 depicts bar graphs quantifying the serum
concentration of IFN-.gamma. (ng/ml) (upper panel) and TGFI.beta.
(ng/ml) (lower panel) in individual rhesus macaques on Day 0, and
on Days 2 and 4 following treatment with the indicated
concentration of rhesus macaque (rm) TL1A-Ig fusion protein or
human (h)TL1A-Ig fusion protein. Data illustrate the mean.+-.S.E.M.
with 2 animals receiving 0.5 mg/kg rmTL1A-Ig, 4 animals receiving
1.5 mg/kg rmTL1A-Ig and 2 animals receiving 1.5 mg/kg hTL1A-Ig.
[0029] FIGS. 14-15 depict line graphs quantifying the frequency (%)
of FoxP3 Treg cells (out of total CD4+ cells) (FIG. 14) and the
frequency (%) of CD28+CD95-naive CD4 T cells (out of total
CD4+CCR7+) (FIG. 15) in rhesus macaques on the indicated day post
treatment with rhTL1A-Ig or hTL1A-Ig fusion protein at the
indicated dose on day 0.
[0030] FIG. 16 is a line graph quantifying the frequency (%) of
FoxP3+CD4+ Treg cells out of total CD4+ cells (cells were pre-gated
on CD3+ cells) on the Y-axis versus the number of days post
treatment with the indicated treatment regimen (Low-dose IL-2
(300,000 units); TL1A-Ig fusion protein+very low dose IL-2 (30,000
units); TL1A-Ig fusion protein+Low Dose IL-2 (300,000 units);
control (IgG); or TL1A-Ig fusion protein) on the X-axis. Data are
illustrated as the mean.+-.SEM using 3 mice per group. Day 5
analysis using one-way ANOVA with Tukey post test demonstrated
significant differences for TL1A-Ig vs control (p<0.05),
TL1A-Ig+very low dose IL-2 vs control (p<0.01) and TL1A-Ig plus
low dose IL-2 versus control (p<0.001).
[0031] FIG. 17 is a line graph quantifying the frequency (%) of
FoxP3+CD4+ Treg cells out of total CD4+ cells (cells were pre-gated
on CD3+ cells) on the Y-axis versus the number of days post
treatment with the indicated treatment regimen (Low-dose IL-2
(300,000 units); 4C12 antibody+Low Dose IL-2 (300,000 units); 4C12
antibody; or control (IgG)) on the X-axis. Data are illustrated as
the mean.+-.SEM using 5 mice per group. Day 5 analysis using
one-way ANOVA with Tukey post test demonstrated significant
differences for 4C12 vs control (p<0.05) and 4C12+ low dose IL-2
vs control (p<0.001).
[0032] FIGS. 18, 19 and 20 depict line graphs quantifying the
frequency (percentage) of adoptively transferred OT-I (CD8+
ovalbumin (ova)-specific) T cells out of total CD8+ T cells (FIG.
18), or of adoptively transferred OT-II (CD4+ ova-specific) T cells
out of total CD4+ T cells (FIG. 19), or of CD4+FoxP3+ Treg cells
out of total CD4+ cells (FIG. 20) in the peripheral blood of mice
from the groups treated as indicated in the graph (Ova/alum+TL1A-Ig
fusion protein+Rapamycin; TL1A-Ig fusion protein+Rapamycin;
ova/alum+Rapamycin; ova/alum+TL1A-Ig fusion protein; or ova/alum).
Data are illustrated as the mean.+-.SEM using 6 mice per group from
a total of two independent experiments.
DETAILED DESCRIPTION
[0033] In some embodiments, the present disclosure provides an
isolated or recombinant nucleic acid comprising a polynucleotide
sequence which encodes a fusion protein. The fusion protein can
comprise (a) a first polypeptide comprising a polypeptide sequence
that specifically binds to Tumor Necrosis Factor Receptor
Superfamily, Member 25 (TNFRSF25), and (b) a second polypeptide
comprising an immunoglobulin (Ig) polypeptide; or a complementary
polynucleotide sequence thereof.
[0034] In some embodiments, disclosed herein are fusion proteins
and nucleic acid encoding the fusion proteins, wherein the fusion
proteins comprise a functionally active fragment of the human or
rhesus macaque TL1A polypeptide. The functionally active fragment
can be, for example, the extracellular domain of TL1A (e.g., human
or rhesus macaque TL1A) or any fragment thereof that retains
specific binding to the TL1A receptor, TNFRSF25. In some
embodiments, the functionally active fragment of TL1A includes
amino acids 68-252 from the human TL1A extracellular domain. The
fusion proteins can also contain one or more Ig molecules, such as
one or more domains of the Ig constant regions, e.g., hinge region,
CH2 domain, and/or CH3 domain.
[0035] It is presently discovered that the fusion proteins
disclosed herein safely and selectively stimulate the proliferation
of cognate T regulatory cells (Treg) in vivo. Based on
epidemiologic data linking TL1A polymorphism to inflammatory bowel
disease (IBD) in humans (see, e.g., International Patent
Publication No. WO 2006/127900), and on murine studies
demonstrating that transgenic overexpression of TL1A predisposes to
IBD susceptibility, it was uncertain whether the fusion proteins
could be safely and effectively administered in vivo. Further, it
was entirely unpredictable whether therapeutically effective doses
of the TL1A fusion proteins could be safely administered, in part
because certain TNFRSF25 agonists have been known to cause harmful
side effects in vivo. For example, prior mouse models examining
transgenic expression of TL1A in mouse models of inflammatory bowel
disease, asthma and arthritis, as described, e.g., by Meylan et al.
Immunity. 2008 Jul. 18; 29(1):79-89; Meylan et al. Mucosal Immunol.
2011 March; 4(2):172-85; Migone et al. Immunity. 2002 March;
16(3):479-92; Fang et al. J Exp Med. 2008 May 12; 205(5):1037-48;
and Bull et al. J Exp Med. 2008 Oct. 27; 205(11):2457-64. These
reports demonstrated that the presence of TL1A, and signaling
through TNFRSF25, resulted in enhanced stimulation of effector T
cells and increased severity of immunopathology observed in the
disease setting being investigated. Further, it was demonstrated
that signaling through TNFRSF25 in the context of vaccination led
to increased proliferation of effector T cells despite a concurrent
expansion of Treg cells (see Schreiber et al. J. Immunol. 2012 Oct.
1; 189(7):3311-8). These studies demonstrated that the specificity
of TNFRSF25 stimulation was governed by the availability of cognate
antigen, and raised serious safety concerns regarding the
specificity of these agents in primates. The reason for this
concern arose from the knowledge that prior studies were performed
in laboratory mice that were housed in pathogen-free settings and
thus did not have a history of exposure to a diverse array of
environmental antigens. Because the environmental setting is very
different in humans and non-human primates (i.e., not
pathogen-free), there was significant concern that exposure of
human or non-human primate cells to TNFRSF25 agonists (e.g.,
TL1A-derived agonists) would lead to stimulation of effector T
cells and enhanced immunopathology, despite enhancing effects on
Treg cells. In particular, based on laboratory data in mice,
enhanced inflammation in the lungs and intestinal system was
predicted due to the high prevalence of `foreign` antigens
(endogenous bacteria) and environmental or food antigens.
[0036] Unexpectedly, however, it is presently demonstrated, in
studies in humanized mice and primates, that treatment with the
TL1A fusion proteins described herein was not only effective for
inducing Treg cell proliferation, but also did not induce weight
loss, cause changes in white blood cell count, or lead to any other
dangerous or unwanted side effects, surprisingly indicating that
the TL1A fusion proteins could be safely administered in vivo,
including to primates (indicating that the TL1A fusion proteins are
expected to be safely administered to humans). Thus, in addition to
the fusion proteins themselves, also described herein are methods
for reducing an adverse event associated with a therapy that
includes the administration of a TNFRSF25 agonist, wherein the
methods include administering a composition containing a TL1A
fusion protein described herein. These methods reduce adverse
events associated with TNFRSF25 agonist therapy, while at the same
are effective for modulating an antigen-specific immune response
and for treating a disease or disorder associated with an
antigen-specific immune response, as discussed below.
[0037] Furthermore, while not intending to be bound by any
particular theory or mechanism of action, the present examples also
demonstrate that the effect of the TL1A fusion proteins was
antigen-specific, both systemically and in the mucosa, indicating
that the fusion proteins could be used to modulate both systemic
and mucosal antigen-specific immune response. Thus, also described
herein are methods of using the fusion proteins for modulating an
antigen-specific immune response (e.g., in a human patient in need
thereof). Also described are methods of treating a disease or
disorder associated with an antigen-specific immune response (e.g.,
autoimmune disease or disorder (e.g. inflammatory bowel disease
(IBD) and rheumatoid arthritis), transplant rejection,
graft-versus-host disease (GVHD), inflammation, asthma, allergies,
and chronic infection), and/or treating one or more symptoms of the
disease or disorder (e.g., in a human patient in need thereof). The
fusion proteins and method of their use are described in detail
below.
[0038] It is also presently discovered that combination therapies
that comprise administering to a subject (1) a TNFRSF25 agonist, (a
TL1A fusion protein disclosed herein, or the agonistic
anti-TNFRSF25 antibody 4C12), and (2) a low dose or very low dose
of interleukin (IL)-2, had a surprising and unexpected synergistic
effect on the expansion of FoxP3+ T regulatory cells. While not
intending to be bound by theory or limited to a particular
mechanism of action, the expansion of Treg cells is thought to have
beneficial effects in diseases and disorders associated with
undesirable antigen-specific immune responses, e.g., autoimmune
disease or disorder (e.g., IBD) and rheumatoid arthritis),
transplant rejection, GVHD, inflammation, asthma, allergies, and
chronic infection. Thus, also provided herein are methods of
treating the above diseases and disorders and others, using the
above-described combination therapies.
[0039] It is also presently discovered that administering the mTOR
inhibitor rapamycin in combination with the agonistic anti-TNFRSF25
antibody 4C12 to a subject resulted in a specific reduction of
effector T cells, but had no effect on the expansion of Treg cells
induced by the agonistic anti-TNFRSF25 antibody. This discovery was
surprising, since it was previously thought that rapamycin would
have a global inhibitory effect on both effector T cells and Treg
cells. Based at least in part on this discovery, also provided
herein is a combination therapy comprising administering to a
subject in need thereof a TNFRSF25 agonist and the administration
of an mTOR inhibitor, e.g., to inhibit unwanted activation and
expansion of CD4+ and/or CD8+ T effector cells.
DEFINITIONS
[0040] As used herein, the term "isolated" means that the
referenced material is removed from the environment in which it is
normally found. Thus, an isolated biological material can be free
of cellular components, i.e., components of the cells in which the
material is found or produced. Isolated nucleic acid molecules
include, for example, and without limitation, a PCR product, an
isolated mRNA, a cDNA, or a restriction fragment. Isolated nucleic
acid molecules also include, for example, sequences inserted into
vectors, plasmids, cosmids, artificial chromosomes, and the like.
An isolated nucleic acid molecule is preferably excised from the
genome in which it may be found, and more preferably is no longer
joined to non-regulatory sequences, non-coding sequences, or to
other genes located upstream or downstream of the nucleic acid
molecule when found within the genome. An isolated nucleic acid has
3' and 5' ends that are different than the 3' and 5' ends of the
nucleic acid when in its natural environment (i.e., in the nucleus
of a cell). An isolated protein may be associated with other
proteins or nucleic acids, or both, with which it associates in the
cell, or with cellular membranes if it is a membrane-associated
protein. An isolated fusion protein may be associated with cellular
components of the cell used to produce the fusion protein in
vitro.
[0041] As used herein, the term "nucleic acid encoding" or
"polynucleotide encoding" a TL1A fusion protein encompasses a
nucleic acid which includes only coding sequence for a TL1A fusion
protein as well as a nucleic acid which includes additional coding
and/or non-coding sequence(s).
[0042] The terms "percent (%) sequence identity," and the like,
generally refer to the degree of identity or correspondence between
different nucleotide sequences of nucleic acid molecules or amino
acid sequences of proteins that may or may not share a common
evolutionary origin. Sequence identity can be determined using any
of a number of publicly available sequence comparison algorithms,
such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group,
Program Manual for the GCG Package, Version 7, Madison, Wis.), etc.
To determine the percent identity between two amino acid sequences
or two nucleic acid molecules, the sequences are aligned for
optimal comparison purposes. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences (i.e., percent identity=number of identical
positions/total number of positions (e.g., overlapping
positions).times.100). In one embodiment, the two sequences are, or
are about, of the same length. The percent identity between two
sequences can be determined using techniques similar to those
described below, with or without allowing gaps. In calculating
percent sequence identity, typically exact matches are counted. The
determination of percent identity between two sequences can be
accomplished using a mathematical algorithm. A non-limiting example
of a mathematical algorithm utilized for the comparison of two
sequences is the algorithm of Karlin and Altschul, Proc. Natl.
Acad. Sci. USA 1990, 87:2264, modified as in Karlin and Altschul,
Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul et
al., J. Mol. Biol. 1990; 215: 403. BLAST nucleotide searches can be
performed with the NBLAST program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to sequences disclosed
herein. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3, to obtain amino acid sequences
homologous to protein sequences disclosed herein. To obtain gapped
alignments for comparison purposes, Gapped BLAST can be utilized as
described in Altschul et al., Nucleic Acids Res. 1997, 25:3389.
Alternatively, PSI-Blast can be used to perform an iterated search
that detects distant relationship between molecules. See Altschul
et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and
PSI-Blast programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used. See
ncbi.nlm.nih.gov/BLAST/on the WorldWideWeb. Another non-limiting
example of a mathematical algorithm utilized for the comparison of
sequences is the algorithm of Myers and Miller, CABIOS1988; 4:
11-17. Such an algorithm is incorporated into the ALIGN program
(version 2.0), which is part of the GCG sequence alignment software
package. When utilizing the ALIGN program for comparing amino acid
sequences, a PAM120 weight residue table, a gap length penalty of
12, and a gap penalty of 4 can be used. The percent identity
between two amino acid sequences can be determined using the
algorithm of Needleman and Wunsch (J. Mol. Biol. 1970, 48:444-453),
which has been incorporated into the GAP program in the GCG
software package (Accelrys, Burlington, Mass.; available at
accelrys.com on the WorldWideWeb), using either a Blossum 62 matrix
or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and
a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred
embodiment, the percent identity between two nucleotide sequences
is determined using the GAP program in the GCG software package
using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or
80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly
preferred set of parameters (and the one that can be used if the
practitioner is uncertain about what parameters should be applied
to determine if a molecule has a certain sequence identity is using
a Blossum 62 scoring matrix with a gap open penalty of 12, a gap
extend penalty of 4, and a frameshift gap penalty of 5.
[0043] The term "substantially identical," at the amino acid
sequence level, means that the sequence identity of two amino acid
sequences has at least about 70% or greater (e.g., 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence
identity.
[0044] As used herein, the TL1A polypeptide that is "capable of
specifically binding to Tumor Necrosis Factor Receptor Superfamily,
Member 25 (TNFRSF25)" is one which interacts with TNFRSF25 such
that at least one function mediated by the natural ligand TL1A is
mediated. This function can be, for example, induction and/or
enhancement of regulatory T cell proliferation. Moreover, the TL1A
polypeptides and fusion proteins described herein are said to be
"specifically binding" if: 1) they exhibit a threshold level of
binding activity, and/or 2) they do not significantly cross-react
with known related receptors. The specificity of binding to
TNFRSF25 is routinely confirmed using the caspase-release assay as
demonstrated in FIG. 6. The control groups for these studies
include cells not transfected with TNFRSF25, which demonstrate no
susceptibility to death following treatment with TNFRSF25 agonists
including TL1A-Ig. The potency and EC.sub.50 of the agonists is
also approximated with this assay, and routinely demonstrated to be
in the ng-.mu.g range as demonstrated in FIG. 6.
[0045] The meaning of the phrase "antigen-specific immune response"
is known in the art. By way of example, an immune response that is
specific for antigen "X" will have activated B and/or T cells that
recognize one or more epitopes present in antigen "X."
[0046] As used herein, the phrase "modulating an antigen-specific
immune response" means that the immune response against the
antigen, as measured by any suitable measure (e.g., frequency of
antigen-specific antibodies, T cells, B cells, antigen-specific T
cell proliferation, etc.) is increased or decreased by at least 5%,
at least 10%, at least 15%, 25%, 50%, 70%, 75%, 80%, 90%, 95%, 99%,
or 100%.
[0047] As used herein, "TNFRSF25 agonist" means a substance that
binds to the TNFRSF25 receptor and triggers a response in the cell
on which the TNFRSF25 receptor is expressed similar to a response
that would be observed by exposing the cell to a natural TNFRSF25
ligand, e.g., TL1A.
[0048] As used herein, "suboptimal" in the context of the expansion
of Treg cells induced by an interleukin (e.g., IL-2 or an analog
thereof) in a combination therapy with another agent (e.g., a
TNFRSF25 agonist, e.g., a TL1A fusion protein, an agonistic
anti-TNFRSF25 antibody, or small molecule agonist of TNFRSF25)
means less than 100% compared to the amount or degree of Treg cell
expansion induced in the presence of that same interleukin or
analog thereof alone (i.e., not in a combination therapy). As used
herein, the term "antibody" is inclusive of all species, including
human and humanized antibodies and the antigenic target, for
example, TNFRSF25, can be from any species. Thus, an antibody, for
example, anti-TNFRSF25 can be mouse anti-human TNFR25, goat
anti-human TNFR25; goat anti-mouse TNFR25; rat anti-human TNFR25;
mouse anti-rat TNFR25 and the like. The combinations of antibody
generated in a certain species against an antigen target, e.g.
TNFRSF25, from another species, or in some instances the same
species (for example, in autoimmune or inflammatory response) are
limitless and all species are embodied in the present disclosure.
The term antibody is used in the broadest sense and includes fully
assembled antibodies, monoclonal antibodies (including human,
humanized or chimeric antibodies), polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), and
antibody fragments that can bind antigen (e.g., Fab', F(ab).sub.2,
Fv, single chain antibodies, diabodies), comprising complementarity
determining regions (CDRs) of the foregoing as long as they exhibit
the desired biological activity.
[0049] Depending on the amino acid sequence of the constant domain
of their heavy chains, human immunoglobulins can be assigned to
different classes. There are five major classes, IgA, IgD, IgE, IgG
and IgM, and several of these may be further divided into
subclasses or isotypes, e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2. The heavy-chain constant domains that correspond to the
different classes of immunoglobulins are called alpha, delta,
epsilon, gamma and mu respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known. Different isotypes have different
effector functions; for example, IgG1 and IgG3 isotypes have ADCC
activity. The invention contemplates that antibodies of any class
or subclass may be prepared, including IgA, IgD, IgE, IgG and IgM,
although IgG is preferred.
[0050] An "immunogenic polypeptide" or "antigen" is a polypeptide
derived from the cell or organism that elicits in a subject an
antibody-mediated immune response (i.e., a "B cell" response or
humoral immunity), a cell-mediated immune response (i.e. a "T cell"
response), or a combination thereof. A cell-mediated response can
involve the mobilization helper T cells, cytotoxic T-lymphocytes
(CTLs), or both. Preferably, an immunogenic polypeptide elicits one
or more of an antibody-mediated response, a CD4+Th1-mediated
response (Th1: type 1 helper T cell), and a CD8+ T cell response.
It should be understood that the term "polypeptide" as used herein
refers to a polymer of amino acids and does not refer to a specific
length of a polymer of amino acids. Thus, for example, the terms
peptide, oligopeptide, and protein are included within the
definition of polypeptide.
[0051] As used herein, "treating" or "treatment" of a state,
disease, disorder or condition includes: (1) preventing or delaying
the appearance of clinical or sub-clinical symptoms of the state,
disease, disorder or condition developing in a mammal that may be
afflicted with or predisposed to the state, disease, disorder or
condition but does not yet experience or display clinical or
subclinical symptoms of the state, disease, disorder or condition;
or (2) inhibiting the state, disease, disorder or condition, i.e.,
arresting, reducing or delaying the development of the state,
disease, disorder or condition, or a relapse thereof (in case of
maintenance treatment) or at least one clinical or sub-clinical
symptom thereof; or (3) relieving the state, disease, disorder or
condition, i.e., causing regression of the state, disease, disorder
or condition or at least one of its clinical or sub-clinical
symptoms. The benefit (e.g., alleviation of at least one symptom of
the state, disease, disorder or condition) to a subject to be
treated is either statistically significant or at least perceptible
to the patient or to the physician. The alleviation (e.g., of at
least one symptom of the state, disease, disorder or condition) is
typically at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90% or greater (compared to before treatment).
[0052] As used herein, "preventing" a state, disease, disorder or
condition (e.g., a state, disease, disorder or condition associated
with an antigen-specific immune response, e.g., an autoimmune
disease or disorder, transplant rejection, graft-versus-host
disease, inflammation, asthma, allergies, and chronic infection) in
a subject means for example, to stop the development of one or more
symptoms of the state, disease, disorder or condition, in a subject
before they occur or are detectable, e.g., by the patient or the
patient's doctor. Preferably, the state, disease, disorder or
condition does not develop at all, i.e., no symptom of the state,
disease, disorder or condition is detectable. However, it can also
result in delaying or slowing of the development of one or more
symptoms of the state, disease, disorder or condition.
Alternatively, or in addition, it can result in the decreasing of
the severity of one or more subsequently developed symptoms.
[0053] As used herein "combination therapy" means the treatment of
a subject (e.g., a subject in need of treatment, e.g., a human
patient) with a TNFRSF25 agonist described herein (e.g., TL1A
fusion protein, agonistic anti-TNFRSF25 antibody, small molecule,
etc.) and one or more other therapies (e.g., drug or therapeutic
treatment) for, e.g., modifying an antigen-specific immune response
and/or treating a disease or disorder (e.g., treating one or more
symptoms of the disease or disorder). Such combination therapy can
be sequential therapy wherein the patient is treated first with one
therapy and then the other, and so on, or all therapies can be
administered simultaneously. In either case, these therapies are
said to be "coadministered." It is to be understood that
"coadministered" does not necessarily mean that the drugs and/or
therapies are administered in a combined form (i.e., they may be
administered separately or together to the same or different sites
at the same or different times).
[0054] The term "pharmaceutically acceptable carrier" means a
diluent, adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water or aqueous solution
saline solutions and aqueous dextrose and glycerol solutions are
preferably employed as carriers, particularly for injectable
solutions. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin.
[0055] The term "pharmaceutically acceptable derivative" as used
herein means any pharmaceutically acceptable salt, solvate or
prodrug, e.g., ester, of a compound disclosed herein, which upon
administration to the recipient is capable of providing (directly
or indirectly) a compound disclosed herein, or an active metabolite
or residue thereof. Such derivatives are recognizable to those
skilled in the art, without undue experimentation. Nevertheless,
reference is made to the teaching of Burger's Medicinal Chemistry
and Drug Discovery, 5th Edition, Vol 1: Principles and Practice,
which is incorporated herein by reference to the extent of teaching
such derivatives. Preferred pharmaceutically acceptable derivatives
are salts, solvates, esters, carbamates, and phosphate esters.
Particularly preferred pharmaceutically acceptable derivatives are
salts, solvates, and esters. Most preferred pharmaceutically
acceptable derivatives are salts and esters. The term "nucleic acid
hybridization" refers to the pairing of complementary strands of
nucleic acids. The mechanism of pairing involves hydrogen bonding,
which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen
bonding, between complementary nucleoside or nucleotide bases
(nucleobases) of the strands of nucleic acids. For example, adenine
and thymine are complementary nucleobases that pair through the
formation of hydrogen bonds. Hybridization can occur under varying
circumstances. Nucleic acid molecules are "hybridizable" to each
other when at least one strand of one nucleic acid molecule can
form hydrogen bonds with the complementary bases of another nucleic
acid molecule under defined stringency conditions. Stringency of
hybridization is determined, e.g., by (i) the temperature at which
hybridization and/or washing is performed, and (ii) the ionic
strength and (iii) concentration of denaturants such as formamide
of the hybridization and washing solutions, as well as other
parameters. Hybridization requires that the two strands contain
substantially complementary sequences. Depending on the stringency
of hybridization, however, some degree of mismatches may be
tolerated. Under "low stringency" conditions, a greater percentage
of mismatches are tolerable (i.e., will not prevent formation of an
anti-parallel hybrid). See Molecular Biology of the Cell, Alberts
et al., 3rd ed., New York and London: Garland Publ., 1994, Ch.
7.
[0056] Typically, hybridization of two strands at high stringency
requires that the sequences exhibit a high degree of
complementarity over an extended portion of their length. Examples
of high stringency conditions include: hybridization to
filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65.degree.
C., followed by washing in 0.1.times.SSC/0.1% SDS (where
1.times.SSC is 0.15 M NaCl, 0.15 M Na citrate) at 68.degree. C. or
for oligonucleotide (oligo) inhibitors washing in 6.times.SSC/0.5%
sodium pyrophosphate at about 37.degree. C. (for 14 nucleotide-long
oligos), at about 48.degree. C. (for about 17 nucleotide-long
oligos), at about 55.degree. C. (for 20 nucleotide-long oligos),
and at about 60.degree. C. (for 23 nucleotide-long oligos).
[0057] Conditions of intermediate or moderate stringency (such as,
for example, an aqueous solution of 2.times.SSC at 65.degree. C.;
alternatively, for example, hybridization to filter-bound DNA in
0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65.degree. C. followed by
washing in 0.2.times.SSC/0.1% SDS at 42.degree. C.) and low
stringency (such as, for example, an aqueous solution of
2.times.SSC at 55.degree. C.), require correspondingly less overall
complementarity for hybridization to occur between two sequences.
Specific temperature and salt conditions for any given stringency
hybridization reaction depend on the concentration of the target
DNA or RNA molecule and length and base composition of the probe,
and are normally determined empirically in preliminary experiments,
which are routine (see Southern, J. Mol. Biol. 1975; 98:503;
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,
vol. 2, ch. 9.50, CSH Laboratory Press, 1989; Ausubel et al.
(eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing Associates, Inc., and John Wiley & Sons, Inc., New
York, at p. 2.10.3). An extensive guide to the hybridization of
nucleic acids is found in, e.g., Tijssen (1993) Laboratory
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Acid Probes part I, chapt 2, "Overview of principles
of hybridization and the strategy of nucleic acid probe assays,"
Elsevier, N.Y. ("Tijssen").
[0058] As used herein, the term "standard hybridization conditions"
refers to hybridization conditions that allow hybridization of two
nucleotide molecules having at least 50% sequence identity.
According to a specific embodiment, hybridization conditions of
higher stringency may be used to allow hybridization of only
sequences having at least 75% sequence identity, at least 80%
sequence identity, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity.
[0059] The definitions of protein, peptide and polypeptide are
well-known in the art. The term "protein", as used herein, is
synonymous with the term "peptide" or "polypeptide," and is
understood to mean a chain of amino acids arranged linearly and
joined together by peptide bonds between the carboxyl and amino
groups of adjacent amino acid residues. Thus, the term polypeptide
can refer to a full length amino acid sequence of a protein, or to
a fragment thereof.
[0060] As used herein, the terms "nucleic acid," "oligonucleotide,"
"polynucleotide" and "polynucleotide sequence" are used
interchangeably, and refer to a deoxyribonucleotide or
ribonucleotide in either single- or double-stranded form. The term
also encompasses nucleic-acid-like structures with synthetic
backbones. DNA backbone analogues provided by the invention include
phosphodiester, phosphorothioate, phosphorodithioate,
methylphosphonate, phosphoramidate, alkyl phosphotriester,
sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate,
morpholino carbamate, and peptide nucleic acids (PNAs); see
Oligonucleotides and Analogues, a Practical Approach, edited by F.
Eckstein, IRL Press at Oxford University Press (1991); Antisense
Strategies, Annals of the New York Academy of Sciences, Volume 600,
Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med.
Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC
Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl)
glycine units. Phosphorothioate linkages are described in WO
97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol.
144:189-197. Other synthetic backbones encompassed by the term
include methyl-phosphonate linkages or alternating
methylphosphonate and phosphodiester linkages (Strauss-Soukup
(1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages
(Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156). The
term nucleic acid is used interchangeably with cDNA, cRNA, mRNA,
oligonucleotide, polynucleotide and amplification product.
[0061] As used herein, "operably linked" with a polynucleotide
sequence means that a target polynucleotide sequence and one or
more expression control sequences (e.g., promoters) are physically
linked so as to permit expression of the polypeptide encoded by the
target polynucleotide sequence within a host cell.
[0062] As used herein, the term "about" or "approximately" usually
means within an acceptable error range for the type of value and
method of measurement. For example, it can mean within 20%, more
preferably within 10%, and most preferably still within 5% of a
given value or range. Alternatively, especially in biological
systems, the term "about" means within about a log(i.e., an order
of magnitude) preferably within a factor of two of a given
value.
[0063] TNFRSF25 Agonists
[0064] The present disclosure provides methods that include the
administration, either alone, or as part of a combination therapy,
of an agonist of TNFRSF25 (also known as DR3). The present
disclosure provides novel TNFRSF25 agonists, such as the TL1A
fusion proteins described herein. However, also provided herein are
novel methods that include the use of other TNFRSF25 agonists which
are known in the art. For example, non-limiting examples of
TNFRSF25 agonists that may be used in the combination therapies
disclosed herein (which include the co-administration of an mTOR
inhibitor, e.g., rapamycin, and/or IL-2 with a TNFRSF25 agonist)
include, e.g., small molecules, antibodies, and fusion proteins.
Non-limiting examples of such TNFRSF25 agonists are described,
e.g., in U.S. pre-grant publication Nos. 2011/0243951,
2012/0029472, and 2012/0135011, all by Podack et al. Methods for
preparing anti-TNFRSF25 antibodies are described in US 2012/0029472
by Podack et al. The present methods, e.g., the combination
therapies disclosed herein, envision the use of any suitable
TNFRSF25 agonist known in the art. In some embodiments, TNFRSF25
agonists are ones which enhance the expansion of Treg cells.
[0065] In some embodiments, a TNFRSF25 agonist is a small molecule.
Chemical agents, referred to in the art as "small molecules" are
typically organic, non-peptide molecules, having a molecular weight
less than 10,000 Da, preferably less than 5,000 Da, more preferably
less than 1,000 Da, and most preferably less than 500 Da. This
class of modulators includes chemically synthesized molecules, for
instance, compounds from combinatorial chemical libraries.
Synthetic compounds may be rationally designed or identified by
screening compound libraries for TNFRSF25-modulating activity
according to methods known in the art. Alternative appropriate
modulators of this class are natural products, particularly
secondary metabolites from organisms such as plants or fungi, which
can also be identified by screening compound libraries for
TNFRSF25-modulating activity. Methods for generating and obtaining
small molecules are well known in the art (see, e.g., Schreiber,
Science 2000; 151:1964-1969; Radmann et al., Science 2000;
151:1947-1948).
[0066] In some embodiments, the present disclosure provides nucleic
acids encoding TL1A fusion proteins and compositions that contain
the TL1A fusion proteins. Also described herein are methods of
producing TL1A fusion proteins. The method can include for example,
introducing into a population of cells a nucleic acid encoding the
TL1A fusion protein, e.g., a nucleic acid described herein (e.g.,
SEQ ID NO: 15), wherein the nucleic acid is operatively linked to a
regulatory sequence effective to produce the fusion protein
polypeptide encoded by the nucleic acid; and culturing the cells in
a culture medium to produce the polypeptide. In some embodiments,
the method can further include isolating the fusion protein
polypeptide from the cells or culture medium. The nucleic acid can
also further contain a third nucleotide sequence that encodes a
secretory or signal peptide operably linked to the fusion protein.
In some embodiments, the fusion protein is secreted from the host
cell as a fusion protein homomultimer (e.g., as a dimer of
trimers). In some embodiments, the fusion protein homomultimer is
recovered from the culture medium, the host cell or host cell
periplasm. Further, the fusion protein homomultimer can contain one
or more covalent disulfide bonds between a cysteine residue of the
first fusion protein and at least one cysteine residue of one or
more additional fusion proteins.
[0067] TL1A is a type II transmembrane protein belonging to the TNF
superfamily and has been designated TNF superfamily member 15
(TNFSF15). TL1A is the natural ligand for TNFRSF25. See U.S. Pat.
No. 6,713,061, and Borysenko, et al., Biochem Biophys Res Commun.
2005 Mar. 18; 328(3):794-9, Sheikh, et al., Curr. Cancer Drug
Targets. 2004 February; 4(1):97-104, and U.S. publication number
2007/0128184. Human TL1A nucleic acid and amino acid sequences are
known and have been described. See, for example GenBank.RTM.
Accession No. CCDS6809.1 (nucleic acid sequence) (SEQ ID NO: 1);
and GenBank.RTM. Accession No. EAW87431 (amino acid sequence) (SEQ
ID NO: 2). Other nucleic acid and amino acid sequences for human
TL1A have been described, including, but not limited to
GenBank.RTM. Accession Nos. NM.sub.--001204344.1/NP 001191273.1,
NM.sub.--005118.3/NP 005109.2,
NM.sub.--001039664.1/NP.sub.--001034753.1, NM.sub.--148970.1/NP
683871.1, NM.sub.--148967.1/NP 683868.1, NM.sub.--148966.1/NP
683867.1, NM.sub.--148965.1/NP 683866.1, and
NM.sub.--003790.2/NP.sub.--003781.1, each of which is incorporated
by reference (including the referenced sequences).
[0068] Rhesus macaque TL1A nucleic acid and amino acid sequences
can be inferred from the Macaca mulatta chromosome 15,
Mmul.sub.--051212, whole genome shotgun sequence (GenBank.RTM.
Accession No. NC.sub.--007872.1). The mRNA sequence having
GenBank.RTM. Accession No. NM.sub.--001194132.1 (SEQ ID NO: 3). An
exemplary rhesus macaque amino acid sequence has GenBank.RTM.
Accession No. NP.sub.--001181061, which is incorporated by
reference (including the referenced sequence). (SEQ ID NO: 4).
[0069] Encompassed herein are non-naturally occurring
polynucleotides encoding fusion proteins that specifically bind to
TNFRSF25. For example, provided herein are isolated or recombinant
nucleic acids containing a polynucleotide sequence which encodes a
fusion protein, the fusion protein containing (a) a first
polypeptide containing a polypeptide sequence that specifically
binds to TNFRSF25, and (b) a second polypeptide containing an Ig
polypeptide; or a complementary polynucleotide sequence thereof. A
fusion protein described herein can also contain a TL1A polypeptide
linked to another second polypeptide that promotes multimerization,
e.g., to for a dimer, a trimer, a dimer of trimers, etc. For
example, the second polypeptide can be a surfactant protein.
[0070] In general, the fusion proteins are agonists of TNFRSF25. In
some embodiments, the fusion protein comprises a TL1A polypeptide
(a "TL1A fusion protein"). Typically, a TL1A fusion protein
encompassed herein induces a signaling response that is similar to
the response induced by the natural ligand, TL1A. For example, in
some embodiments, TL1A fusion proteins encompassed herein induce
proliferation of Treg cells in vitro and/or in vivo. In some
embodiments, the TL1A fusion proteins encompassed herein have a T
effector cells costimulation effect. Suitable assays for measuring
T cell proliferation in vitro and in vivo are known in the art and
described in Example 1 (materials and methods). The activity of the
TL1A fusion proteins can be measured as described in detail in Khan
et al. J. Immunol. 2013 Feb. 15; 190(4):1540-50. In some
embodiments, the TL1A fusion protein comprises: a first polypeptide
that is capable of binding to TNFRSF25; and at least a second
polypeptide. In some embodiments the polypeptide comprises or
consists of the extracellular domain of TL1A (e.g., human TL1A
extracellular domain) or a fragment thereof that is capable of
binding to TNFRSF25 (i.e., a "functionally active fragment"). In
some embodiments, the polypeptide is a variant or ortholog of human
TL1A or a functionally active fragment thereof. In some
embodiments, the human TL1A polypeptide comprises or consists of
amino acid residues 68-252 from the TL1A extracellular domain. In
some embodiments, the second polypeptide can be an Ig molecule. For
example the immunoglobulin molecule can be the constant region of
an antibody (e.g., IgG, IgA, IgM or IgD). The Ig heavy chains can
be divided into three functional regions: Fd (containing V.sub.H
and CH1 domains), hinge, and Fc. Fd in combination with the light
chain forms the "Fab" portion of an antibody. The hinge region is
found in IgG, IgA, and IgD classes, and acts as a flexible spacer,
allowing the Fab portion to move freely in space. The hinge domains
are structurally diverse, varying in both sequence and length among
immunoglobulin classes and subclasses. Three human IgG subclasses,
IgG1, IgG2, and IgG4, have hinge regions of 12-15 amino acids while
IgG3 has approximately 62 amino acids, including 21 proline
residues and 11 cysteine residues. The structure of the hinge
region is described in detail in Shin et al., Immunological Reviews
130:87 (1992) and in U.S. Patent Application Publication No.
2013/0142793.
[0071] For an immunoglobulin fusion protein which is intended for
use in humans, the constant regions may be of human sequence origin
in order to minimize a potential anti-human immune response. The
constant region may also be of human sequence origin in order to
provide appropriate effector functions. In some embodiments, the
constant region may facilitate multimerization of the fusion
protein. Manipulation of sequences encoding antibody constant
regions is described in the PCT publication of Morrison and Oi, WO
89/007142. For example, the CH1 domain can be deleted and the
carboxyl end of the binding domain is joined to the amino terminus
of CH2 through the hinge region.
[0072] In some embodiments, the Ig molecule comprises a CH2 domain
and/or a CH3 domain and/or a hinge region of an immunoglobulin. In
some embodiments the second polypeptide is an Ig molecule
comprising a hinge region, a CH2 domain and a CH3 domain of an IgG
molecule (e.g., human IgG). In some embodiments, the second
polypeptide is an Ig molecule comprising a CH2 domain and a CH3
domain of an IgG molecule (e.g., human IgG). In some embodiments,
the second polypeptide is an Ig molecule comprising a hinge region
and one or more of: a CH2 domain and a CH3 domain of an IgG
molecule (e.g., human IgG). In some embodiments, the second
polypeptide is an Ig molecule comprising a CH2 domain and at least
one of: a hinge region and a CH3 domain of an IgG molecule (e.g.,
human IgG). In some embodiments, the human immunoglobulin hinge
region is an IgG1 hinge region comprising 0, 1, 2, 3, or more
cysteine residues.
[0073] Fusion proteins encompassed herein can also contain other
polypeptides instead of or in addition to the Ig molecules
described above. For example, a fusion protein can contain a
polypeptide that binds to TNFRSF25 and a surfactant protein, or
other polypeptide that facilitates multimerization of the fusion
protein.
[0074] The nucleic acids disclosed herein, also referred to herein
as polynucleotides, may be in the form of RNA or in the form of
DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The
DNA may be double-stranded or single-stranded, and if single
stranded may be the coding strand or non-coding (anti-sense)
strand. The sense and anti-sense strands are "complementary" to
each other. The nucleic acids which encode TL1A fusion proteins for
use according to the compositions and methods disclosed herein may
include, but are not limited to: only the coding sequence for the
TL1A fusion protein; the coding sequence for the TL1A fusion
protein and additional coding sequence; the coding sequence for the
TL1A fusion protein (and optionally additional coding sequence) and
non-coding sequence, such as introns or non-coding sequences 5'
and/or 3' of the coding sequence for the TL1A fusion polypeptide,
which for example may further include but need not be limited to
one or more regulatory nucleic acid sequences that may be a
regulated or regulatable promoter, enhancer, other transcription
regulatory sequence, repressor binding sequence, translation
regulatory sequence or any other regulatory nucleic acid sequence.
Thus, as defined above, the term "nucleic acid encoding" or
"polynucleotide encoding" a TL1A fusion protein encompasses a
nucleic acid which includes only coding sequence for a TL1A fusion
polypeptide as well as a nucleic acid which includes additional
coding and/or non-coding sequence(s).
[0075] Exemplary fusion proteins and the nucleic acid molecules
encoding the fusion proteins are described below. It is to be
understood that the sequences described below are not limiting. As
discussed in more detail below, other TL1A fusion proteins (e.g.
those containing fragments, variants, and orthologs of TL1A are
also encompassed by the present disclosure, as well as various
second polypeptides and/or other functional domains. For example,
also encompassed herein are fusion proteins that comprise TL1A and
a surfactant protein. In some embodiments, the human TL1A
polypeptide comprises or consists of amino acid residues 68-252
from the TL1A extracellular domain.
[0076] By way of non-limiting example, in some embodiments the
nucleic acid and amino acid sequences of the TL1A portion of the
rhesus macaque TL1A fusion protein are:
TABLE-US-00001 (SEQ ID NO: 5)
aaaggacaggagtttgcaccttcacatcagcaagtttatgcacctcttagagcagacggagataagccaagggc-
aca
cctgacagttgtgacacaaactcccacacagcactttaaaaatcagttcccagctctgcactgggaacatgaac-
taggcctggcc
ttcaccaagaaccgaatgaactataccaacaaattcctgctgatcccagagtcgggagactacttcatttactc-
ccaggtcacattc
cgtgggatgacctctgagtgcagtgaaatcagacaagcaggccgaccaaacaagccagactccatcactgtggt-
catcaccaa
ggtaacagacagctaccctgagccaacccagctcctcatggggaccaagtctgtgtgcgaagtaggtagcaact-
ggttccagc
ccatctacctcggacccatgttctccttgcaagaaggggacaagctaatggtgaacgtcagtgacatctccttg-
gtggattacaca aaagaagataaaaccttctttggagccttcttactatag; and (SEQ ID
NO: 6)
kgqefapshqqvyaplradgdkprahltvvtqtptqhfknqfpalhwehelglaftknrmnytnkfllipesgd-
y
fiysqvtfrgmtsecseirqagrpnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwfqpiylgpmfslq-
egdklmv nvsdislvdytkedktffgafll.
[0077] Furthermore, in some embodiments the nucleic acid of the
rhesus macaque Ig sequence (IgG1 hinge-CH2-CH3 sequence) is:
[0078]
ataaaaacatgtggtggtggcagcaaacctcccacgtgcccaccgtgcccagcacctgaactcctgg-
ggggac
cgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcac-
atgcgtggtggtagacgt
gagccaggaagaccccgatgtcaagttcaactggtacgtaaacggcgcggaggtgcatcatgcccagacgaag-
ccacggga
gacgcagtacaacagcacatatcgtgtggtcagcgtcctcaccgtcacgcaccaggactggctga-
acggcaaggagtacacgt
gcaaggtctccaacaaagccctcccggtccccatccagaaaaccatctccaaagacaaagggcagccccgaga-
gcctcagg
tgtacaccctgcccccgtcccgggaggagctgaccaagaaccaggtcagcctgacctgcctggtc-
aaaggcttctacccca
gcgacatcgtcgtggagtgggagaacagcgggcagccggagaacacctacaagaccaccccgcccgtgctgga-
ctccg
acggctcctacttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttc-
tcatgctccgtgat gcatgaggctctgcacaaccactacacgcag (SEQ ID NO: 7),
wherein residues 1-52 (bold text) are the hinge region, residues
53-382 are the CH2 domain, and residues 383-675 (bold, italicized
text) are the CH3 domain; and the amino acid sequence of the rhesus
macaque Ig sequence (IgG1 hinge-CH2-CH3 sequence) is:
[0079]
iktcgggskpptcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpdvkfnwyvn-
gaevh
haqtkpretqynstyrvvsvltvthqdwlngkeytckvsnkalpvpiqktiskdkgqpreppytlpps-
reelknqvsltcl
vkgfypsdivvewensgpentykttppvldsdgsyflyskltvdksrwqqgnvfscsvmhealhnhytq
(SEQ ID NO: 8), wherein residues 1-17 (bold text) are the hinge
region, residues 18-127 are the CH2 domain, and residues 128-225
(bold, italicized text) are the CH3 domain.
[0080] In some embodiments, the nucleic acid and amino acid
sequences of the rhesus macaque TL1A-Ig fusion protein are:
TABLE-US-00002 (SEQ ID NO: 9)
atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgac
ataaaaacatg
tggtggtggcagcaaacctcccacgtgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcc-
tcttcccccc
aaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtagacgtgagccaggaag-
accccgat
gtcaagttcaactggtacgtaaacggcgcggaggtgcatcatgcccagacgaagccacgggagacgcagtacaa-
cagcacat
atcgtgtggtcagcgtcctcaccgtcacgcaccaggactggctgaacggcaaggagtacacgtgcaaggtctcc-
aacaaagcc
ctcccggtccccatccagaaaaccatctccaaagacaaagggcagccccgagagcctcaggtgtacaccctgcc-
cccgtccc
gggaggagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgtcgtg-
gagtggga
gaacagcgggcagccggagaacacctacaagaccaccccgcccgtgctggactccgacggctcctacttcctct-
acagcaag
ctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaa-
ccactacac gcag
aaaggacaggagtttgcaccttcacatcagcaagtttatgcacctcttagagcagacggagataagcca-
agggc
acacctgacagttgtgacacaaactcccacacagcactttaaaaatcagttcccagctctgcactgggaacatg-
aactaggcctg
gccttcaccaagaaccgaatgaactataccaacaaattcctgctgatcccagagtcgggagactacttcattta-
ctcccaggtcac
attccgtgggatgacctctgagtgcagtgaaatcagacaagcaggccgaccaaacaagccagactccatcactg-
tggtcatcac
caaggtaacagacagctaccctgagccaacccagctcctcatggggaccaagtctgtgtgcgaagtaggtagca-
actggttcc
agcccatctacctcggacccatgttctccttgcaagaaggggacaagctaatggtgaacgtcagtgacatctcc-
ttggtggattac acaaaagaagataaaaccttctttggagccttcttactatag; and (SEQ
ID NO: 10) metdtlllwvlllwvpgstgd
iktcgggskpptcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvsqed
pdvkfnwyvngaevhhaqtkpretqynstyrvvsvltvthqdwlngkeytckvsnkalpvpiqktiskdkgqpr-
epqvyt
lppsreeltknqvsltclvkgfypsdivvewensgqpentykttppvldsdgsyflyskltvdksrwqqgnvfs-
csvmheal hnhytq
kgqefapshqqvyaplradgdkprahltvvtqtptqhfknqfpalhwehelglaftknrmnytnkfl-
lipesgd
yfiysqvtfrgmtsecseirqagrpnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwfqpiylgpmfsl-
qegdklm vnvsdislvdytkedktffgafll.
[0081] In the above fusion protein sequences (DNA and amino acid,
SEQ ID NOs: 9 and 10), the italicized and underlined residues
correspond to the mouse kappa leader sequence (residues 1-63 of SEQ
ID NO: 9 and residues 1-21 of SEQ ID NO: 10); the bold and
italicized text corresponds to restriction enzyme cloning sites
(residues 64-69 and 745-750 of SEQ ID NO: 9 and residues 22-23 and
249-250 of SEQ ID NO: 10); the plain text corresponds to the rhesus
macaque IgG1 hinge-CH2-CH3 sequence (residues 70-744 of SEQ ID NO:
9 and residues 24-248 of SEQ ID NO: 10); and the underlined text
corresponds to rhesus macaque TL1A extracellular domain sequence
(residues 751-1290 of SEQ ID NO: 9 and residues 251-429 of SEQ ID
NO: 10).
[0082] In some embodiments, the human TL1A polypeptide comprises or
consists of amino acid residues 68-252 from the extracellular
domain of human TL1A.
[0083] Also, by way of non-limiting example, in some embodiments
the nucleic acid and amino acid sequences of the TL1A portion of
the human TL1A-Ig fusion protein are:
TABLE-US-00003 (SEQ ID NO: 11)
cgggcccagggagaggcctgtgtgcagttccaggctctaaaaggacaggagtttgcaccttcacatcagcaagt-
ttatgca
cctcttagagcagacggagataagccaagggcacacctgacagttgtgagacaaactcccacacagcactttaa-
aaatcagttc
ccagctctgcactgggaacatgaactaggcctggccttcaccaagaaccgaatgaactataccaacaaattcct-
gctgatcccag
agtcgggagactacttcatttactcccaggtcacattccgtgggatgacctctgagtgcagtgaaatcagacaa-
gcaggccgacc
aaacaagccagactccatcactgtggtcatcaccaaggtaacagacagctaccctgagccaacccagctcctca-
tggggacca
agtctgtgtgcgaagtaggtagcaactggttccagcccatctacctcggagccatgttctccttgcaagaaggg-
gacaagctaat
ggtgaacgtcagtgacatctctttggtggattacacaaaagaagataaaaccttctttggagccttcttactat-
ag (encoding the extracellular domain of human TL1A); and (SEQ ID
NO: 12)
raqgeacvqfqalkgqefapshqqvyaplradgdkprahltvvrqtptqhfknqfpalhwehelglaftknrmn
ytnkfllipesgdyfiysqvtfrgmtsecseirqagrpnkpdsitvvitkvtdsypeptql1mgtksvcevgsn-
wfqpiylga mfslqegdklmvnvsdislvdytkedktffgafll (the extracellular
domain of human TL1A).
[0084] In some embodiments the nucleic acid and amino acid
sequences of the human Ig molecule (IgG1 hinge-CH2-CH3 sequence)
are:
TABLE-US-00004 (SEQ ID NO: 13)
tgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccc-
ccca
aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaaga-
ccctgagg
tcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaac-
agcacgta
ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctcca-
acaaagccc
tcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccc-
ccatcccg
ggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtgg-
agtgggaga
gcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctac-
agcaagctc
accgtggacaagagcaggtggcagcaggggaacgtatctcatgctccgtgatgcatgaggctctgcacaaccac-
tacacgca gaagagcctctccctgtctccgggtaaa; and (SEQ ID NO: 14)
cdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpr
eeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvslt-
clvkgfyp
sdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk
(the hinge, CH2 and CH3 region of human IgG).
[0085] In some embodiments the nucleic acid and amino acid
sequences of the human TL1A fusion protein are:
TABLE-US-00005 (SEQ ID NO: 15)
tgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccc-
cccaaaa
cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccc-
tgaggtca
agttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagc-
acgtacc
gtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaac-
aaagccctc
ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccc-
atcccgg
gatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtgga-
gtgggagag
caatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctaca-
gcaagctcac
cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccact-
acacgcaga agagcctctccctgtctccgggtaaa
cgggcccagggagaggcctgtgtgcagttccaggctctaaaaggacaggag
tttgcaccttcacatcagcaagtttatgcacctcttagagcagacggagataagccaagggcacacctgacagt-
tgtgagacaaa
ctcccacacagcactttaaaaatcagttcccagctctgcactgggaacatgaactaggcctggccttcaccaag-
aaccgaatgaa
ctataccaacaaattcctgctgatcccagagtcgggagactacttcatttactcccaggtcacattccgtggga-
tgacctctgagtg
cagtgaaatcagacaagcaggccgaccaaacaagccagactccatcactgtggtcatcaccaaggtaacagaca-
gctaccctg
agccaacccagctcctcatggggaccaagtctgtgtgcgaagtaggtagcaactggttccagcccatctacctc-
ggagccatgtt
ctccttgcaagaaggggacaagctaatggtgaacgtcagtgacatctctttggtggattacacaaaagaagata-
aaaccttctttg gagccttcttactatag; and (SEQ ID NO: 16)
cdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpre-
eq
ynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclv-
kgfypsdia
vewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk
raqgeacvq
fqalkgqefapshqqvyaplradgdkprahltvvrqtptqhfknqfpalhwehelglaftknrmnytnkfllip-
esgdyfiys
qvtfrgmtsecseirqagrpnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwfqpiylgamfslqegdk-
lmvnvsd islvdytkedktffgafll.
[0086] In the above exemplary fusion protein sequences (human
nucleic acid and amino acid sequences, SEQ ID NOs: 15 and 16), the
bold and italicized residues correspond to the restriction enzyme
cloning site (residues 685-690 of SEQ ID NO: 15 and residues
229-230 of SEQ ID NO: 16), the residues occurring before the
restriction enzyme cloning site (plain text) correspond to the
human IgG1 hinge-CH2-CH3 sequence (residues 1-684 of SEQ ID NO: 15
and residues 1-228 of SEQ ID NO: 16), and the residues following
the restriction enzyme cloning site (underlined text) correspond to
the human TL extracellular domain sequence (residues 691-1269 of
SEQ ID NO: 15 and residues 231-422 of SEQ ID NO: 16).
[0087] Murine TL1A fusion proteins were also constructed and
encompassed herein. The methods for their construction, and the
functional characterization of an exemplary murine fusion protein,
are described in detail in Khan, S. Q., et al. (2013) "Cloning,
expression, and functional characterization of TL1A-Ig." J Immunol
190:1540-1550.
[0088] In addition to the polynucleotide sequences described above,
polynucleotide sequences comprising nucleotide sequences having
certain percent sequence identities to any of the aforementioned
sequences are also encompassed. For example, polynucleotides
encompassed herein can have about, e.g., 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any
of the aforementioned polynucleotides (e.g., encoding human or
rhesus macaque TL1A and/or immunoglobulin polynucleotides). Such
polynucleotide sequences can include, e.g., variants and species
orthologs, and preferably hybridize under conditions of moderate or
high stringency as described above. A variant, e.g. of a TL1A
polynucleotide, is a modified or altered gene or DNA sequence,
e.g., mutant. "Mutant" and "mutation" refer to any detectable
change in genetic material (e.g., DNA) or any process, mechanism,
or result of such a change. This includes gene mutations, in which
the structure (e.g., DNA sequence) of a gene is altered, any gene
or DNA arising from any mutation process, and any expression
product (e.g., protein, e.g., TL1A) expressed by a modified gene or
DNA sequence.
[0089] Modification of the polypeptide may be effected by any means
known to those of skill in this art. Such methods may rely on
modification of DNA encoding the fusion protein and expression of
the modified DNA. DNA encoding one of the TL1A fusion proteins
discussed above may be mutagenized using standard methodologies,
including those described below. For example, cysteine residues
that may otherwise facilitate multimer formation or promote
particular molecular conformations can be deleted from a
polypeptide or replaced, e.g., cysteine residues that are
responsible for aggregate formation. Conversely, where aggregation
is desired, e.g., to produce dimers or trimers and/or dimers of
trimers, additional cysteine residues can be introduced, e.g., to
the hinge region of an Ig molecule. If necessary, the identity of
cysteine residues that contribute to aggregate formation may be
determined empirically, by deleting and/or replacing a cysteine
residue and ascertaining whether the resulting protein aggregates
in solutions containing physiologically acceptable buffers and
salts. Moreover, conservative substitutions of amino acids are
well-known and may be made generally without altering the
biological activity of the resulting TL1A fusion protein molecule.
For example, such substitutions are generally made by interchanging
within the groups of polar residues, charged residues, hydrophobic
residues, small residues, and the like. If necessary, such
substitutions may be determined empirically merely by testing the
resulting modified protein for the ability to bind to the
appropriate cell surface receptors (e.g., TNFRSF25) and/or trigger
desired effects (e.g., Treg cell proliferation) in in vitro
biological assays.
[0090] Orthologs are genes in different species that apparently
evolved from a common ancestral gene by speciation. Normally,
orthologs retain the same function through the course of evolution.
Identification of orthologs can provide reliable prediction of gene
function in newly sequenced genomes. Sequence comparison algorithms
that can be used to identify orthologs include without limitation
BLAST, FASTA, DNA Strider, and the GCG pileup program. Orthologs
often have high sequence similarity. Contemplated for use herein
are all orthologs of TL1A that retain the ability to specifically
bind to TNFRSF25, and preferably, to human TNFRSF25. In some
embodiments, truncated components (i.e., fragments) of TL1A
polypeptide, hinge region polypeptide, linker, etc., for use in a
TL1A fusion protein, are provided. Also provided are nucleic acids
encoding a TL1A fusion protein having such truncated components. A
truncated molecule may be any molecule that contains less than a
full length version of the molecule. Truncated molecules provided
by the present disclosure may include truncated biological polymers
and, in some embodiments of the disclosure, such truncated
molecules may be truncated nucleic acid molecules or truncated
polypeptides. Truncated nucleic acid molecules have less than the
full length nucleotide sequence of a known or described nucleic
acid molecule, where such a known or described nucleic acid
molecule may be a naturally occurring, a synthetic or a recombinant
nucleic acid molecule. Thus, for example, truncated nucleic acid
molecules that correspond to a gene sequence contain less than the
full length gene where the gene contains coding and non-coding
sequences, promoters, enhancers and other regulatory sequences,
flanking sequences and the like, and other functional and
non-functional sequences that are recognized as part of the gene.
In another example, truncated nucleic acid molecules that
correspond to a mRNA sequence contain less than the full length
mRNA transcript, which may include various translated and
non-translated regions as well as other functional and
non-functional sequences.
[0091] Truncated molecules can be polypeptides that contain less
than the full length amino acid sequence of a particular protein or
polypeptide component. As used herein "deletion" has its common
meaning as understood by those familiar with the art, and may refer
to molecules that lack one or more of a portion of a sequence from
either terminus or from a non-terminal region, relative to a
corresponding full length molecule, for example, as in the case of
truncated molecules provided herein. Truncated molecules that are
linear biological polymers such as nucleic acid molecules or
polypeptides may have one or more of a deletion from either
terminus of the molecule or a deletion from a non-terminal region
of the molecule, where such deletions may be deletions of 1-550,
1-500, 1-450, 1-400, 1-350, 1-300, 1-250, 1-200, 1-150, 1-100,
1-50, 1-25, 1-20, 15, 1-10, or 1-5 contiguous nucleotide residues.
Truncated polypeptide molecules may have a deletion of 1-250,
1-200, 1-150, 1-100, 1-50, 1-25, 1-20, 1-15, 1-10, or 1-5
contiguous amino acid residues. Truncation molecules (i.e.,
fragments) can include, e.g., a portion of the extracellular domain
of TL1A that specifically bind to TNFRSF25.
[0092] Exemplary truncated TL1A polypeptides that can be used in
the compositions and methods disclosed herein include, e.g., a
fragment containing, e.g. 421 or fewer continuous amino acids of
SEQ ID NO: 16, e.g., 400-420, 350-400, 300-350, 250-300, 200-250,
150-200, 100-150, 50-100, 25-75, 50-75, 25-75, 15-25, or 10-20
contiguous amino acids of SEQ ID NO: 16. Any fragment of TL1A
polypeptide (or nucleic acid) is contemplated for use as described
herein, provided that the fragment is functionally active, i.e.,
retains the ability to specifically bind to TNFRSF25 (or, for
nucleic acid fragments, encodes a polypeptide that is functionally
active).
[0093] The present disclosure further relates to variants of the
herein referenced nucleic acids which encode fragments, analogs
and/or derivatives of a TL1A fusion protein. The variants of the
nucleic acids encoding TL1A fusion proteins may be naturally
occurring allelic variants of the nucleic acids or non-naturally
occurring variants. As is known in the art, an allelic variant is
an alternate form of a nucleic acid sequence which may have at
least one of a substitution, a deletion or an addition of one or
more nucleotides, any of which does not substantially alter the
function of the encoded TL1A fusion protein.
[0094] Variants and derivatives of TL1A fusion protein may be
obtained by mutations of nucleotide sequences encoding TL1A fusion
proteins. Alterations of the native amino acid sequence may be
accomplished by any of a number of conventional methods. Mutations
can be introduced at particular loci by synthesizing
oligonucleotides containing a mutant sequence, flanked by
restriction sites enabling ligation to fragments of the native
sequence. Following ligation, the resulting reconstructed sequence
encodes an analog having the desired amino acid insertion,
substitution, or deletion.
[0095] Alternatively, oligonucleotide-directed site-specific
mutagenesis procedures can be employed to provide an altered gene
wherein predetermined codons can be altered by substitution,
deletion or insertion. Exemplary methods of making such alterations
are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al.
(Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19);
Smith et al. (Genetic Engineering: Principles and Methods
BioTechniques, January 1985, 12-19); Smith et al. (Genetic
Engineering: Principles and Methods, Plenum Press, 1981); Kunkel
(Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods
in Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and
4,737,462.
[0096] As an example, modification of DNA may be performed by
site-directed mutagenesis of DNA encoding the protein combined with
the use of DNA amplification methods using primers to introduce and
amplify alterations in the DNA template, such as PCR splicing by
overlap extension (SOE). Site-directed mutagenesis is typically
effected using a phage vector that has single- and double-stranded
forms, such as M13 phage vectors, which are well-known and
commercially available. Other suitable vectors that contain a
single-stranded phage origin of replication may be used (see, e.g.,
Veira et al., Meth. Enzymol. 15:3, 1987). In general, site-directed
mutagenesis is performed by preparing a single-stranded vector that
encodes the protein of interest (e.g., all or a component portion
of a given TL1A fusion protein). An oligonucleotide primer that
contains the desired mutation within a region of homology to the
DNA in the single-stranded vector is annealed to the vector
followed by addition of a DNA polymerase, such as E. coli DNA
polymerase I (Klenow fragment), which uses the double stranded
region as a primer to produce a heteroduplex in which one strand
encodes the altered sequence and the other the original sequence.
The heteroduplex is introduced into appropriate bacterial cells and
clones that include the desired mutation are selected. The
resulting altered DNA molecules may be expressed recombinantly in
appropriate host cells to produce the modified protein.
[0097] Equivalent DNA constructs that encode various additions or
substitutions of amino acid residues or sequences, or deletions of
terminal or internal residues or sequences not needed for
biological activity are also encompassed herein. For example, and
as discussed above, sequences encoding Cys residues that are not
desirable or essential for biological activity can be altered to
cause the Cys residues to be deleted or replaced with other amino
acids, preventing formation of incorrect intramolecular disulfide
bridges upon renaturation. Alternatively, additional Cys residues
can be introduced to facilitate multimerization.
[0098] Binding domains for TL and binding interactions of TL with
its receptor, TNFRSF25 have been characterized, such that one
having ordinary skill in the art may readily select appropriate
polypeptide domains for inclusion in the encoded products of the
instant expression constructs, and will understand which nucleic
acid and/or amino acid residues are amenable to modification (e.g.,
substitution, deletion, addition, etc.). See e.g., Zhan et al.
Biochemistry. 2009 Aug. 18; 48(32):7636-45. Further, assays for
determining whether any of the above described polypeptides
specifically bind to TNFRSF25 are known in the art and described in
Example 1, as discussed above.
[0099] Nucleic acids and oligonucleotides for use as described
herein can be synthesized by any method known to those of skill in
this art (see, e.g., WO 93/01286, U.S. application Ser. No.
07/723,454; U.S. Pat. No. 5,218,088; U.S. Pat. No. 5,175,269; U.S.
Pat. No. 5,109,124). Identification of oligonucleotides and nucleic
acid sequences for use in the present disclosure involves methods
well known in the art. For example, the desirable properties,
lengths and other characteristics of useful oligonucleotides are
well known. In certain embodiments, synthetic oligonucleotides and
nucleic acid sequences may be designed that resist degradation by
endogenous host cell nucleolytic enzymes by containing such
linkages as: phosphorothioate, methylphosphonate, sulfone, sulfate,
ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and
other such linkages that have proven useful in antisense
applications (see, e.g., Agrawal and Goodchild; Tetrehedron Lett.
28:3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665
(1971); Stec et al., Tetrehedron Lett. 26:2191-2194 (1985); Moody
et al., Nucl. Acids Res. 12:4769-4782 (1989); Uznanski et al.,
Nucl. Acids Res. (1989); Letsinger et al., Tetrahedron 40:137-143
(1984); Eckstein, Annu Rev. Biochem. 54:367-402 (1985); Eckstein,
Trends Biol. Sci. 14:97-100 (1989); Stein In:
Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression,
Cohen, Ed, Macmillan Press, London, pp. 97-117 (1989); Jager et
al., Biochemistry 27:7237-7246 (1988)).
[0100] Host organisms include those organisms in which recombinant
production of TL1A fusion products encoded by the recombinant
constructs of the present disclosure may occur, such as bacteria
(for example, E. coli), yeast (for example, Saccharomyces
cerevisiae and Pichia pastoris), insect cells and mammals,
including in vitro and in vivo expression. Host organisms thus may
include organisms for the construction, propagation, expression or
other steps in the production of the compositions provided herein;
hosts also include subjects in which immune responses take place,
as described above. Presently preferred host organisms are E. coli
bacterial strains, inbred murine strains and murine cell lines,
non-human primate subjects and cell lines, and human cells,
subjects and cell lines.
[0101] The DNA construct encoding the desired TL1A fusion protein
is introduced into a plasmid for expression in an appropriate host.
The host can be a bacterial host. The sequence encoding the ligand
or nucleic acid binding domain is preferably codon-optimized for
expression in the particular host. Thus, for example, if a human
TL1A fusion protein is expressed in bacteria, the codons would be
optimized for bacterial usage. For small coding regions, the gene
can be synthesized as a single oligonucleotide. For larger
proteins, splicing of multiple oligonucleotides, mutagenesis, or
other techniques known to those in the art may be used. The
sequences of nucleotides in the plasmids that are regulatory
regions, such as promoters and operators, are operationally
associated with one another for transcription. The sequence of
nucleotides encoding a TL1A fusion protein may also include DNA
encoding a secretion signal, whereby the resulting peptide is a
precursor protein. The resulting processed protein may be recovered
from the periplasmic space or the fermentation medium.
[0102] The DNA plasmids can also include a transcription terminator
sequence. As used herein, a "transcription terminator region" is a
sequence that signals transcription termination. The entire
transcription terminator may be obtained from a protein-encoding
gene, which may be the same or different from the inserted TL1A
fusion protein encoding gene or the source of the promoter.
Transcription terminators are optional components of the expression
systems herein.
[0103] The plasmids used herein include a promoter in operative
association with the DNA encoding the protein or polypeptide of
interest and are designed for expression of proteins in a suitable
host as described above (e.g., bacterial, murine or human)
depending upon the desired use of the plasmid (e.g., administration
of a vaccine containing TL1A fusion protein encoding sequences).
Suitable promoters for expression of proteins and polypeptides
herein are widely available and are well known in the art.
Inducible promoters or constitutive promoters that are linked to
regulatory regions are preferred. Such promoters include, but are
not limited to, the T7 phage promoter and other T7-like phage
promoters, such as the T3, T5 and SP6 promoters, the trp, 1 pp, and
lac promoters, such as the lacUV5, from E. coli; the P10 or
polyhedrin gene promoter of baculovirus/insect cell expression
systems (see, e.g., U.S. Pat. Nos. 5,243,041, 5,242,687, 5,266,317,
4,745,051, and 5,169,784) and inducible promoters from other
eukaryotic expression systems. This may also include the human
ferritin promoter. For expression of the proteins such promoters
are inserted in a plasmid in operative linkage with a control
region such as the lac operon.
[0104] Preferred promoter regions are those that are inducible and
functional in E. coli. Examples of suitable inducible promoters and
promoter regions include, but are not limited to: the E. coli lac
operator responsive to isopropyl 13-D-thiogalactopyranoside (IPTG;
see Nakamura et al., Cell 18:1109-1117, 1979); the metallothionein
promoter metal-regulatory-elements responsive to heavy-metal (e.g.,
zinc) induction (see, e.g., U.S. Pat. No. 4,870,009 to Evans et
al.); the phage T7lac promoter responsive to IPTG (see, e.g., U.S.
Pat. No. 4,952,496; and Studier et al., Meth. Enzymol. 185:60-89,
1990) and the TAC promoter.
[0105] The plasmids may optionally include a selectable marker gene
or genes that are functional in the host. A selectable marker gene
includes any gene that confers a phenotype on bacteria that allows
transformed bacterial cells to be identified and selectively grown
from among a vast majority of untransformed cells. Suitable
selectable marker genes for bacterial hosts, for example, include
the ampicillin resistance gene (Amp.sup.r), tetracycline resistance
gene (Tc.sup.r) and the kanamycin resistance gene (Kan.sup.r).
[0106] The plasmids may also include DNA encoding a signal for
secretion of the operably linked protein. Secretion signals
suitable for use are widely available and are well known in the
art. Prokaryotic and eukaryotic secretion signals functional in E.
coli may be employed. The presently preferred secretion signals
include, but are not limited to, those encoded by the following E.
coli genes: ompA, ompT, ompF, ompC, beta-lactamase, and alkaline
phosphatase, and the like (von Heijne, J. Mol. Biol. 184:99-105,
1985). In addition, the bacterial pelB gene secretion signal (Lei
et al., J. Bacteriol. 169:4379, 1987), the phoA secretion signal,
and the cek2 functional in insect cell may be employed. Other
prokaryotic and eukaryotic secretion signals known to those of
skill in the art may also be employed (see, e.g., von Heijne, J.
Mol. Biol. 184:99-105, 1985). Using the methods described herein,
one of skill in the art can substitute secretion signals that are
functional in either yeast, insect or mammalian cells to secrete
proteins from those cells.
[0107] In some embodiments preferred plasmids for transformation of
E. coli cells include the pET expression vectors (e.g., pET-11a,
pET-12a-c, pET-15b; see U.S. Pat. No. 4,952,496; available from
Novagen, Madison, Wis.). Other preferred plasmids include the pKK
plasmids, particularly pKK 223-3, which contains the tac promoter
(Brosius et al., Proc. Natl. Acad. Sci. 81:6929, 1984; Ausubel et
al., Current Protocols in Molecular Biology; U.S. Pat. Nos.
5,122,463, 5,173,403, 5,187,153, 5,204,254, 5,212,058, 5,212,286,
5,215,907, 5,220,013, 5,223,483, and 5,229,279). Plasmid pKK has
been modified by replacement of the ampicillin resistance gene with
a kanamycin resistance gene. (Available from Pharmacia; obtained
from pUC4K, see, e.g., Vieira et al. (Gene 19:259-268, 1982; and
U.S. Pat. No. 4,719,179.) Baculovirus vectors, such as pBlueBac
(also called pJVETL and derivatives thereof), particularly pBlueBac
III (see, e.g., U.S. Pat. Nos. 5,278,050, 5,244,805, 5,243,041,
5,242,687, 5,266,317, 4,745,051, and 5,169,784; available from
Invitrogen, San Diego) may also be used for expression of the
polypeptides in insect cells. Other plasmids include the
pIN-IIIompA plasmids (see U.S. Pat. No. 4,575,013; see also Duffaud
et al., Meth. Enz. 153:492-507, 1987), such as pIN-IIIompA2.
[0108] In some embodiments, the DNA molecule is replicated in
bacterial cells, preferably in E. coli. The preferred DNA molecule
also includes a bacterial origin of replication, to ensure the
maintenance of the DNA molecule from generation to generation of
the bacteria. In this way, large quantities of the DNA molecule can
be produced by replication in bacteria. Preferred bacterial origins
of replication include, but are not limited to, the fl-ori and col
E1 origins of replication. Preferred hosts contain chromosomal
copies of DNA encoding T7 RNA polymerase operably linked to an
inducible promoter, such as the lacUV promoter (see U.S. Pat. No.
4,952,496). Such hosts include, but are not limited to, lysogens E.
coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS, HMS174(DE3) and
BL21(DE3). Strain BL21(DE3) is preferred. The pLys strains provide
low levels of T7 lysozyme, a natural inhibitor of T7 RNA
polymerase.
[0109] The DNA molecules provided may also comprise a gene coding
for a repressor protein. The repressor protein is capable of
repressing the transcription of a promoter that contains sequences
of nucleotides to which the repressor protein binds. The promoter
can be de-repressed by altering the physiological conditions of the
cell. For example, the alteration can be accomplished by adding to
the growth medium a molecule that inhibits the ability to interact
with the operator or with regulatory proteins or other regions of
the DNA or by altering the temperature of the growth media.
Repressor proteins include, but are not limited to the E. coli lad
repressor responsive to IPTG induction, the temperature sensitive
Xc1857 repressor, and the like.
[0110] In general, recombinant constructs will also contain
elements necessary for transcription and translation. In
particular, such elements are preferred where the recombinant
expression construct containing nucleic acid sequences encoding
TL1A fusion proteins is intended for expression in a host cell or
organism. In certain embodiments of the present disclosure, cell
type preferred or cell type specific expression of a cell TL1A
fusion protein encoding gene may be achieved by placing the gene
under regulation of a promoter. The choice of the promoter will
depend upon the cell type to be transformed and the degree or type
of control desired. Promoters can be constitutive or active and may
further be cell type specific, tissue specific, individual cell
specific, event specific, temporally specific or inducible.
Cell-type specific promoters and event type specific promoters are
preferred. Examples of constitutive or nonspecific promoters
include the SV40 early promoter (U.S. Pat. No. 5,118,627), the SV40
late promoter (U.S. Pat. No. 5,118,627), CMV early gene promoter
(U.S. Pat. No. 5,168,062), and adenovirus promoter. In addition to
viral promoters, cellular promoters can also be used. In
particular, cellular promoters for the so-called housekeeping genes
are useful. Viral promoters are preferred, because generally they
are stronger promoters than cellular promoters. Promoter regions
have been identified in the genes of many eukaryotes including
higher eukaryotes, such that suitable promoters for use in a
particular host can be readily selected by those skilled in the
art.
[0111] Inducible promoters may also be used. These promoters
include MMTV LTR (PCT WO 91/13160), inducible by dexamethasone;
metallothionein promoter, inducible by heavy metals; and promoters
with cAMP response elements, inducible by cAMP. By using an
inducible promoter, the nucleic acid sequence encoding a TL1A
fusion protein may be delivered to a cell by the expression
construct and will remain quiescent until the addition of the
inducer. This allows further control on the timing of production of
the gene product.
[0112] Event-type specific promoters are active or up-regulated
only upon the occurrence of an event, such as tumorigenicity or
viral infection. The HIV LTR is a well-known example of an
event-specific promoter. The promoter is inactive unless the tat
gene product is present, which occurs upon viral infection. Some
event-type promoters are also tissue-specific.
[0113] Additionally, promoters that are coordinately regulated with
a particular cellular gene may be used. For example, promoters of
genes that are coordinately expressed may be used when expression
of a particular TL1A fusion protein-encoding gene is desired in
concert with expression of one or more additional endogenous or
exogenously introduced genes. This type of promoter is especially
useful when one knows the pattern of gene expression relevant to
induction of an immune response in a particular tissue of the
immune system, so that specific immunocompetent cells within that
tissue may be activated or otherwise recruited to participate in
the immune response.
[0114] In addition to the promoter, repressor sequences, negative
regulators, or tissue-specific silencers may be inserted to reduce
non-specific expression of TL1A fusion protein encoding genes in
certain situations, such as, for example, a host that is
transiently immunocompromised as part of a therapeutic strategy.
Multiple repressor elements may be inserted in the promoter region.
Repression of transcription is independent on the orientation of
repressor elements or distance from the promoter. One type of
repressor sequence is an insulator sequence. Such sequences inhibit
transcription (Dunaway et al., Mol Cell Biol 17: 182-9, 1997; Gdula
et al., Proc Natl Acad Sci USA 93:9378-83, 1996, Chan et al., J
Virol 70: 5312-28, 1996; Scott and Geyer, EMBO J. 14:6258-67, 1995;
Kalos and Fournier, Mol Cell Biol 15:198-207, 1995; Chung et al.,
Cell 74: 505-14, 1993) and will silence background
transcription.
[0115] Repressor elements have also been identified in the promoter
regions of the genes for type II (cartilage) collagen, choline
acetyltransferase, albumin (Hu et al., J. Cell Growth Differ.
3(9):577-588, 1992), phosphoglycerate kinase (PGK-2) (Misuno et
al., Gene 119(2):293-297, 1992), and in the
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.
(Lemaigre et al., Mol. Cell. Biol. 11(2):1099-1106.) Furthermore,
the negative regulatory element Tse-1 has been identified in a
number of liver specific genes, and has been shown to block cAMP
response element--(CRE) mediated induction of gene activation in
hepatocytes. (Boshart et al., Cell 61(5):905-916, 1990).
[0116] In some embodiments, elements that increase the expression
of the desired product are incorporated into the construct. Such
elements include internal ribosome binding sites (IRES; Wang and
Siddiqui, Curr. Top. Microhiol. Immunol 203:99, 1995; Ehrenfeld and
Semler, Curr. Top. Microhiol. Immunol. 203:65, 1995; Rees et al.,
Biotechniques 20:102, 1996; Sugimoto et al., Biotechnology 12:694,
1994). IRES increase translation efficiency. Other sequences may
also enhance expression, e.g., for some genes, sequences especially
at the 5' end inhibit transcription and/or translation. These
sequences are usually palindromes that can form hairpin structures.
Any such sequences in the nucleic acid to be delivered are
generally deleted. Expression levels of the transcript or
translated product are assayed to confirm or ascertain which
sequences affect expression. Transcript levels may be assayed by
any known method, including Northern blot hybridization, RNase
probe protection and the like. Protein levels may be assayed by any
known method, including ELISA, western blot, immunocytochemistry or
other well-known techniques.
[0117] Other elements may be incorporated into the TL1A fusion
protein encoding constructs of the present disclosure. For example,
the construct can include a transcription terminator sequence,
including a polyadenylation sequence, splice donor and acceptor
sites, and an enhancer. Other elements useful for expression and
maintenance of the construct in mammalian cells or other eukaryotic
cells may also be incorporated (e.g., origin of replication).
Because the constructs can be conveniently produced in bacterial
cells, elements that are necessary for, or that enhance,
propagation in bacteria can be incorporated. Such elements include
an origin of replication, a selectable marker and the like.
[0118] As provided herein, an additional level of controlling the
expression of nucleic acids encoding TL1A fusion proteins delivered
to cells using the constructs disclosed herein may be provided by
simultaneously delivering two or more differentially regulated
nucleic acid constructs. The use of such a multiple nucleic acid
construct approach may permit coordinated regulation of an immune
response such as, for example, spatiotemporal coordination that
depends on the cell type and/or presence of another expressed
encoded component. Those familiar with the art will appreciate that
multiple levels of regulated gene expression may be achieved in a
similar manner by selection of suitable regulatory sequences,
including but not limited to promoters, enhancers and other well
known gene regulatory elements.
[0119] The present disclosure also relates to vectors, and to
constructs prepared from known vectors that include nucleic acids
of the present disclosure, and in particular to "recombinant
expression constructs" that include any nucleic acids encoding TL1A
fusion proteins and polypeptides as provided above; to host cells
which are genetically engineered with vectors and/or constructs and
to methods of administering expression constructs containing
nucleic acid sequences encoding such TL1A fusion proteins disclosed
herein, or fragments, orthologs or variants thereof, by recombinant
techniques. TL1A fusion proteins can be expressed in virtually any
host cell under the control of appropriate promoters, depending on
the nature of the construct (e.g., type of promoter, as described
above), and on the nature of the desired host cell (e.g., whether
postmitotic terminally differentiated or actively dividing; e.g.,
whether the expression construct occurs in host cell as an episome
or is integrated into host cell genome). Appropriate cloning and
expression vectors for use with prokaryotic and eukaryotic hosts
are described by Sambrook, et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor, N.Y., (1989); as noted
above, in some embodiments, recombinant expression is conducted in
mammalian cells that have been transfected or transformed with the
recombinant expression constructs described herein.
[0120] Typically, the constructs are derived from plasmid vectors.
A preferred construct is a modified pNASS vector (Clontech, Palo
Alto, Calif.), which has nucleic acid sequences encoding an
ampicillin resistance gene, a polyadenylation signal and a T7
promoter site. Other suitable mammalian expression vectors are well
known (see, e.g., Ausubel et al., 1995; Sambrook et al., supra; see
also, e.g., catalogues from Invitrogen, San Diego, Calif.; Novagen,
Madison, Wis.; Pharmacia, Piscataway, N.J.; and others). Presently
preferred constructs may be prepared that include a dihydrofolate
reductase (DHFR) encoding sequence under suitable regulatory
control, for promoting enhanced production levels of the TL1A
fusion protein, which levels result from gene amplification
following application of an appropriate selection agent (e.g.,
methotrexate).
[0121] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, and a promoter derived from a
highly-expressed gene to direct transcription of a downstream
structural sequence, as described above. The heterologous
structural sequence is assembled in appropriate phase with
translation initiation and termination sequences. Thus, for
example, the TL1A fusion protein encoding nucleic acids as provided
herein may be included in any one of a variety of expression vector
constructs as a recombinant expression construct for expressing a
TL1A fusion protein in a host cell. In certain preferred
embodiments the constructs are included in formulations that are
administered in vivo. Such vectors and constructs include
chromosomal, nonchromosomal and synthetic DNA sequences, e.g.,
derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids;
vectors derived from combinations of plasmids and phage DNA, viral
DNA, such as vaccinia, adenovirus, fowl pox virus, and
pseudorabies, or replication deficient retroviruses as described
below. However, any other vector may be used for preparation of a
recombinant expression construct, and in preferred embodiments such
a vector will be replicable and viable in the host.
[0122] The appropriate DNA sequence(s) may be inserted into the
vector by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Standard techniques for cloning, DNA
isolation, amplification and purification, for enzymatic reactions
involving DNA ligase, DNA polymerase, restriction endonucleases and
the like, and various separation techniques are those known and
commonly employed by those skilled in the art. A number of standard
techniques are described, for example, in Ausubel et al. (1993
Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc.
& John Wiley & Sons, Inc., Boston, Mass.); Sambrook et al.
(1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory,
Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning, Cold
Spring Harbor Laboratory, Plainview, N.Y.); Glover (Ed.) (1985 DNA
Cloning Vol. I and II, IRL Press, Oxford, UK); Hames and Higgins
(Eds.), (1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK);
and elsewhere.
[0123] The DNA sequence in the expression vector is operatively
linked to at least one appropriate expression control sequences
(e.g., a constitutive promoter or a regulated promoter) to direct
mRNA synthesis. Representative examples of such expression control
sequences include promoters of eukaryotic cells or their viruses,
as described above. Promoter regions can be selected from any
desired gene using CAT (chloramphenicol transferase) vectors or
other vectors with selectable markers. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art, and preparation of certain particularly
preferred recombinant expression constructs containing at least one
promoter or regulated promoter operably linked to a nucleic acid
encoding an TL1A fusion protein is described herein.
[0124] Transcription of the DNA encoding the polypeptides of the
present disclosure by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 by that
act on a promoter to increase its transcription. Examples including
the SV40 enhancer on the late side of the replication origin by 100
to 270, a cytomegalovirus early promoter enhancer, the polyoma
enhancer on the late side of the replication origin, and adenovirus
enhancers.
[0125] As provided herein, in some embodiments the vector may be a
viral vector such as a retroviral vector. (Miller et al., 1989
BioTechniques 7:980; Coffin and Varmus, 1996 Retroviruses, Cold
Spring Harbor Laboratory Press, NY.) For example, retroviruses from
which the retroviral plasmid vectors may be derived include, but
are not limited to, Moloney Murine Leukemia Virus, spleen necrosis
virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma
virus, avian leukosis virus, gibbon ape leukemia virus, human
immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma
Virus, and mammary tumor virus.
[0126] Retroviruses are RNA viruses which can replicate and
integrate into the genome of a host cell via a DNA intermediate.
This DNA intermediate, or provirus, may be stably integrated into
the host cell DNA. According to certain embodiments of the present
disclosure, an expression construct may contain a retrovirus into
which a foreign gene that encodes a foreign protein is incorporated
in place of normal retroviral RNA. When retroviral RNA enters a
host cell coincident with infection, the foreign gene is also
introduced into the cell, and may then be integrated into host cell
DNA as if it were part of the retroviral genome. Expression of this
foreign gene within the host results in expression of the foreign
protein.
[0127] Most retroviral vector systems which have been developed for
gene therapy are based on murine retroviruses. Such retroviruses
exist in two forms, as free viral particles referred to as virions,
or as proviruses integrated into host cell DNA. The virion form of
the virus contains the structural and enzymatic proteins of the
retrovirus (including the enzyme reverse transcriptase), two RNA
copies of the viral genome, and portions of the source cell plasma
membrane containing viral envelope glycoprotein. The retroviral
genome is organized into four main regions: the Long Terminal
Repeat (LTR), which contains cis-acting elements necessary for the
initiation and termination of transcription and is situated both 5'
and 3' of the coding genes, and the three coding genes gag, pol,
and env. These three genes gag, pol, and env encode, respectively,
internal viral structures, enzymatic proteins (such as integrase),
and the envelope glycoprotein (designated gp70 and p15e) which
confers infectivity and host range specificity of the virus, as
well as the "R" peptide of undetermined function.
[0128] Separate packaging cell lines and vector producing cell
lines have been developed because of safety concerns regarding the
uses of retroviruses, including their use in expression constructs
as provided by the present disclosure. Briefly, this methodology
employs the use of two components, a retroviral vector and a
packaging cell line (PCL). The retroviral vector contains long
terminal repeats (LTRs), the foreign DNA to be transferred and a
packaging sequence (y). This retroviral vector will not reproduce
by itself because the genes which encode structural and envelope
proteins are not included within the vector genome. The PCL
contains genes encoding the gag, pol, and env proteins, but does
not contain the packaging signal "y". Thus, a PCL can only form
empty virion particles by itself. Within this general method, the
retroviral vector is introduced into the PCL, thereby creating a
vector-producing cell line (VCL). This VCL manufactures virion
particles containing only the retroviral vector's (foreign) genome,
and therefore has previously been considered to be a safe
retrovirus vector for therapeutic use.
[0129] "Retroviral vector construct" refers to an assembly which is
capable of directing the expression of a sequence(s) or gene(s) of
interest, such as TL1A fusion protein encoding nucleic acid
sequences. Briefly, the retroviral vector construct must include a
5' LTR, a tRNA binding site, a packaging signal, an origin of
second strand DNA synthesis and a 3' LTR. A wide variety of
heterologous sequences may be included within the vector construct,
including for example, sequences which encode a protein (e.g.,
cytotoxic protein, disease-associated antigen, immune accessory
molecule, or replacement gene), or which are useful as a molecule
itself (e.g., as a ribozyme or antisense sequence).
[0130] Retroviral vector constructs of the present disclosure may
be readily constructed from a wide variety of retroviruses,
including for example, B, C, and D type retroviruses as well as
spumaviruses and lentiviruses (see, e.g., RNA Tumor Viruses, Second
Edition, Cold Spring Harbor Laboratory, 1985). Such retroviruses
may be readily obtained from depositories or collections such as
the American Type Culture Collection ("ATCC"; Rockville, Md.), or
isolated from known sources using commonly available techniques.
Any of the above retroviruses may be readily utilized in order to
assemble or construct retroviral vector constructs, packaging
cells, or producer cells of the present disclosure given the
disclosure provided herein, and standard recombinant techniques
(e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d
ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS
82:488, 1985).
[0131] Suitable promoters for use in viral vectors generally may
include, but are not limited to, the retroviral LTR; the SV40
promoter; and the human cytomegalovirus (CMV) promoter described in
Miller, et al., Biotechniques 7:980-990 (1989), or any other
promoter (e.g., cellular promoters such as eukaryotic cellular
promoters including, but not limited to, the histone, pol III, and
(.beta.-actin promoters). Other viral promoters which may be
employed include, but are not limited to, adenovirus promoters,
thymidine kinase (TK) promoters, and B19 parvovirus promoters. The
selection of a suitable promoter will be apparent to those skilled
in the art from the teachings contained herein, and may be from
among either regulated promoters or promoters as described
above.
[0132] As described above, the retroviral plasmid vector is
employed to transduce packaging cell lines to form producer cell
lines. Examples of packaging cells which may be transfected
include, but are not limited to, the PE501, PA317, .Psi-2, Psi-AM,
PA12, T19-14X, VT-19-17-H2, Psi-CRE, Psi-CRIP, GP+E-86, GP+envAm12,
and DAN cell lines as described in Miller, Human Gene Therapy,
1:5-14 (1990). The vector may transduce the packaging cells through
any means known in the art. Such means include, but are not limited
to, electroporation, the use of liposomes, and CaPO.sub.4
precipitation. In one alternative, the retroviral plasmid vector
may be encapsulated into a liposome, or coupled to a lipid, and
then administered to a host.
[0133] The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the TL1A fusion proteins. Such retroviral vector particles
then may be employed, to transduce eukaryotic cells, either in
vitro or in vivo. The transduced eukaryotic cells will express the
nucleic acid sequence(s) encoding the TL1A fusion protein.
Eukaryotic cells which may be transduced include, but are not
limited to, embryonic stem cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, circulating peripheral blood
mononuclear and polymorphonuclear (PMN) cells including
myelomonocytic cells, lymphocytes, myoblasts, tissue macrophages,
dendritic cells, Kupffer cells, lymphoid and reticuloendothelia
cells of the lymph nodes and spleen, keratinocytes, endothelial
cells, and bronchial epithelial cells.
[0134] As another example in which a viral vector is used to
prepare the recombinant TL1A fusion expression construct, host
cells transduced by a recombinant viral construct directing the
expression of TL1A fusion proteins or fusion proteins may produce
viral particles containing expressed TL1A fusion proteins or fusion
proteins that are derived from portions of a host cell membrane
incorporated by the viral particles during viral budding.
[0135] In some embodiments, the present disclosure relates to host
cells containing the above described recombinant TL1A fusion
expression constructs. Host cells are genetically engineered
(transduced, transformed or transfected) with the vectors and/or
expression constructs disclosed herein, which may be, for example,
a cloning vector, a shuttle vector or an expression construct. The
vector or construct may be, for example, in the form of a plasmid,
a viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying
particular genes such as genes encoding TL1A fusion proteins or
TL1A fusion proteins. The culture conditions for particular host
cells selected for expression, such as temperature, pH and the
like, will be readily apparent to the ordinarily skilled
artisan.
[0136] The host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell,
or the host cell can be a prokaryotic cell, such as a bacterial
cell. Representative examples of appropriate host cells according
to the present disclosure include, but need not be limited to,
bacterial cells, such as E. coli, Streptomyces, Salmonella
typhimurium; fungal cells, such as yeast; insect cells, such as
Drosophila S2 and Spodoptera 519; animal cells, such as CHO, COS or
293 cells; adenoviruses; plant cells, or any suitable cell already
adapted to in vitro propagation or so established de novo. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein.
[0137] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will contain an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences, for example as described herein regarding
the preparation of TL1A fusion expression constructs. DNA sequences
derived from the SV40 splice, and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
Introduction of the construct into the host cell can be effected by
a variety of methods with which those skilled in the art will be
familiar, including but not limited to, for example, calcium
phosphate transfection, DEAE Dextran mediated transfection, or
electroporation (Davis et al., 1986 Basic Methods in Molecular
Biology).
[0138] mTOR Inhibitors
[0139] In some embodiments, provided herein are combination
therapies for, e.g., modulating an antigen-specific immune
response, and/or for treating a disease or disorder associated with
an antigen-specific immune response, and/or for treating one or
more symptoms of the disease or disorder, in a human patient in
need thereof. In some embodiments, the methods comprise
administering to the patient in need thereof a composition
comprising a TNFRSF25 agonist (e.g., TL1A fusion protein, agonistic
anti-TNFRSF25 antibody, small molecule agonist of TNFR25 agonist,
etc.) and an effective amount of an mTOR inhibitor. In some
embodiments, the above methods comprise administering to the
patient a combination therapy comprising TNFRSF25 agonist and an
mTOR inhibitor.
[0140] The mammalian target of rapamycin, commonly known as mTOR,
is a serine/threonine protein kinase that regulates cell growth,
cell proliferation, cell motility, cell survival, protein
synthesis, and transcription. mTOR is a key intermediary in
multiple mitogenic signaling pathways and plays a central role in
modulating proliferation and angiogenesis in normal tissues and
neoplastic processes. mTOR exists within two complexes, mTORC1 and
mTORC2. mTORC1 is sensitive to rapamycin analogs (such as
temsirolimus or everolimus) and mTORC2 is largely
rapamycin-insensitive.
[0141] As used herein, the term "mTOR inhibitor" refers to a
compound or a ligand that inhibits at least one activity of an
mTOR, such as the serine/threonine protein kinase activity on at
least one of its substrates (e.g., p70S6 kinase 1, 4E-BP1, AKT/PKB
and eEF2). A person skilled in the art can readily determine
whether a compound, such as rapamycin or an analogue or derivative
thereof, or other compound, antibody, or small molecule, etc., is
an mTOR inhibitor. Methods of identifying mTOR inhibitors are known
in the art. Examples of mTOR inhibitors include, without
limitation, rapamycin (sirolimus), rapamycin derivatives, CI-779,
everolimus (Certican.TM.), ABT-578, tacrolimus (FK 506), ABT-578,
AP-23675, BEZ-235, OSI-027, QLT-0447, ABI-009, BC-210, salirasib,
TAFA-93, deforolimus (AP-23573), temsirolimus (Torisel.TM.),
2-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol
(PP242) and AP-23841.
[0142] As used herein, the term "selective mTOR inhibitor" refers
to a compound or a ligand that inhibits mTOR activity but does not
inhibit PI3K activity. Suitable selective mTOR inhibitors include
RAD001. Accordingly, in some embodiments, provided herein is a
combination therapy comprising the administration of a TNFRSF25
agonist and the administration of a selective mTOR inhibitor.
[0143] Rapamycin is a known macrolide antibiotic produced by
Streptomyces hygroscopicus. Suitable derivatives of rapamycin are
disclosed, e.g., in WO 94/09010, WO 95/16691, WO 96/41807, U.S.
Pat. No. 5,362,718 and WO 99/15530. They may be prepared using the
procedures described in these references. Representative rapamycin
derivatives are, e.g., 32-deoxorapamycin,
16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32(S or
R)-dihydro-rapamycin, 16-pent-2-ynyloxy-32(S or
R)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (also
called CCI779) or 40-epi-(tetrazolyl)-rapamycin (also called
ABT578). Rapamycin derivatives may also include the so-called
rapalogs, e.g., as disclosed in WO 98/02441 and WO 01/14387, e.g.
AP23573, AP23464, AP23675 or AP23841. Further, non-limiting
examples of a rapamycin derivative are those disclosed under the
name TAFA-93 (a rapamycin prodrug), biolimus-7 or biolimus-9.
[0144] In some embodiments, the mTOR inhibitor used in a
composition and/or combination therapy provided herein is
Everolimus (RAD001) or
2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol
(PP242) (see, e.g., Apsel et al., Nature Chemical to Biology 4,
691-699 (2008)).
[0145] Interleukins
[0146] In any of the compositions and methods disclosed herein
comprising an interleukin, the interleukin can be any interleukin
that achieves the desired synergistic effect on the expansion of
Treg cells, e.g. when administered in a combination therapy with an
agonist of TNFRSF25. In some embodiments, the interleukin is IL-2.
In some embodiments, the interleukin is IL-7. In some embodiments,
the interleukin is IL-15.
[0147] Also encompassed herein are analogs of IL-2, e.g., agonist
and partial agonist IL-2 analogs (e.g., IL-2 muteins). Such analogs
are known in the art. A non-limiting example of an agonist IL-2
analog includes, e.g., BAY 50-4798 (see Margolin et al. Clin Cancer
Res Jun. 1, 2007 13; 3312; for other examples, see also, Imler and
Zurawski. J Biol. Chem. 1992 Jul. 5; 267(19):13185-90. Furthermore,
in vitro screening assays for determining whether a compound is an
IL-2 analog (i.e., maintains the ability to bind to the high
affinity IL-2 receptor and initiate T cell proliferation) are known
in the art. See, e.g., Zurawski and Zurawski. EMBO J. 1992
November; 11(11): 3905-3910; "The Interleukin 2 Receptor" Annual
Review of Cell Biology; Vol. 5: 397-425 (Volume publication date
November 1989; and "The Biology of Interleukin-2"; Annual Review of
Immunology; Vol. 26: 453-479 (Volume publication date April
2008).
[0148] Compositions and Pharmaceutical Compositions
[0149] In some embodiments, provided herein are compositions
comprising a human TL1A-Ig fusion protein, wherein the fusion
protein comprises (a) a first polypeptide comprising a polypeptide
that specifically binds to TNFRSF25; and (b) a second polypeptide
comprising an immunoglobulin (Ig) polypeptide. In some embodiments,
the first polypeptide comprises the extracellular domain of a human
TL1A polypeptide or a fragment thereof, and wherein the fragment is
capable of specifically binding to TNFRSF25. In some embodiments,
when administered to a human in need thereof, the composition
reduces the frequency of naive CD4 T cells in the human.
[0150] In some embodiments, provided herein are compositions
comprising a TNFRSF25 agonist and one or both of an interleukin
(e.g., IL-2, IL-7, IL-15, or an analog thereof) and an mTOR
inhibitor.
[0151] In some embodiments, provided herein are compositions
comprising a TL1A fusion protein as described herein and an
effective amount of IL-2.
[0152] In some embodiments, provided herein are compositions
comprising an agonistic TNFRSF25 antibody as described herein and
an effective amount of IL-2.
[0153] In some embodiments, provided herein are compositions
comprising a small molecule agonist of TNFRSF25 as described herein
and an effective amount of IL-2.
[0154] In some embodiments, provided herein are compositions
comprising a TL1A fusion protein as described herein and an
effective amount of an mTOR inhibitor.
[0155] In some embodiments, provided herein are compositions
comprising an agonistic TNFRSF25 antibody as described herein and
an effective amount of an mTOR inhibitor.
[0156] In some embodiments, provided herein are compositions
comprising a small molecule agonist of TNFRSF25 as described herein
and an effective amount of an mTOR inhibitor.
[0157] In some embodiments, provided herein are compositions
comprising a TL1A fusion protein as described herein and an
effective amount of rapamycin.
[0158] In some embodiments, provided herein are compositions
comprising an agonistic TNFRSF25 antibody as described herein and
an effective amount of rapamycin.
[0159] In some embodiments, provided herein are compositions
comprising a small molecule agonist of TNFRSF25 as described herein
and an effective amount of rapamycin.
[0160] While it is possible to use a composition disclosed herein
(a composition containing a TL1A fusion protein) for therapy as is,
in some embodiments it may be preferable to formulate the
composition in a pharmaceutical formulation, e.g., in admixture
with a suitable pharmaceutical excipient, diluent, or carrier
selected with regard to the intended route of administration and
standard pharmaceutical practice. Accordingly, pharmaceutical
compositions or formulations containing at least one active
composition disclosed herein (e.g., TL1A fusion protein, agonistic
anti-TNFRSF25 antibody, small molecule agonist of TNFRSF25, etc.)
in association with a pharmaceutically acceptable excipient,
diluent, and/or carrier are provided herein. The excipient, diluent
and/or carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
deleterious to the recipient thereof.
[0161] The compositions can be formulated for administration in any
convenient way for use in human or veterinary medicine. For in vivo
administration to humans, the compositions disclosed herein can be
formulated according to known methods used to prepare
pharmaceutically useful compositions. The TNFRSF25 agonists (e.g.,
TL1A fusion proteins, agonistic anti-TNFRSF25 antibodies, small
molecule agonist of TNFRSF25, etc.) can be combined in admixture,
either as the sole active material or with other known active
materials, (e.g., one or more therapies useful for combination
therapy, as described below) with pharmaceutically suitable
diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g.,
Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers,
adjuvants and/or carriers. Suitable carriers and their formulations
are described in Remington's Pharmaceutical Sciences, 16th ed.
1980, Mack Publishing Co. The TNFRSF25 agonists (e.g., TL1A fusion
proteins, antibodies) described herein, as well as interleukins
(IL-2, or an analog thereof etc.) and mTOR inhibitors (e.g.,
rapamycin, etc.) can be formulated together or separately as a
sustained release composition. A "sustained release composition"
can include any suitable vehicle that releases the TL1A fusion
protein over a period of time. Non-limiting examples of sustained
release compositions include microspheres (e.g.,
poly(DL-lactide-co-glycolide) (PLGA) microspheres), anhydrous
poly-vinyl alcohol (PVA), millicylinders, alginate gels,
biodegradable hydrogels, complexing agents and nanoparticles. [See,
e.g., Ashton, et al. (2007) Biomaterials, 28, 36, 5518; Drury, J.
L. et al. (2003) Biomaterials; 24:4337-4351; U.S. Pat. No.
7,226,617 to Ding et al.; Simmons, C. A. et al. (2004) Bone;
35:562-569; Zhu, G. et al. (2000) Nat Biotech; 18:52-57,
Biodegradable Hydrogels for Drug Delivery, K. Park et al, 1993,
Technomic Publishing, Trans Am Ophthalmol Soc, K. Derwent et al,
2008; 106:206-13.]
[0162] Administration and Dosage
[0163] The compositions described herein can be administered by any
suitable route of administration known in the art. For example, the
TNFRSF25 agonists, interleukins, and mTOR inhibitors can be
formulated, together or separately, for parenteral administration
(e.g., intravenous, intraperitoneal, epidural, intrathecal,
intramuscular, intraluminal, intratracheal, intradermal or
subcutaneous).
[0164] In an exemplary embodiment, an TL1A fusion protein,
agonistic anti-TNFRSF25 antibody, or a small molecule agonist of
TNFRSF25 is administered to a patient via an immunization route,
e.g., intra-venously, intra-muscularly, intra-peritoneally, and the
like.
[0165] For any composition or formulation used in the methods
described herein, the therapeutically effective dose can be
estimated initially from animal models. Dose-response curves
derived from animal systems are then used to determine testing
doses for the initial clinical studies in humans. In safety
determinations for each composition, the dose and frequency of
administration should meet or exceed those anticipated for use in
the clinical studies. The data obtained from the animal studies can
be used in formulating a range of doses for use in humans. The
therapeutically effective doses for use in humans is preferably
within a range of circulating concentrations that include the
ED.sub.50 with little or no toxicity. The dosage can vary within
this range depending upon the dosage form employed and the route of
administration utilized.
[0166] The compositions described herein will typically contain an
effective amount of the compositions for achieving the desired
effect. As used herein the terms "therapeutically effective amount"
and "effective amount," used interchangeably, applied to a dose or
amount refers to a quantity of a composition, compound or
pharmaceutical formulation that is sufficient to result in a
desired activity upon administration to an animal in need thereof.
Within the context of the present disclosure, the term
"therapeutically effective amount" refers to that quantity of a
composition, compound or pharmaceutical formulation that is
sufficient to reduce or eliminate at least one symptom of a disease
or condition specified herein. When a combination of active
ingredients is administered, the effective amount of the
combination may or may not include amounts of each ingredient that
would have been effective if administered individually. The dosage
of the therapeutic formulation will vary, depending upon the nature
of the disease or condition, the patient's medical history, the
frequency of administration, the manner of administration, the
clearance of the agent from the host, and the like. The initial
dose may be larger, followed by smaller maintenance doses. The dose
may be administered, e.g., weekly, biweekly, daily, semi-weekly,
etc., to maintain an effective dosage level. As used herein, an
"effective amount of an interleukin" (e.g., IL-2, IL-7, IL-15, or
an analog thereof) is the amount that, when administered to a
subject as part of a combination therapy with a TNFRSF25 agonist
(e.g., an agonistic anti-TNFRSF25 antibody, a TL1A fusion protein,
or a small molecule agonist of TNFRSF25 as described herein), is
sufficient to achieve a synergistic effect on the expansion of Treg
cells. Typically, the effective amount of IL-2 or other suitable
interleukin or analog thereof used in the methods (e.g.,
combination therapies) disclosed herein is an amount (dose) that
would induce suboptimal, or fail to induce, expansion of Treg cells
if administered alone (i.e., not in a combination therapy) to a
human patient. Typically, a dose of IL-2 that would induce
suboptimal, or fail to induce, expansion of Treg cells in a human
patient is a dose that is less than 1 million units per square
meter per day (see, e.g., Koreth, J.; N Engl J. Med. 2011 Dec. 1;
365(22):2055-66; and Matsuoka, K. Sci Transl Med. 2013 Apr. 3;
5(179):179ra43). In some embodiments, IL-2 is administered to a
human patient in an amount that is considered to be a "low dose" of
IL-2 or a "very low dose" of IL-2. As used herein, a "low dose of
IL-2" is a dose of approximately 300,000 units per square meter per
day. As used herein a "very low dose of IL-2" is a dose of
approximately 30,000 units per square meter per day. In some
embodiments, an effective amount of IL-2 is an amount in the range
of 30,000 to 300,000 units per square meter per day.
[0167] As used herein, an "effective amount of an mTOR inhibitor"
(e.g., rapamycin) is the amount that, when administered to a
subject as part of a combination therapy with a TNFRSF25 agonist
(e.g., an agonistic anti-TNFRSF25 antibody, a TL1A fusion protein,
or a small molecule agonist of TNFRSF25 as described herein), is
sufficient to reduce the frequency and/or expansion of effector T
cells. Typically, the effective amount of an mTOR inhibitor is an
amount that inhibits the expansion and/or reduced the frequency of
effector T cells in a subject, e.g., by at least 1%, at least 2%,
at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at
least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99%. In other embodiments, the amount of inhibition of
effector T cells expansion and/or reduction in the frequency of
effector T cells is at 2-fold, at least 3-fold, at least 4-fold, at
least 5-fold, at least 10-fold, or more. Therapeutically effective
dosages can be determined stepwise by combinations of approaches
such as (i) characterization of effective doses of the composition
or compound in in vitro cell culture assays using, e.g., T
regulatory cell proliferation as a read-out (ii) characterization
in animal studies using T regulatory cell proliferation and/or
animal survival and/or improvement in the modeled condition (e.g.,
IBD, asthma, etc.) as a readout, followed by (iii) characterization
in human trials using improvement in condition (e.g., disease or
disorder, e.g., autoimmune disease, asthma, graft-versus host
disease, chronic infection, inflammation, etc.) and/or enhanced
survival rates as a readout.
[0168] The appropriate dose and dosage times under certain
conditions can be determined by the test based on the
above-described indices but may be refined and ultimately decided
according to the judgment of the practitioner and each patient's
circumstances (age, general condition, severity of symptoms, sex,
etc.) according to standard clinical techniques.
[0169] Typical dosages of a TL1A fusion protein in a composition
described herein range from about 0.001-100 milligram per kilogram
body weight per day (mg/kg/day), from about 0.001-50 mg/kg/day,
from about 0.0025-40 mg/kg/day, from about 0.005-30 mg/kg/day, from
about 0.01-25 mg/kg/day, from about 0.025-20 mg/kg/day, from about
0.05-15 mg/kg/day, from about 0.05-10 mg/kg/day, 0.05-5 mg/kg/day,
or from about 1-2 mg/kg/day.
[0170] Typical dosages of an agonistic anti-TNFRSF25 antibody per
day are between about 0.05 mg/kg and 10 mg/kg, between about 0.1
mg/kg and 8 mg/kg, between about 0.2 mg/kg and about 5 mg/kg, or
between about 0.4 mg/kg and about 4 mg/kg. In some embodiments, a
typical dosage of the agonistic anti-TNFRSF25 antibody is about 0.4
mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2
mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4
mg/kg, etc.
[0171] Typical dosages of IL-2 or other cytokine (e.g., IL-7,
IL-15) for use in a combination therapy disclosed herein, e.g., a
combination therapy with a TNFRSF25 agonist (e.g., TL1A fusion
protein, agonistic anti-TNFRSF25 antibody, etc.) is a dosage
between about 10,000 and 1,000,000 units per square meter per day,
between about 15,000 and 900,000 units per square meter per day,
between about 20,000 and 800,000 units per square meter per day,
between about 25,000 and 700,000 units per square meter per day,
between about 30,000 and 500,000 units per square meter per day,
between about 30,000 and 400,000 units per square meter per day, or
between about 30,000 and 300,000 units per square meter per
day.
[0172] Typical dosages of rapamycin per day are between about 25
.mu.g/kg and about 500 .mu.g/kg, between about 50 .mu.g/kg and
about 400 .mu.g/kg, or between about 75 .mu.g/kg and about 300
.mu.g/kg.
[0173] A TNFRSF25 agonist described herein (e.g., a TL1A fusion
protein, an agonistic anti-TNFRSF25 antibody, a small molecule
inhibitor of TNFRSF25, etc.), and/or one or more compositions
comprising one or more TNFRSF25 agonists and/or comprising an
interleukin (e.g., IL-2, IL-7, IL-15) can be administered to a
subject in one or more dosages sufficient to increase proliferation
of Treg cells in the subject by at least about 2-fold, at least
about 3-fold, at least about 5-fold, at least about 10-fold, or
more. According to the present invention, IL-2 and a TNFRSF25
agonist are administered in amounts that together result in a
synergistic effect on the expansion of Treg cells. Methods for
measuring Treg cell proliferation are known in the art. For
example, for monitoring Treg proliferation in vivo, peripheral
blood cells can be collected from treated subjects, stained (e.g.,
immunostaining) for cell markers including CD4, CD25, IL7R and
FoxP3, and analyzed by flow cytometry. Numbers of circulating FoxP3
Treg cells can be quantified and compared to starting numbers
(e.g., before treatment).
[0174] Further, in some embodiments, an effective amount of an mTOR
inhibitor (e.g., rapamycin) is administered to a subject in one or
more dosages sufficient to inhibit effector T cells expansion
and/or to decrease the frequency of effector T cells in the subject
by at least 5%, at least 10%, at least 20%, at least 25%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
or at least 99%. In some embodiments, the amount of inhibition of
effector T cells expansion and/or the reduction in the effector T
cell frequency is at 2-fold, at least 3-fold, at least 4-fold, at
least 5-fold, at least 10-fold, or more.
[0175] Methods for measuring the frequency and/or expansion (e.g.,
proliferation) of effector T cells are known in the art. For
example, for monitoring CD4 and/or CD8 T cell proliferation in
vivo, peripheral blood cells can be collected from treated
subjects, stained (e.g., immunostaining) for cell markers including
CD4, CD25, FoxP3, Ki67, and other suitable cell markers of
proliferation, and analyzed by flow cytometry. Numbers and
frequencies of circulating T effector cells can be quantified and
compared to starting numbers (e.g., before treatment).
[0176] Uses of TL1a Fusion Proteins and Other TNFRSF25 Agonists
[0177] Described herein are methods of modulating an
antigen-specific immune response in a human patient in need
thereof. In some embodiments, the method can include administering
to the patient a composition comprising a TL1A fusion protein
described herein. Typically, the composition comprising the TL1A
fusion protein contains a therapeutically effective amount of the
TL1A fusion protein. In a specific embodiment, the antigen-specific
immune response is inhibited.
[0178] In some embodiments, an antigen-specific immune response is
determined to be modulated if the immune response against the
antigen, as measured by any suitable measure (e.g., frequency of
antigen-specific antibodies, T cells, B cells, antigen-specific T
cell proliferation, etc.) is increased or decreased by at least 5%,
at least 10%, at least 20%, at least 25%, at least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, or at least
99%. In other embodiments, an antigen-specific immune response is
determined to be modulated if the immune response against the
antigen, as measured by any suitable measure (e.g., frequency of
antigen-specific antibodies, T cells, B cells, antigen-specific T
cell proliferation, etc.) is increased or decreased by at 2-fold,
at least 3-fold, at least 4-fold, at least 5-fold, at least
10-fold, or more. Also described herein are methods of treating a
disease or disorder associated with an antigen-specific immune
response, or treating one or more symptoms of the disease or
disorder, in a human patient in need thereof. The methods can
include administering to the patient a composition comprising a
TL1A fusion protein as described herein. Typically, the composition
comprising the TL1A fusion protein contains a therapeutically
effective amount of the TL1A fusion protein.
[0179] Also described herein are methods of reducing the severity
and/or frequency of one or more adverse events associated with
and/or caused by the administration of a TNFRSF25 agonist to a
subject (e.g., patient). In some embodiments, the method of
reducing the severity and/or frequency of one or more adverse
events comprises administering to a human patient in need thereof a
composition comprising a TL1A fusion protein as described herein in
a physiologically acceptable carrier. In some embodiments, an
adverse event that is reduced or inhibited is one or more symptoms
of inflammatory bowel disease, development of inflammatory bowel
disease, weight loss, rash, diarrhea, myalgias, decreased platelet
counts, elevated liver enzyme levels, and death.
[0180] Also provided herein are methods of modulating an
antigen-specific immune response, and/or for treating a disease or
disorder associated with an antigen-specific immune response,
and/or for treating one or more symptoms of the disease or
disorder, in a human patient in need thereof, comprising
administering a combination therapy comprising a TNFRSF2 agonist
and an interleukin (e.g., an effective amount of an interleukin,
e.g., IL-2) to a subject (patient) in need thereof. Also provided
herein are methods of modulating an antigen-specific immune
response, and/or for treating a disease or disorder associated with
an antigen-specific immune response, and/or for treating one or
more symptoms of the disease or disorder, in a human patient in
need thereof, comprising administering a combination therapy
comprising a TNFRSF25 agonist and an mTOR inhibitor (e.g.,
rapamycin) to a subject (patient) in need thereof. Also provided
herein are methods of modulating an antigen-specific immune
response, and/or for treating a disease or disorder associated with
an antigen-specific immune response, and/or for treating one or
more symptoms of the disease or disorder, in a human patient in
need thereof, comprising administering a combination therapy
comprising a TNFRSF25 agonist and an interleukin (e.g., an
effective amount of an interleukin (e.g., IL-2, IL-7, IL-15, or an
analog thereof)) and an mTOR inhibitor (e.g., rapamycin) to a
subject (patient) in need thereof.
[0181] Exemplary effective amounts of TNFRSF25 agonists,
interleukins, and mTOR inhibitors for use in the methods disclosed
herein are described above.
[0182] In any of the above-described methods, a patient in need of
treatment can be, for example and without limitation, a patient
undergoing or about to undergo induction therapy in preparation for
a solid organ or stem cell transplant, a patient who is a solid
organ or stem cell transplant recipient and is undergoing or is
about to undergo maintenance therapy, a patient who is a solid
organ or stem cell transplant recipient, an allergic patient; a
patient who is receiving or about to receive a vaccine, or a
patient being treated or about to be treated with an immune
checkpoint inhibitor (e.g., CTLA-4 or PD-1 inhibitor).
[0183] In any of the above methods, the disease or disorder that
can be treated can be an autoimmune disease or disorder (e.g.,
inflammatory bowel disease, rheumatoid arthritis), transplant
rejection, graft-versus-host disease, inflammation, asthma,
allergies, and chronic infection.
[0184] In some embodiments the above methods reduce an
antigen-specific immune response in the patient by at least 5%, at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 99%. In other
embodiments, the antigen-specific immune response is reduced by at
least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at
least 10-fold, or more.
[0185] In some embodiments, the above-described methods result in
significantly increased proliferation of Treg cells in a patient
following administration of a composition (e.g., comprising a TL1A
fusion protein) or combination therapy (e.g., administration of a
TNFRSF25 agonist and IL-2 and/or an mTOR inhibitor) described
herein. For example in some embodiments, the methods described
herein result in increased proliferation of Treg cells by at least
2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at
least 10-fold or more, in the patient following administration of
the method.
[0186] In any of the above embodiments, the methods can comprise
one or multiple administrations of one or more of the compositions
to be administered to the patient. For example, when a subject
(e.g., patient) is to be administered a composition comprising a
TL1A fusion protein, the method can comprise a single
administration or more. Exemplary dosing regimens are described
above. When a TNFRSF25 agonist is administered in a combination
therapy with an interleukin (e.g., IL-2, IL-7, IL-15) and/or an
mTOR inhibitor (e.g., rapamycin), the TNFRSF25 agonist can be
administered on the same or a different day than the interleukin
and/or mTOR inhibitor. Each active agent can be administered in a
separate composition or two or more active agents can be
administered in combination.
[0187] As described above, in some embodiments, the methods
described herein are useful for treating autoimmune diseases,
alloimmune responses, or any other disease, disorder or condition
that involves a T cell response (e.g., in a patient in need
thereof). Generally, these are conditions in which the immune
system of an individual (e.g., activated T cells) attacks the
individual's own tissues and cells, or implanted tissues, cells, or
molecules (as in a graft or transplant). Non-limiting examples of
diseases and disorders that can be treated according to the methods
described herein, include, e.g., autoimmune disease or disorder
(e.g., IBD and rheumatoid arthritis), transplant rejection,
graft-versus-host disease (GVHD), inflammation, asthma, allergies,
and chronic infection.
[0188] For transplant rejection and GVHD associated disorders, a
patient in need of treatment can be a patient who is undergoing or
about to undergo induction therapy in preparation for a solid organ
or stem cell transplant, a patient who is a solid organ or stem
cell transplant recipient and is undergoing or is about to undergo
maintenance therapy, a patient who is a solid organ or stem cell
transplant recipient (and the therapy, e.g., TL1A fusion protein or
combination therapy comprising administration of a TNFRSF25 agonist
and an interleukin (e.g., IL-2, IL-7, IL-15, or an analog thereof)
and/or an mTOR inhibitor (e.g., rapamycin), is administered in
order to facilitate early withdrawal of maintenance
immunosuppressive therapy), an allergic patient (and the therapy,
e.g., TL1A fusion protein or combination therapy comprising
administration of a TNFRSF25 agonist and an interleukin (e.g.,
IL-2, IL-7, IL-15) and/or an mTOR inhibitor (e.g., rapamycin), is
administered to reduce symptoms of a specific allergic reaction); a
patient who is receiving or about to receive a vaccine (and the
therapy, e.g., TL1A fusion protein or combination therapy
comprising administration of a TNFRSF25 agonist and an interleukin
(e.g., IL-2, IL-7, IL-15, or an analog thereof) and/or an mTOR
inhibitor (e.g., rapamycin), is administered in order to enhance
antigen-specific T cell responses stimulated by the vaccine or in
order to enhance T cell memory immune responses), or a patient
being treated or about to be treated with an immune checkpoint
inhibitor (e.g., CTLA-4 or PD-1 inhibitor) (and the therapy, e.g.,
TL1A fusion protein or combination therapy comprising
administration of a TNFRSF25 agonist and an interleukin (e.g.,
IL-2, IL-7, IL-15, or an analog thereof) and/or an mTOR inhibitor
(e.g., rapamycin), is administered in order to enhance T cell
immune responses).
[0189] Exemplary autoimmune diseases that can be treated with the
methods of the present disclosure include, e.g., type I diabetes,
multiple sclerosis, thyroiditis (such as Hashimoto's thyroiditis
and Ord's thyroiditis), Grave's disease, systemic lupus
erythematosus, scleroderma, psoriasis, arthritis, rheumatoid
arthritis, alopecia greata, ankylosing spondylitis, autoimmune
hemolytic anemia, autoimmune hepatitis, Behcet's disease, Crohn's
disease, dermatomyositis, glomerulonephritis, Guillain-Barre
syndrome, IBD, lupus nephritis, myasthenia gravis, myocarditis,
pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa,
polymyositis, primary biliary cirrhosis, rheumatic fever,
sarcoidosis, Sjogren's syndrome, ulcerative colitis, uveitis,
vitiligo, and Wegener's granulomatosis.
[0190] Exemplary alloimmune responses that can be treated with the
methods of the present disclosure include GVHD and transplant
rejection. Thus, for example, the fusion proteins disclosed herein
can be administered as an "induction therapy" in preparation for a
solid organ or stem cell transplant, or as "maintenance therapy" in
solid organ or stem cell transplant recipients, and can also be
administered to a solid organ or stem cell transplant recipient in
order to facilitate early withdrawal of maintenance
immunosuppressive therapy.
[0191] The methods described herein, e.g., comprising
administration of a TL1A fusion protein or combination therapy
comprising administration of a TNFRSF25 agonist and an interleukin
(e.g., IL-2, IL-7, IL-15, or an analog thereof) and/or an mTOR
inhibitor (e.g., rapamycin) can also be administered to an allergic
patient to reduce one or more symptoms of a specific allergic
reaction. Examples of allergic reaction include, e.g., allergic
asthma, nut (e.g., peanut) allergy, celiac disease (wheat gluten)
allergy, tolerization protocol for drug allergy (e.g., penicillins,
sulfonamides). Many substances can act as allergens; however, some
substances are very common allergens, such as, pollen and mold,
dust mite droppings, pet allergens, various foods, insect stings,
and cockroach antigens.
[0192] The compositions and combination therapies described herein
can also be administered to a patient in conjunction with a vaccine
in order to enhance antigen specific T cell responses stimulated by
the vaccine. As described above, the TL1A fusion proteins described
herein can enhance antigen-specific immune responses by having an
effect on T effector cell co-stimulation.
[0193] The TL1A fusion proteins and the combination therapies
comprising the administration of a TNFRSF25 agonist and the
administration of an interleukin (e.g., IL-2, IL-7, IL-15, or an
analog thereof) and/or an mTOR inhibitor (e.g., rapamycin)
described herein can also be administered to a patient in
conjunction with a vaccine in order to enhance T cell memory immune
responses, or in conjunction with an immune checkpoint inhibitor
(e.g., CTLA-4 or PD-1 inhibitor) in order to enhance T cell immune
responses. As described above, the TL1A fusion proteins and the
combination therapies comprising the administration of a TNFRSF25
agonist and the administration of an interleukin (e.g., IL-2, IL-7,
IL-15, or an analog thereof) and/or an mTOR inhibitor (e.g.,
rapamycin) described herein can enhance antigen-specific immune
responses by having an effect on T effector cell costimulation.
[0194] Also contemplated herein are combination therapies
comprising the administration of a TL1A fusion protein and/or
another TNFRSF25 agonist and an anti-inflammatory and/or
immunosuppressive antibody or other anti-inflammatory or
immunosuppressive agent. By way of example, in some embodiments, a
TL1A fusion protein or other TNFRSF25 agonist described herein can
be administered to a subject in an induction therapy in preparation
for a solid organ or stem cell transplant, or as maintenance
therapy in solid organ or stem cell transplant recipients, and can
be administered to a solid organ or stem cell transplant recipient
in order to facilitate early withdrawal of maintenance
immunosuppressive therapy. In some embodiments it may be
advantageous to coadminister the TL1A fusion protein in a
combination therapy with agents such as rapamycin, tacrolimus,
other mTOR inhibitors, MEK inhibitors, CTLA4-Ig molecules, CD80 or
CD86 blocking antibodies or molecules, CD40 or CD40L blocking
antibodies or molecules, PTEN blocking molecules, OX40 or OX40L
blocking antibodies or molecules, prednisone, methylprednisone,
fluticasone or combinations thereof. Alternatively, or in addition,
the TL1A fusion protein or other TNFRSF25 agonist can be
administered with an interleukin (e.g., a low dose or very low dose
of IL-2 as described in the Examples below).
[0195] As another example, in some embodiments, a TL1A fusion
protein or other TNFRSF25 agonist described herein can be
administered to a subject patient to reduce one or more symptoms of
a specific allergic reaction (e.g., asthma, celiac disease, drug
allergies). It may be advantageous to coadminister the TL1A fusion
protein or other TNFRSF25 agonist in a combination therapy with
rapamycin, tacrolimus, other mTOR inhibitors, CTLA4-Ig molecules,
CD80 or CD86 blocking antibodies or molecules, CD40 or CD40L
blocking antibodies or molecules, PTEN blocking molecules, OX40 or
OX40L blocking antibodies or molecules, prednisone,
methylprednisone, fluticasone or calcineurin inhibitors.
Alternatively, or in addition, the TL1A fusion protein or other
TNFRSF25 agonist can be administered with an interleukin (e.g., a
low dose or very low dose of IL-2, as described in the Examples
below).
[0196] Furthermore, as discussed above, it is presently discovered
that the TL1A fusion proteins disclosed herein safely and
selectively stimulate the proliferation of cognate T regulatory
cells (Treg) in vivo. In particular, in contrast to certain
previously described attempts at modulating immune responses with
TNFRSF25 modulating agents, it is presently demonstrated in studies
in humanized mice and primates, that treatment with the TL1A fusion
proteins described herein did not induce weight loss, cause changes
in white blood cell count, or lead to any other dangerous or
unwanted side effects, indicating that the TL1A fusion proteins
could be safely administered in vivo, including to primates. The
studies thus also demonstrated that the TL1A fusion proteins are
expected to be safely administered to human patients. Thus, in
conjunction with these discoveries, also contemplated herein are
methods of reducing an adverse event associated with a therapy that
includes the administration of a TNFRSF25 agonist in a human
patient. The methods include administering to a patient in need
thereof a TL1A fusion protein-containing composition described
herein in a physiologically acceptable carrier. For example, the
adverse event can be development of one or more symptoms of
inflammatory bowel disease. The adverse event can also be weight
loss, rash, diarrhea, myalgias, decreased platelet counts, elevated
liver enzyme levels, and/or death.
[0197] In accordance with the present disclosure, there may be
employed conventional molecular biology, microbiology, recombinant
DNA, immunology, cell biology and other related techniques within
the skill of the art. See, e.g., Sambrook et al., (2001) Molecular
Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory
Press: Cold Spring Harbor, N.Y.; Sambrook et al., (1989) Molecular
Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory
Press: Cold Spring Harbor, N.Y.; Ausubel et al., eds. (2005)
Current Protocols in Molecular Biology. John Wiley and Sons, Inc.:
Hoboken, N.J.; Bonifacino et al., eds. (2005) Current Protocols in
Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et
al., eds. (2005) Current Protocols in Immunology, John Wiley and
Sons, Inc.: Hoboken, N.J.; Coico et al., eds. (2005) Current
Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken,
N.J.; Coligan et al., eds. (2005) Current Protocols in Protein
Science, John Wiley and Sons, Inc.: Hoboken, N.J.; Enna et al.,
eds. (2005) Current Protocols in Pharmacology John Wiley and Sons,
Inc.: Hoboken, N.J.; Hames et al., eds. (1999) Protein Expression:
A Practical Approach. Oxford University Press: Oxford; Freshney
(2000) Culture of Animal Cells: A Manual of Basic Technique. 4th
ed. Wiley-Liss; among others. The Current Protocols listed above
are updated several times every year.
EXAMPLES
Example 1
Materials and Methods
[0198] The following are the materials and methods used in the
Examples set forth below.
[0199] Mice and Adoptive Transfer Model
[0200] Foxp3+RFP+ (FIR mice) and Foxp3+GFP+ reporter mice on a B6
background (generously provided by Dr. Richard Flavell and Dr.
Alexander Rudensky [see, Wan, Y. Y., and R. A. Flavell. 2005.
Identifying Foxp3-expressing suppressor T cells with a bicistronic
reporter. Proc. Natl. Acad. Sci. USA 102: 5126-5131]), CD4-/- mice,
NOD.SCID/.gamma.c-/- (NSG), OT-II and OT-II/FIR mice were bred in
an animal facility. Mice were used at 6-12 weeks of age and were
maintained in pathogen-free conditions. Treg adoptive transfer
models were established as previously reported (see, Schreiber et
al. Oncoimmunology. 2012 Aug. 1; 1(5):642-648).
[0201] Cloning of Rhesus Macaque and Human TL1A-Ig
[0202] Total RNA was isolated from preparations of rhesus macaque
peripheral blood mononuclear cells using RNeasy kits (Qiagen). A
rhesus macaque cDNA library was then generated by PCR amplification
of 5' capped and 3' poly-A tailed RNA using 5'RACE (Invitrogen).
The extracellular domain of rhesus macaque TL1A (amino acids
73-252) were amplified using the following primers: forward
5'-AAAGGACAGGAGTTTGCACC-3' (SEQ ID NO: 17), reverse
5'-CTATAGTAAGAAGGCTCCAAA-3' (SEQ ID NO: 18), and fused to the
hinge-CH2-CH3 domain of rhesus macaque IgG1, which was amplified
using the following primers: forward 5'-ATAAAAACATGTGGTGGTGG-3'
(SEQ ID NO; 19), and reverse 5'-CTGCGTGTAGTGGTTGTGCA-3' (SEQ ID NO:
20), in cloned into the second multiple cloning site of the
mammalian expression vector pVITRO2-hygro-mcs (Invivogen).
[0203] For the human TL1A-Ig, a human cDNA library was generated by
PCR amplification of 5' capped and 3' poly-A tailed RNA using
5'RACE (Invitrogen). The extracellular domain of human TL1A (amino
acids 60-251) was amplified and fused to the hinge-CH2-CH3 domain
of human IgG1, and cloned into the pVITRO2-hygro (InvivoGen, San
Diego, Calif.).
[0204] The nucleic acid and amino acid sequences of the TL1A
portion of the rhesus macaque fusion protein were as follows:
TABLE-US-00006 Nucleic Acid Sequence: (SEQ ID NO: 5)
aaaggacaggagtttgcaccttcacatcagcaagtttatgcacctcttagagcagacggagataagccaagggc-
aca
cctgacagttgtgacacaaactcccacacagcactttaaaaatcagttcccagctctgcactgggaacatgaac-
taggcctggcc
ttcaccaagaaccgaatgaactataccaacaaattcctgctgatcccagagtcgggagactacttcatttactc-
ccaggtcacattc
cgtgggatgacctctgagtgcagtgaaatcagacaagcaggccgaccaaacaagccagactccatcactgtggt-
catcaccaa
ggtaacagacagctaccctgagccaacccagctcctcatggggaccaagtctgtgtgcgaagtaggtagcaact-
ggttccagc
ccatctacctcggacccatgttctccttgcaagaaggggacaagctaatggtgaacgtcagtgacatctccttg-
gtggattacaca aaagaagataaaaccttctttggagccttcttactatag; and Amino
Acid Sequence: (SEQ ID NO: 6)
kgqefapshqqvyaplradgdkprahltvvtqtptqhfknqfpalhwehelglaftknrmnytnkfllipesgd-
y
fiysqvtfrgmtsecseirqagrpnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwfqpiylgpmfslq-
egdklmv nvsdislvdytkedktffgafll.
[0205] The nucleic acid and amino acid sequences of the rhesus
macaque IgG1 hinge-CH2-CH3 sequence were as follows:
TABLE-US-00007 Nucleic Acid Sequence: (SEQ ID NO: 7)
ataaaaacatgtggtggtggcagcaaacctcccacgtgcccaccgtgcccagcacctgaactcctggggggacc-
gt
cagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtg-
gtagacgtgag
ccaggaagaccccgatgtcaagttcaactggtacgtaaacggcgcggaggtgcatcatgcccagacgaagccac-
gggagac
gcagtacaacagcacatatcgtgtggtcagcgtcctcaccgtcacgcaccaggactggctgaacggcaaggagt-
acacgtgca
aggtctccaacaaagccctcccggtccccatccagaaaaccatctccaaagacaaagggcagccccgagagcct-
caggtgta
caccctgcccccgtcccgggaggagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctacc-
ccagcgac
atcgtcgtggagtgggagaacagcgggcagccggagaacacctacaagaccaccccgcccgtgctggactccga-
cggctcc
tacttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat-
gcatgaggct ctgcacaaccactacacgcag; and Amino Acid Sequence: (SEQ ID
NO: 8)
iktcgggskpptcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpdvkfnwyvngaevhh
aqtkpretqynstyrvvsvltvthqdwlngkeytckvsnkalpvpiqktiskdkgqprepqvytlppsreeltk-
nqvsltclv
kgfypsdivvewensgqpentykttppvldsdgsyflyskltvdksrwqqgnvfscsvmhealhnhytq.
[0206] The nucleic acid and amino acid sequences of the complete
rhesus macaque TL1A-Ig fusion protein were:
TABLE-US-00008 DNA Sequence: (SEQ ID NO: 9)
atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgac
ataaaaacatg
tggtggtggcagcaaacctcccacgtgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcc-
tcttcccccc
aaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtagacgtgagccaggaag-
accccgat
gtcaagttcaactggtacgtaaacggcgcggaggtgcatcatgcccagacgaagccacgggagacgcagtacaa-
cagcacat
atcgtgtggtcagcgtcctcaccgtcacgcaccaggactggctgaacggcaaggagtacacgtgcaaggtctcc-
aacaaagcc
ctcccggtccccatccagaaaaccatctccaaagacaaagggcagccccgagagcctcaggtgtacaccctgcc-
cccgtccc
gggaggagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgtcgtg-
gagtggga
gaacagcgggcagccggagaacacctacaagaccaccccgcccgtgctggactccgacggctcctacttcctct-
acagcaag
ctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaa-
ccactacac gcag
aaaggacaggagtttgcaccttcacatcagcaagtttatgcacctcttagagcagacggagataagcc-
aagggc
acacctgacagttgtgacacaaactcccacacagcactttaaaaatcagttcccagctctgcactgggaacatg-
aactaggcctg
gccttcaccaagaaccgaatgaactataccaacaaattcctgctgatcccagagtcgggagactacttcattta-
ctcccaggtcac
attccgtgggatgacctctgagtgcagtgaaatcagacaagcaggccgaccaaacaagccagactccatcactg-
tggtcatcac
caaggtaacagacagctaccctgagccaacccagctcctcatggggaccaagtctgtgtgcgaagtaggtagca-
actggttcc
agcccatctacctcggacccatgttctccttgcaagaaggggacaagctaatggtgaacgtcagtgacatctcc-
ttggtggattac acaaaagaagataaaaccttctttggagccttcttactatag; and Amino
Acid Sequence: (SEQ ID NO: 10) metdtlllwvlllwvpgstgd
iktcgggskpptcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvsqed
pdvkfnwyvngaevhhaqtkpretqynstyrvvsvltvthqdwlngkeytckvsnkalpvpiqktiskdkgqpr-
epqvyt
lppsreeltknqvsltclvkgfypsdivvewensgqpentykttppvldsdgsyflyskltvdksrwqqgnvfs-
csvmheal hnhytq
kgqefapshqqvyaplradgdkprahltvvtqtptqhfknqfpalhwehelglaftknrmnytnkf-
llipesgd
yfiysqvtfrgmtsecseirqagrpnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwfqpiylgpmfsl-
qegdklm vnvsdislvdytkedktffgafll.
[0207] In each of the above complete fusion protein sequences (DNA
and amino acid), the italicized and underlined residues correspond
to the mouse kappa leader sequence; the bold and italicized text
corresponds to restriction enzyme cloning sites; the plain text
corresponds to the rhesus macaque IgG1 hinge-CH2-CH3 sequence; and
the underlined text corresponds to rhesus macaque TL1A
extracellular domain sequence.
[0208] The nucleic acid and amino acid sequences of the TL1A
portion of the human TL1A-Ig fusion protein were as follows:
TABLE-US-00009 Nucleic Acid Sequence: (SEQ ID NO: 11)
cgggcccagggagaggcctgtgtgcagttccaggctctaaaaggacaggagtttgcaccttcacatcagcaagt-
ttatgca
cctcttagagcagacggagataagccaagggcacacctgacagttgtgagacaaactcccacacagcactttaa-
aaatcagttc
ccagctctgcactgggaacatgaactaggcctggccttcaccaagaaccgaatgaactataccaacaaattcct-
gctgatcccag
agtcgggagactacttcatttactcccaggtcacattccgtgggatgacctctgagtgcagtgaaatcagacaa-
gcaggccgacc
aaacaagccagactccatcactgtggtcatcaccaaggtaacagacagctaccctgagccaacccagctcctca-
tggggacca
agtctgtgtgcgaagtaggtagcaactggttccagcccatctacctcggagccatgttctccttgcaagaaggg-
gacaagctaat
ggtgaacgtcagtgacatctctttggtggattacacaaaagaagataaaaccttctttggagccttcttactat-
ag; and Amino Acid Sequence: (SEQ ID NO: 12)
racqgeacvqfqalkgqefapshqqvyaplradgdkprahltvvrqtptqhfknqfpalhwehelglaftknrm-
nytnk
fllipesgdyfiysqvtfrgmtsecseirqagrpnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwfqp-
iylgamfslq egdklmvnvsdislvdytkedktffgafll.
[0209] The nucleic acid and amino acid sequences of the human IgG1
hinge-CH2-CH3 sequence were as follows:
TABLE-US-00010 Nucleic Acid Sequence: (SEQ ID NO: 13)
tgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccc-
cccaaaa
cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccc-
tgaggtca
agttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagc-
acgtacc
gtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaac-
aaagccctc
ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccc-
atcccgg
gatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggettctatcccagcgacatcgccgtgga-
gtgggagag
caatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctaca-
gcaagctcac
cgtggacaagagcaggtggcagcaggggaacgtettctcatgctccgtgatgcatgaggctctgcacaaccact-
acacgcaga agagcctctccctgtctccgggtaaa; and Amino Acid Sequence: (SEQ
ID NO: 14)
cdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpre-
eq
ynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclv-
kgfypsdia
vewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk
[0210] The nucleic acid and amino acid sequences of the complete
hTL1A-Ig fusion protein were as follows:
TABLE-US-00011 Nucleic Acid Sequence (SEQ ID NO: 15)
tgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccc-
cccaaaa
cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccc-
tgaggtca
agttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagc-
acgtacc
gtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaac-
aaagccctc
ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccc-
atcccgg
gatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtgga-
gtgggagag
caatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctaca-
gcaagctcac
cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccact-
acacgcaga agagcctctccctgtctccgggtaaa
cgggcccagggagaggcctgtgtgcagttccaggctctaaaaggacaggag
tttgcaccttcacatcagcaagtttatgcacctcttagagcagacggagataagccaagggcacacctgacagt-
tgtgagacaaa
ctcccacacagcactttaaaaatcagttcccagctctgcactgggaacatgaactaggcctggccttcaccaag-
aaccgaatgaa
ctataccaacaaattcctgctgatcccagagtcgggagactacttcatttactcccaggtcacattccgtggga-
tgacctctgagtg
cagtgaaatcagacaagcaggccgaccaaacaagccagactccatcactgtggtcatcaccaaggtaacagaca-
gctaccctg
agccaacccagctcctcatggggaccaagtctgtgtgcgaagtaggtagcaactggttccagcccatctacctc-
ggagccatgtt
ctccttgcaagaaggggacaagctaatggtgaacgtcagtgacatctctttggtggattacacaaaagaagata-
aaaccttctttg gagccttcttactatag; and Amino Acid Sequence (SEQ ID NO:
16)
cdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpre-
eq
ynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclv-
kgfypsdia
vewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk
raqgeacvq
fqalkgqefapshqqvyaplradgdkprahltvvrqtptqhfknqfpalhwehelglaftknrmnytnkfllip-
esgdyfiys
qvtfrgmtsecseirgagrpnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwfqpiylgamfslqegdk-
lmvnvsd islvdytkedktffgafll.
[0211] In each of the above complete fusion protein sequences
(human nucleic acid and amino acid sequences), the bold and
italicized residues correspond to the restriction enzyme cloning
site, the residues occurring before the restriction enzyme cloning
site (plain text) correspond to the human IgG1 hinge-CH2-CH3
sequence, and the residues following the restriction enzyme cloning
site (underlined text) correspond to the human TL1A extracellular
domain sequence.
[0212] A murine TL1A-Ig fusion protein was also constructed. The
method for its construction, and the functional characterization of
the murine fusion protein are described in detail in Khan, S. Q.,
et al. (2013) "Cloning, expression, and functional characterization
of TL1A-Ig. J Immunol 190:1540-1550," the content of which is
herein incorporated by reference in its entirety.
[0213] Cell Culture
[0214] Transfections of NIH-CHO cells were performed using standard
electroporation and lipid-based transfection methods. Transfected
cells were selected with an appropriate antibiotic (hygromycin) and
further selected by limiting dilution single-cell cloning
techniques to identify high-titer producing clones. Selected clones
were then weaned off of serum and adapted to grow in serum-free
conditions. The stable clone, growing in serum free media, was then
loaded into a hollow-fiber cartridge system for fusion protein
manufacturing. NIH-CHO-hTL1A-Ig cells were maintained in OPTI-CHO
media and cell culture supernatants containing hTL1A-Ig were
collected. Purification of hTL1A-Ig was performed by binding to a
Protein A or Protein G column using standard methods and eluted
from the column using a basic elution buffer to maintain
appropriate functionality of the fusion protein. Elution with a
basic buffer was essential, as elution with acidic buffers
destroyed functional activity. Following elution, the protein
fractions were pooled and quantitated using both standard protein
assays (Bradford) and specific ELISA assays for detection of the
IgG tail. Purified protein was then dialyzed to PBS and stored at
-80 degrees C.
[0215] Reagents, Antibodies and Flow Cytometry
[0216] Commercial antibodies for use in flow cytometry, ELISA and
in vivo studies were purchased from BD Pharmingen, eBioscience or
BioLegend. The Armenian Hamster IgG Isotype control was bought from
eBioscience. Armenian hamster hybridomas producing antibodies to
mouse TNFRSF25 (4C12, agonistic) were generated as described in
Fang, et al. 2008. Essential role of TNF receptor superfamily 25
(TNFRSF25) in the development of allergic lung inflammation. J Exp
Med 205:1037-1048. Briefly, 4C12, hTL1A-Ig and rmTL1A-Ig were
produced in hollow fiber bioreactors (Fibercell Systems, Frederick,
Md.) and purified from serum-free supernatants on a protein G
(4C12) or protein A (TL1A-Ig) column (GE Healthcare, UK). For flow
cytometry analysis, single cell suspensions were prepared from
spleen and lymph nodes. 10.sup.6 cells were pre-blocked with
anti-mouse CD16/CD32 and stained with different antibody
combinations. Intracellular staining was performed according to
standard procedures. Flow cytometric acquisition was performed on a
Becton Dickinson Fortessa instrument and FACSDIVA or FlowJo
software was used for analysis.
[0217] Western Blot
[0218] Standard methods were used including loading of 4-12%
SDS-PAGE gels with 10-40 ng per well, gel running using standard
buffers and voltages, transferring to PVDF membranes using standard
methods and one-step staining using a-hIgG-HRP and subsequent
detection using Pico or Femto alkaline phosphatase based detection
reagents (Pierce).
[0219] Caspase Detection Assay
[0220] Human TNFRSF25 (hTNFRSF25) was cloned and used to transfect
p815 cells (purchased from ATCC). Cells were transfected using
standard electroporation or lipid based transfection methods. The
transfected p815 cells were then co-incubated with increasing
concentrations of purified hTL1A-Ig (1 pg/ml-1 .mu.g/ml) and
caspase detection assay was performed according to manufacturer
protocol (Roche, Caspase 3 Activity Assay, Product
12012952001).
[0221] T Cell Proliferation Assay
[0222] These assays were performed as described in Khan et al. J.
Immunol. 2013 Feb. 15; 190(4):1540-50.
[0223] Generation of Humanized Mice
[0224] Human fetal livers from elective terminations (12-20 weeks
of gestational age, Advanced Bioscience Resources) were acquired on
a fee for service basis. CD34+ cells were enriched using
immunomagnetic beads according to the manufacturer's instructions
following density gradient centrifugation of single cell
suspensions (CD34+ selection kit; Miltenyi Biotec, Auburn, Calif.).
Purity of CD34+ cells isolated was evaluated by flow cytometry and
was >85%. Cells were aliquotted and frozen for later HLA typing
and transplanted into sub-lethally irradiated neonatal NSG mice.
HLA typed CD34+ cells hematopoietic stem cells (HSC) were placed
into culture containing 10% human serum, 10 ng/ml each of
interleukin (IL)-3, IL-6 and stem cell factor (SCF) in IMDM for 3-5
days. On the day of transplant, cells were harvested from culture
dishes and washed in HBSS and CFU and LTC-IC assays were performed
to evaluate proliferation and differentiation potential. These
assays were performed using standard reagents (StemCell Inc.) and
methods as published by the manufacturer. CFU potential was
identical to bone marrow progenitor cells following 14 day culture
in methylcellulose media supplemented with SCF, GM-CSF, IL-3 and
EPO. These assays readily reveal whether gross morphological
characteristics and colony numbers of isolated HSC are within
expected limits. One-day old NSG mice generated from timed matings
were housed with foster-dams for 24 hours post-irradiation
(sub-lethal, 1 Gy, whole body irradiation) at which time they are
transplanted with 1.times.10.sup.6-2.times.10.sup.6 pre-cultured
HSC intra-hepatically (i.h.) in a volume of 20 .mu.l using a
Hamilton syringe and a 30 gauge 1/2 inch needle. The pups were
immediately returned to their foster-dams and allowed to nurse
until weaning at 28 days. Human/mouse chimerism was evaluated in
peripheral blood at 15 weeks of age by determining the relative
percentages of murine and human CD45+ cells by flow cytometry. Once
human CD45+ cells were detected, analysis was extended to include
human CD3, CD4, CD8, CD11c and CD19 on all blood collections.
Successfully engrafted mice were selected when the fraction of
human CD45+ cells in peripheral blood exceeded 60% (NSG-hu).
[0225] Safety and Activity of TL1A-Ig in Rhesus Macaque
[0226] The safety and efficacy of TL1A-Ig in non-human primates
(NHP) (Indian-origin macaca mulatta) were determined. Following
routine screening and 60-day quarantine, rhesus macaque (rm)TL1A-Ig
or human (h)TL1A-Ig were administered to the NHP on Day 0 by
intravenous (IV) bolus infusion (15 minutes). Animal weights and
cage-side observations were performed daily and peripheral blood
was collected for analysis of complete blood counts, routine
chemistries, CD4 TruCounts, serum isolation and flow cytometry
analysis. For flow cytometry analysis, peripheral blood cells were
counted on a Countess automated cell counter and aliquots of
1.5.times.10.sup.6 cells were distributed per sample tube, FMO
(Fluorescent Minus One), controls, and compensation tubes. Test
samples were stained in D-PBS and 0.5% FBS with 1 .mu.L/mL live
dead aqua blue discriminator (Life Technologies). Test samples and
appropriate controls were surface stained for 30 minutes with the
indicated cocktails of antibodies (purchased from BD, Becton
Dickinson, eBioscience or Life Technologies). For intracellular
staining, cells were permeabilized with Fix/perm solution
(eBioscience) for 30 minutes at 4.degree. C. Cells were washed and
stained intracellularly with anti-FoxP3 antibody for 30 minutes.
Following staining, cells were washed and resuspended in 300 .mu.L,
FACs wash buffer for acquisition on a BD.TM. LSRII flow cytometer
(Beckton Dickinson (BD)). Cells were acquired and analysis was done
of FlowJo software (Tree Star, Inc., Ashland, Oreg.). For CD4/CD8
Trucounts, CD45, CD3, CD4 and CD8 antibodies were dispensed in
TruCount tubes (BD, Cat. No. 340334); 50 uL samples were added and
allowed to stain for 15 minutes at room temperature in the dark.
Subsequently, samples are lysed with 450 FACS lysing solution, and
incubated for 15 minutes. Sample tubes were analyzed in the
cytometer (Calibur, BD). The events were gated on lymphocytes in
side scatter (SSC) dot plot and the CD45-positive population was
selected, and then reported: CD3+CD4+ T cells, and CD3+CD8+T cells.
For multiplex analysis of serum cytokines, serum from the indicated
days was collected and samples and controls for standard curves
were placed in filter plates, and then diluted 1/4, and incubated
for 2 hour at room temperature with anti-cytokine beads. The
content was removed and washed 2 times with the buffer before
adding a mixture of biotinylated detection antibodies to each well.
After incubating with detection antibody for 1 hour, plates were
washed 2 times and incubated for 30 minutes with streptavidin-PE.
Plates were again washed and wells were resuspended with 150 sheath
fluid; plates were then read and analyzed on the Luminex.RTM. SD
Analyzer (Life Technologies, Inc.). The PE signal (Median
Fluorescent Intensity, MFI) is proportional to the amount of each
cytokine present in the sample; concentrations were calculated from
the standard curve.
[0227] Statistical Analysis
[0228] All graphing and statistical analyses were performed using
the ABI Prism.RTM. program (Applied Biosystems). Paired analysis
was performed using the Student's t-test. Analysis of conditions
with more than two conditions was performed using one-way ANOVA
with Tukey's post-hoc test. Significance is indicated throughout
Figures as *(p<0.05); **(p<0.01); and ***(p<0.001).
Example 2
Connate Antigen-Dependent Tree Proliferation
[0229] This Example demonstrates that Treg proliferation stimulated
by TNFRSF25 is dependent on cognate antigen.
[0230] A mouse model was utilized wherein CD4.sup.-/- mice were
adoptively transferred with a mixed population of
CD4.sup.+FoxP3.sup.GFP+ (tTreg), which are specific for self
antigen, and ovalbumin (ova)-specific
CD4.sup.+V.alpha.2.sup.+V.beta.5.sup.+FoxP3.sup.RFP-
(OTII.sub.conv, generated by crossing OT-II mice to FIR mice) as
described in Schreiber, T. H., et al. 2012. Oncoimmunology
1:642-648. The model is represented as a schematic outline in FIG.
1. In these studies, tTreg are presumed to include all
thymic-derived Treg cells recognizing endogenous (germline encoded)
self antigens. pTreg are presumed to include all Treg that are
generated from peripheral T cells that exited the thymus without
expressing FoxP3, and therefore were presumed to recognize foreign
(non-germline encoded) antigens. pTreg are therefore presumed to be
important for regulation of immune response in tissue that
regularly encounter endogenous environmental or microbial antigens
including the gastrointestinal tract, skin and lungs.
OT-II.sub.conv are cells derived from transgenic mice expressing a
MHC class II restricted T cell receptor specific for the foreign
antigen ovalbumin ("ova"). These cells were isolated from OT-II
transgenic mice on the basis of expression of CD4 and
non-expression of FoxP3.
[0231] Following a five-day oral administration of 0.5% ovalbumin
in drinking water, the mice were treated with either IgG isotype
control antibody or TNFRSF25 agonistic antibody, clone 4C12. After
5 days, splenocytes and mesenteric lymph node (mLN) cells were
harvested and analyzed for OT-II pTreg cells and FoxP3-RFP positive
nTreg cells. This model provided a tractable model in which
predominantly self-antigen specific tTreg could be distinguished
from OVA-specific pTreg.sub.OTII on the basis of FoxP3.sup.GFP and
FoxP3.sup.RFP expression, respectively.
[0232] In another experiment, following a five-day oral
administration of 0.5% ovalbumin in drinking water, CD4.sup.-/-
mice in which a mixed population of tTreg and OTII.sub.conv had
been adoptively transferred, as above, were treated with either IgG
isotype control antibody or TNFRSF25 agonistic antibody, clone
4C12. In FIG. 2, the proportion of OT-II-iTreg cells and nTreg
cells undergoing proliferation (Ki67+) in the mLN and spleen is
illustrated. 6.6.+-.1.6% of splenic OTII.sub.conv were induced to
express FoxP3.sup.RFP and became pTreg.sub.OTII. In a separate
experiment, to determine the antigen-dependence of OT-II iTreg cell
or nTreg cell proliferation in the mLN (FIG. 3) and spleen (FIG.
4), 1% ova in drinking water was either continued (left panels) or
replaced with normal water to `washout` ovalbumin (ova) for the
indicated number of days (right panels). After the indicated
treatment period (0, 10 or 20 days), groups were treated with
either IgG isotype control antibody or 4C12 antibody and OT-II
iTreg cells and nTreg cells were analyzed as above, to determine
the percentage of FoxP3+Ki67+ cells, in each tissue 5 days later.
Following induction of pTreg.sub.OTII with oral administration of
ovalbumin, individual cages were either maintained on
ovalbumin-containing drinking water or switched to regular drinking
water to determine the relationship between cognate antigen
availability and sensitivity to TNFRSF25 stimulation.
[0233] Administration of the TNFRSF25 agonistic antibody, clone
4C12, prior to antigen withdrawal (day 0) led to proliferation of
both pTreg.sub.OTII and tTreg in all mice (FIG. 3). For all groups,
proliferation of tTreg served as the internal control because of
persistent availability of cognate `self` antigen for tTreg.
Following 10 days of ovalbumin antigen withdrawal ("Days ova
washout"), pTreg.sub.OTII continued to proliferate following
administration of 4C12, indicating that ovalbumin persists for at
least 10 days following its withdrawal from the drinking water.
Following 20 days of ovalbumin antigen withdrawal, however, no
proliferation of pTreg.sub.OTII was observed following
administration of 4C12, despite the continued proliferation of
tTreg in both the mesenteric lymph nodes (FIG. 3) and spleen (FIG.
4). If drinking water containing ova was provided for the same 20
day period, pTreg.sub.OTII continued to proliferate in response to
4C12 in both tissues (mLN and spleen). These data demonstrated that
withdrawal of cognate antigen prevented responsiveness of
pTreg.sub.OTII to 4C12 stimulated proliferation, indicating an
antigen-specific response.
Example 3
Human TL1A-I.sub.2 Stimulates Proliferation of Human tTreg in
Humanized Mice
[0234] This Example demonstrates that human TL1A-Ig fusion protein
induced strong proliferation of human Tregs systemically and in the
mucosa of humanized mice.
[0235] A fusion protein containing the extracellular domain of
human TL1A and the hinge-CH2-CH3 domain of human IgG1 was cloned
and purified from cell-culture supernatants, as described above.
Monomeric and multimeric hTL1A-Ig complexes were identified by
Western blot (FIG. 5). The in vitro activity of human TL1A-Ig
(hTL1A-Ig) was demonstrated using a caspase detection assay in
which human TNFRSF25 (hTNFRSF25) transfected p815 cells were
co-incubated with increasing concentrations of purified hTL1A-Ig
(FIG. 6).
[0236] To determine the in vivo activity of hTL1A-Ig on human
Tregs, humanized mice were generated using human fetal liver
CD34.sup.+ cells transferred into NOD, SCID, common .gamma.-chain
deficient (NSG) recipient mice, as described above. Human
CD45.sup.+ cell engraftment in the experimental mice (NSG-hu)
showed .gtoreq.60% chimerism in splenocytes and .gtoreq.90%
chimerism in lymph nodes in all mice at 15 weeks of age. The
majority of human CD45+ lymphocytes in the spleen were T cells
(.about.50%) followed by B cells (.about.35%), NK cells
(.about.2-3%) and dendritic cells (.about.1%).
[0237] Administration of hTL1A-Ig did not significantly alter the
overall balance of hCD45.sup.+, hCD4.sup.+ or hCD8.sup.+ cells in
any of the spleen, lymph nodes, or small intestine in recipient
mice analyzed on day 5 after injection (FIGS. 7-9). However,
analysis of human Treg cells as a percentage of FoxP3.sup.+ cells
out of total CD4.sup.+CD25.sup.hiCD127.sup.- cells demonstrated a
significant increase both in the total frequency of Treg and the
proportion of Treg in active proliferation (Ki67.sup.+) on day 5
after treatment in spleen, lymph node and small intestine (FIGS.
7-9). In spleen, the mean Treg value (.+-.SEM) for the controls is
0.72.+-.0.11% and for hTL1A-Ig 1.71.+-.0.24% (FIG. 7). In lymph
nodes, the control mean Treg value was 0.91.+-.0.12 whereas for
hTL1A-Ig it was 1.94.+-.0.21 (FIG. 8). In the small intestine, the
mean Treg value (.+-.SEM) for the controls is 1.56.+-.0.31% and for
the hTL1A-Ig treated is 4.54.+-.0.69% (FIG. 9). Phenotypically,
Treg cells had a mainly central memory
(CCR7.sup.+CD45.sup.-RA.sup.-; .about.70-80%) phenotype whereas
non-Treg cells show a mainly naive (CCR7.sup.+CD45.sup.-RA.sup.+;
.about.70-80%) phenotype. Analysis of activation markers (CD25,
CD69) expressed by conventional CD4+ and CD8+ T cells demonstrated
increased expression of CD69 in both CD4.sup.+ (p=0.0363) and
CD8.sup.+ (p=0.0064) conventional cells in the spleen but not in
lymph nodes of hTL1A-Ig treated NSG-hu. These data demonstrated
that hTL1A-Ig induced strong proliferation of human Tregs
systemically and in the mucosa of humanized mice.
[0238] Similar results were obtained using the murine fusion
protein, as described in detail in Khan, S. Q. et al. (supra).
Example 4
Safety and Activity of TL1A-I.sub.2 in Rhesus Macaques
[0239] This Example demonstrates that TL1A-Ig was capable of safely
and selectively stimulating the proliferation of cognate Treg cells
in vivo in humanized mice and primates.
[0240] An inherent limitation of rodent animal models is related to
the highly controlled and restricted history of foreign antigen
challenge of laboratory animals bred and housed under pathogen-free
conditions. Given the antigen-dependent activity of TNFRSF25
agonists, this limitation has important consequences for
translational studies of these agents into NHP, wherein the history
of foreign antigen exposure is dramatically more diverse. To
address this question, rhesus macaque TL1A-Ig (rmTL1A-Ig) was
produced and tested in NHP. Treatment-naive Indian-origin rhesus
macaques (nonhuman primates (NHP)) were procured, housed and
treated by expert personnel at Advanced Bioscience Laboratories
(ABL, Rockville, Md.). Rhesus macaque TL1A-Ig (rmTL1A-Ig) and human
(h)TL1A-Ig were manufactured and purified as described above and
shipped to ABL personnel at the indicated concentration in blinded
tubes diluted in a total volume of 10 ml PBS. After clearing 60-day
quarantine, baseline complete blood counts (CBC) and serum
chemistries were obtained 14 days prior to the scheduled
intravenous injection of rmTL1A-Ig and hTL1A-Ig. 2 animals received
0.5 mg/kg rmTL1A-Ig, 4 animals received 1.5 mg/kg rmTL1A-Ig, and 2
animals received 1.5 mg/kg hTL1A-Ig by a single IV injection on day
0 of the study. TL1A-Ig half-life, Treg expansion, T.sub.conv
subset and activation analysis and cytokine profiles were monitored
by serial blood draws over the 21 day course of the experiment
together with tissue histopathology from animals sacrificed on day
21 of the study.
[0241] No acute toxicities were observed either in the immediate
twelve hours post-injection in any NHP or during the remainder of
the study. Daily cage-side observations noted normal behavior of
all NHP throughout the course of the study and no evidence of
wheezing, somnolence, diarrhea, vomiting, anaphylaxis, skin rashes
or irritation, peripheral edema, joint effusions or mucous membrane
discharge. There was no diarrhea or weight loss observed in any of
the NHP over the course of the study (FIG. 10). Serum chemistries
indicated no changes in electrolyte levels: sodium, potassium,
phosphate, chloride, calcium, glucose, creatinine, blood urea
nitrogen, total serum protein, albumin, total cholesterol or
globulin. Complete blood counts indicated an increase in total
white blood cells (FIG. 11A) and neutrophils (FIG. 11B) on the
first day after treatment but no changes in total hemoglobin,
hematocrit, total red blood cells, MCV, MCH, platelets,
lymphocytes, monocytes, eosinophils or basophils over the course of
the study were observed. A liver enzyme panel indicated no changes
in alkaline phosphatase, total bilirubin or ALT. However, in all
animals, approximate 10-fold increases in AST and creatinine
phosphokinase were observed on the first day after treatment,
increases which are routinely observed in NHP following
intramuscular injection of anesthetic prior to the treatment
protocol. The levels of AST and CPK returned to baseline by day 4
of the study in all animals.
[0242] Analysis of serially collected serum samples indicated a
half-life of 12.5 hours for hTL1A-Ig in NHP, calculated using a
one-phase exponential decay model (FIG. 12). Multiplex analysis
(Luminex.RTM.) of serum cytokines at baseline (day 0), day 2 and at
the time of peak Treg expansion (day 4) demonstrated no detectable
changes in the levels of IL-2, IL-4, IL-5, IL-10 or TNF-.alpha.,
and a trend toward decreased levels of IFN-.gamma. and TGF-.beta.
by day 4 after treatment (FIG. 13). Analysis of peripheral blood
Treg cells by flow cytometry demonstrated nearly identical relative
magnitude and kinetics of in vivo Treg expansion in rhesus macaque
as compared to humanized mice (FIG. 14). The relative
fold-expansion peaked at 4 days post-treatment with approximately
3-fold expansion of the Treg compartment. Similar responses were
observed using 1.5 mg/kg of both rmTL1A-Ig and hTL1A-Ig, with the
0.5 mg/kg rmTL1A-Ig dose demonstrating a non-significantly reduced
trend for Treg expansion.
[0243] To monitor for CD4+ and CD8+ T.sub.conv cell activation,
naive (CCR7+CD28+CD95-), central memory (CCR7+CD28+CD95+), effector
memory (CCR7-CD28-CD95+) and transitional effector memory
(CCR7-CD28+CD95+) T cell subsets were monitored over the course of
the experiment in the peripheral blood. This analysis demonstrated
no significant fluctuations in the CD8 naive, central memory,
effector memory or transitional effector memory T cell
compartments. Within the CD4 compartment, there was a significant
reduction in the frequency of peripheral blood naive CD4 cells by
day 11 of the study, which then rapidly rebounded to baseline by
day 15
[0244] (FIG. 15). Although not significant, there was a trend
toward increased frequencies of CD4 central memory by day 11 of the
study, which, when adjusted for absolute cell numbers, demonstrated
no change; indicating that this relative difference was likely
related to the relative reduction in naive cells rather than a
proliferative effect on central memory cells.
[0245] Although there was no evidence of CD4+ or CD8+ effector T
cell activation or increased concentrations of inflammatory
cytokines immediately following TL1A-Ig administration, it remained
possible that signs of sub-clinical immunopathology may be present
within individual tissues. To investigate signs of immunopathology
within individual tissues, 2 animals receiving rmTL1A-Ig (1.5
mg/kg) and the 2 animals receiving hTL1A-Ig (1.5 mg/kg) were
selected for necropsy and end-organ histopathology on day 21 of the
study. Analysis of hematoxylin and eosin stained sections of the
midbrain, brainstem, liver and pancreas demonstrated no evidence of
inflammation or pathology in any of the animals analyzed. In one
animal, lung sections demonstrated evidence of minimal, multifocal
perivascular lymphocytic aggregates, with the remaining three
animals interpreted as essentially normal lung tissue. In two
animals, sections of haired skin were shown to exhibit mild to
moderate areas of focally extensive dermal edema, with the
remaining two animals interpreted as essentially normal skin
tissue. In three of the four animals, jejunal sections demonstrated
evidence of mild, diffuse submucosal edema. In four out of four
animals, sections of the terminal ileum demonstrated evidence of
mild, diffuse mucosal and submucosal edema. In four out of four
animals, sections of sigmoid colon demonstrated evidence of mild,
multifocal, lymphoplasmacytic infiltrates. Mesenteric lymph node
sections were interpreted as essentially normal tissue in three or
four animals, with one animal showing evidence of mild, diffuse
lymphocytosis.
[0246] Together, these results provide evidence that stimulation of
TNFRSF25 with receptor agonistic antibodies and ligand fusion
proteins provides a unique and specific method for in vivo
modulation of human and NHP Treg cells. The kinetics and
specificity of Treg stimulation are remarkably similar in mice, in
NSG-hu and in NHP following treatment with mouse, rhesus macaque
and human-specific TL1A-Ig; which may indicate that the underlying
mechanism involving cognate antigen/TCR engagement, IL-2 receptor
and Akt activation is also conserved in humans as was also
demonstrated in mice (Khan et al. (supra)). The observation in NHP
of a decrease in naive CD4 T cells immediately following the peak
in Treg expansion in the peripheral blood may indicate evidence of
in vivo suppression of CD4 naive cells.
[0247] Discussion
[0248] The data in Examples 2-4 indicate that TL1A-Ig is a molecule
capable of safely and selectively stimulating the proliferation of
cognate Treg cells in vivo in mice, NSG-hu and NHP. Of particular
concern were possible susceptibilities to inflammatory bowel
disease (IBD) due both to epidemiologic data linking TL1A
polymorphism to IBD in humans and to murine studies demonstrating
that transgenic expression of TL1A predisposes to IBD
susceptibility. Because tolerance to endogenous `foreign` antigens
in the gut is particularly dependent upon the immunosuppressive
activity of Treg, it was predicted that modulation of TNFRSF25 in
NHP would lead to similar immunopathology. No such toxicities were
observed in these studies as demonstrated by behavioral changes,
diarrhea or weight loss over the course of these studies, and there
was no evidence of diffuse effector cell activation or inflammatory
cytokine production in the peripheral blood. End-organ
histopathology demonstrated only mild accumulation of lymphoid
cells within the terminal ileum and sigmoid colon, without overt
signs of tissue immunopathology.
Example 5
Combination Therapy with TNFRSF25 Agonists and IL-2
[0249] This Example demonstrates the surprising and unexpected
discovery that the combination of a low or a very dose of IL-2 with
a TNFRSF25 agonist, such as TL1A-Ig fusion protein or the agonistic
anti-TNFRSF25 antibody 4C12, had a synergistic effect on the
expansion of Treg cells in vivo.
[0250] In a first set of experiments, wild type mice were treated
with low-dose IL-2 (300,000 units/m.sup.2), control (IgG), TL1A-Ig
(0.5 mg/kg), or a combination treatment with TL1A-Ig and a single
injection of very low-dose IL-2 (30,000 units/m.sup.2) or with a
combination treatment with TL1A-Ig and a single injection of low
dose IL-2 (300,000 units/m.sup.2). The frequency of CD4+FoxP3+
cells out of total CD4+ cells was monitored in the peripheral blood
on the indicated days.
[0251] As shown in FIG. 16, treatment with TL1A-Ig and either dose
of IL-2 (very lose dose IL-2 (30,000 units) or low dose (300,000
units) had a synergistic effect on the Treg cell expansion compared
to treatment with TL1A-Ig or IL-2 alone. For example, 5 days
post-treatment, the percentage of FoxP3+ Treg cells was 60% in mice
treated with TL1A-Ig and Low Dose IL-2, and 50% in mice treated
with TL1A-Ig and very lose dose IL-2, compared to 40% in mice
treated with TL1A-Ig and less than 20% in mice treated with Low
dose IL-2 alone. The synergistic effect was even more pronounced 6
days after treatment, when the frequency of Treg cells was 55% and
about 40% following combination treatment with TL1A-Ig and low or
very low dose IL-2, respectively, compared to less than 25% and
less than 20% following treatment with TL1A-Ig or Low dose IL-2,
respectively.
[0252] In a second set of experiments, wild type mice were treated
with low-dose IL-2 (300,000 units/m2), control (IgG), 4C12 antibody
(0.4 mg/kg), or with a combination treatment with 4C12 antibody and
a single injection of low dose IL-2 (300,000 units/m2). The
frequency of CD4+FoxP3+ cells out of total CD4+ cells was monitored
in the peripheral blood on the indicated days.
[0253] As shown in FIG. 17, treatment with a combination of the
4C12 antibody and the Low Dose IL-2 had a dramatic, synergistic
effect on expansion of Treg cells. Six days after treatment with
the 4C12/IL-2 combination, the frequency of Treg cells was about
70% of all CD4+ CD3+ peripheral blood cells, compared to about 30%
of all CD4+ CD3+ peripheral blood cells, and less than 20% of all
CD4+ CD3+ peripheral blood cells, in mice that received only 4C12
antibody or only low-dose IL-2, respectively.
[0254] The results achieved with the above-described combination
therapies were surprising and unexpected, for a number of reasons.
For example: 1) the TNFRSF25 agonist/IL-2 combination achieved Treg
expansion at a dose of IL-2 (300,000 units) not previously shown to
expand Treg cells; 2) the combination treatments achieved Treg
expansion at a 10-fold lower dose than what had been considered
"low-dose" IL-2 (30,000 units); and 3) the magnitude of Treg
expansion was unprecedented. This is believed to be the first
description of obtaining 50% Treg cells in the CD4 compartment
(i.e., 50% of all CD4+ cells were Treg cells), and in the case of
4C12/low dose IL-2, that fraction even reached 70%. Furthermore,
these high numbers of Treg cells were achieved using both TL1A-Ig
and TNFRSF25 agonistic antibodies in the combination therapy with
IL-2, indicating that this is a property of the receptor itself and
not of a specific reagent.
Example 6
Combination Therapy with TL1A-I.sub.2 and Rapamycin
[0255] This Example demonstrates the surprising and unexpected
discovery that the combination of rapamycin with TL1A-Ig fusion
protein preserved Treg cell expansion in vivo while eliminating
concurrent effector T cell activation.
[0256] Wild-type mice were adoptively transferred with ovalbumin
specific CD8 (OT-I) or CD4 (OT-II) T cells on day -2. Mice were
then immunized with Aluminum hydroxide ("alum")-adjuvanted
ovalbumin together with either control IgG, TL1A-Ig (0.5 mg/kg)
and/or a 6-day course of low-dose rapamycin (75 .mu.g/kg). The
frequency of OT-I cells out of total CD8+ cells, OT-II cells out of
total CD4+ cells, and the frequency of CD4+FoxP3+ cells (Treg
cells) out of total CD4+ cells were monitored by flow cytometry
over 18 days.
[0257] As shown in FIG. 18 and FIG. 19, the overall frequency of
CD8+ (OT-I) and CD4+ (OT-II) cells following treatment that
included rapamycin was markedly reduced. What was most striking,
however, was that the frequency of Treg cells following treatment
with OVA/alum, TL1A-Ig, and Rapamycin was not affected (FIG. 20),
indicating the effect of rapamycin was specifically on activated T
effector cells and not Treg cells.
[0258] This finding was surprising and indicates that therapies
that involve the administration of TNFRSF25 agonists (such as
TL1A-Ig fusion protein, and other agonists, e.g., 4C12 antibody)
benefit from co-administration of rapamycin to prevent unwanted
activation and expansion of CD4 and CD8 T effector cells.
Prophetic Example 1
Administration to Human Subjects of Human TL1A-I.sub.2 Fusion
Protein
[0259] The hTL1A-Ig fusion protein is formulated in buffered saline
for intravenous administration. Increasing dosages, ranging from
0.1 mg/kg/day to 10 mg/kg/day of hTL1A-Ig fusion protein, prepared
and isolated as described in Example 1, above, are administered to
human patients as an induction agent several days prior to solid
organ or stem cell transplantation. The efficacy of this treatment
is then measured by serial blood draws over a period of several
weeks wherein the frequencies of Treg cells, T effector (T.sub.eff)
cells and inflammatory cytokines are measured in the peripheral
blood of treated subjects. Long-term benefit of this treatment is
also measured in treated subjects based on the ability for early
weaning of standard immunosuppressive maintenance therapy
including, but not limited to, tacrolimus or other mTOR inhibitors,
cyclosporine inhibitors and steroid regimens including prednisone
and methylprednisone.
[0260] An initial safety study may also be performed in healthy
subjects wherein 0.1-10 mg/kg/day hTL1A-Ig is administered in
buffered saline intravenously. The function of hTL1A-Ig is then
measured in treated subjects by serial blood draws over a period of
several weeks wherein the frequencies of Treg cells, Teff cells and
inflammatory cytokines are measured in the peripheral blood of
treated subjects. Safety is also monitored using standard
observational methods.
Prophetic Example 2
Combination Therapy with Human TL1A-12 Fusion Protein and
Interleukin-2
[0261] The hTL1A-Ig fusion protein is formulated in buffered saline
for intravenous administration in a dosage found to be effective in
Prophetic Example 1, above (e.g., in the range from 0.1 mg/kg/day
to 10 mg/kg/day), prepared and isolated as described in Example 1,
above. The hTL1A fusion protein is administered to human patients
as an induction agent several days prior to solid organ or stem
cell transplantation. One or two days prior to, on the same day as,
or one or two days following the administration of the hTL1A-Ig
fusion protein, the patients are also administered either low dose
(300,000 units) or very low dose (30,000 units) or a dose between
30,000 and 300,000 units per square meter of IL-2
intravenously.
[0262] The efficacy of this treatment is then measured by serial
blood draws over a period of several weeks wherein the frequencies
of Treg cells, T effector (T.sub.eff) cells and inflammatory
cytokines are measured in the peripheral blood of treated subjects.
Long-term benefit of this treatment is also measured in treated
subjects based on the ability for early weaning of standard
immunosuppressive maintenance therapy including, but not limited
to, tacrolimus or other mTOR inhibitors, cyclosporine inhibitors
and steroid regimens including prednisone and methylprednisone.
Prophetic Example 3
Combination Therapy with TNFRSF25 Agonist and Rapamycin
[0263] The human TL1A-Ig fusion protein described in, e.g.,
Examples 1 and 3, above, or an agonistic anti-TNFRSF25 antibody is
formulated in buffered saline for intravenous administration in an
effective dosage (e.g., for TL1A-Ig fusion protein, a dosage found
to be effective in Prophetic Example 1, above (e.g., in the range
from 0.1 mg/kg/day to 10 mg/kg/day), prepared and isolated as
described in Example 1, above. The human TL1A fusion protein or
agonistic anti-TNFRSF25 antibody is administered to human patients
as an induction agent several days prior to solid organ or stem
cell transplantation. One or two days prior to, on the same day as,
or one or two days following the administration of the TNFRSF25
agonist, the patients are also administered rapamycin at a dosage
of between 75 and 300 micrograms per kg body weight per day. The
efficacy of this treatment is then measured by serial blood draws
over a period of several weeks wherein the frequencies of Treg
cells, T effector (T.sub.eff) cells and inflammatory cytokines are
measured in the peripheral blood of treated subjects. Long-term
benefit of this treatment is also measured in treated subjects
based on the ability for early weaning of standard
immunosuppressive maintenance therapy including, but not limited
to, tacrolimus or other mTOR inhibitors, cyclosporine inhibitors
and steroid regimens including prednisone and methylprednisone.
[0264] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. It is further to be understood that all
values are approximate, and are provided for description.
Accordingly, other embodiments are within the scope of the
following claims.
Sequence CWU 1
1
161756DNAHomo sapiens 1atggccgagg atctgggact gagctttggg gaaacagcca
gtgtggaaat gctgccagag 60cacggcagct gcaggcccaa ggccaggagc agcagcgcac
gctgggctct cacctgctgc 120ctggtgttgc tccccttcct tgcaggactc
accacatacc tgcttgtcag ccagctccgg 180gcccagggag aggcctgtgt
gcagttccag gctctaaaag gacaggagtt tgcaccttca 240catcagcaag
tttatgcacc tcttagagca gacggagata agccaagggc acacctgaca
300gttgtgagac aaactcccac acagcacttt aaaaatcagt tcccagctct
gcactgggaa 360catgaactag gcctggcctt caccaagaac cgaatgaact
ataccaacaa attcctgctg 420atcccagagt cgggagacta cttcatttac
tcccaggtca cattccgtgg gatgacctct 480gagtgcagtg aaatcagaca
agcaggccga ccaaacaagc cagactccat cactgtggtc 540atcaccaagg
taacagacag ctaccctgag ccaacccagc tcctcatggg gaccaagtct
600gtatgcgaag taggtagcaa ctggttccag cccatctacc tcggagccat
gttctccttg 660caagaagggg acaagctaat ggtgaacgtc agtgacatct
ctttggtgga ttacacaaaa 720gaagataaaa ccttctttgg agccttctta ctatag
7562251PRTHomo sapiens 2Met Ala Glu Asp Leu Gly Leu Ser Phe Gly Glu
Thr Ala Ser Val Glu1 5 10 15 Met Leu Pro Glu His Gly Ser Cys Arg
Pro Lys Ala Arg Ser Ser Ser 20 25 30 Ala Arg Trp Ala Leu Thr Cys
Cys Leu Val Leu Leu Pro Phe Leu Ala 35 40 45 Gly Leu Thr Thr Tyr
Leu Leu Val Ser Gln Leu Arg Ala Gln Gly Glu 50 55 60 Ala Cys Val
Gln Phe Gln Ala Leu Lys Gly Gln Glu Phe Ala Pro Ser65 70 75 80 His
Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg 85 90
95 Ala His Leu Thr Val Val Arg Gln Thr Pro Thr Gln His Phe Lys Asn
100 105 110 Gln Phe Pro Ala Leu His Trp Glu His Glu Leu Gly Leu Ala
Phe Thr 115 120 125 Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu Leu
Ile Pro Glu Ser 130 135 140 Gly Asp Tyr Phe Ile Tyr Ser Gln Val Thr
Phe Arg Gly Met Thr Ser145 150 155 160 Glu Cys Ser Glu Ile Arg Gln
Ala Gly Arg Pro Asn Lys Pro Asp Ser 165 170 175 Ile Thr Val Val Ile
Thr Lys Val Thr Asp Ser Tyr Pro Glu Pro Thr 180 185 190 Gln Leu Leu
Met Gly Thr Lys Ser Val Cys Glu Val Gly Ser Asn Trp 195 200 205 Phe
Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu Gln Glu Gly Asp 210 215
220 Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp Tyr Thr
Lys225 230 235 240 Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 245
250 31659DNARhesus macaque 3ggaaaaggga aggaggagac tgagtgatta
agtcacccac tgtgagagct ggtcttctat 60ttaatggggg ctctctctgc ccaggagtca
gaggtgcctc caggagcagc aagagcatgg 120ccgaggatct gggactgagc
tttggggaga cagccagtgt ggaaatgctg ccagagcacg 180gcagctgcag
gcccaaggcc aggagcagca gcgcatgctg ggctctcacc tgctgcctgg
240tgttgctccc cttccttgca gggctcacca cctacctgct tgtcagccag
ctccgggccc 300aaggagaggc ctgtgtgcag ctccaggatc taaaaggaca
ggagtttgca ccttcacatc 360agcaagttta tgcacctctt agagcagatg
gagataagcc aagggcacac ctgacagttg 420tgagacaaac tcccacacag
cacttaaaaa atcagttccc agctctgcac tgggaacatg 480aactaggcct
ggccttcacc aagaaccgaa tgaactatac caacaaattc ctgctgatcc
540cagagtcggg agactacttc gtttactccc aggtcacatt ccgtgggatg
acctctgagt 600gcagtgaaat cagacaagca ggccgaccaa acaagccaga
ctccatcact gtggtcatca 660ccaaggtaac agacagctac cctgagccaa
cccagctcct catggggacc aagtctgtgt 720gtgaagtagg cagtaactgg
ttccagccca tctacctcgg agccatgttc tccttgcaag 780aaggggacaa
gctcatggtg aacgtcagtg acatctcttt ggtggattac acaaaagaag
840ataaaacctt ctttggagcc ttcttactat aggaggagag caaatatcat
tatgtgaagt 900cctctgccac cgagttccta attttcttcg ttcaaatgta
attacaacca ggggttttct 960tggggccggg agtagggggc attccgcagg
gacaatggtt tagctatgaa atttggggcc 1020caaaatttca cacttcatgt
gccttactga tgaaagtact aactggaaaa aggctgaaga 1080gagcaaatat
attattatgg tgggttggag gattggtgag tttctaaata ttaagacact
1140gatcactaaa cgaatggatg atctactcag gtcaggattg aaagagaaat
atttcaacac 1200cttcctgcta cacaatggtc accagtggtc cagttattgt
tcaatttgat cataaatttg 1260cttcaattca ggagctttga aggaagtcca
aggaaagctc tagaaaacag tataaacctt 1320cagaggcaaa atccttcacc
aatttttccg catactttca tgccttgcct aaaaaaatta 1380acagagagtt
ggtatgtctc atgaatgctc tcacagaagg agttgctttt catgtcatct
1440acagcatatg agaaaagcta cctttctttt gattatatac acagatatca
aaataagcaa 1500ggatgagttt tacgtgtata tcaaaaatac aacagttgct
tgtattcagc cgagttttct 1560tgaccaccta ttatgttctg ggtgctacct
taacccagaa gacactatga aaaacaagac 1620agacttcact caaaacttac
atgaacacca ctagatgct 16594251PRTRhesus macaque 4Met Ala Glu Asp Leu
Gly Leu Ser Phe Gly Glu Thr Ala Ser Val Glu1 5 10 15 Met Leu Pro
Glu His Gly Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser 20 25 30 Ala
Cys Trp Ala Leu Thr Cys Cys Leu Val Leu Leu Pro Phe Leu Ala 35 40
45 Gly Leu Thr Thr Tyr Leu Leu Val Ser Gln Leu Arg Ala Gln Gly Glu
50 55 60 Ala Cys Val Gln Leu Gln Asp Leu Lys Gly Gln Glu Phe Ala
Pro Ser65 70 75 80 His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly
Asp Lys Pro Arg 85 90 95 Ala His Leu Thr Val Val Arg Gln Thr Pro
Thr Gln His Leu Lys Asn 100 105 110 Gln Phe Pro Ala Leu His Trp Glu
His Glu Leu Gly Leu Ala Phe Thr 115 120 125 Lys Asn Arg Met Asn Tyr
Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser 130 135 140 Gly Asp Tyr Phe
Val Tyr Ser Gln Val Thr Phe Arg Gly Met Thr Ser145 150 155 160 Glu
Cys Ser Glu Ile Arg Gln Ala Gly Arg Pro Asn Lys Pro Asp Ser 165 170
175 Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu Pro Thr
180 185 190 Gln Leu Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly Ser
Asn Trp 195 200 205 Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu
Gln Glu Gly Asp 210 215 220 Lys Leu Met Val Asn Val Ser Asp Ile Ser
Leu Val Asp Tyr Thr Lys225 230 235 240 Glu Asp Lys Thr Phe Phe Gly
Ala Phe Leu Leu 245 250 5540DNArhesus macaque 5aaaggacagg
agtttgcacc ttcacatcag caagtttatg cacctcttag agcagacgga 60gataagccaa
gggcacacct gacagttgtg acacaaactc ccacacagca ctttaaaaat
120cagttcccag ctctgcactg ggaacatgaa ctaggcctgg ccttcaccaa
gaaccgaatg 180aactatacca acaaattcct gctgatccca gagtcgggag
actacttcat ttactcccag 240gtcacattcc gtgggatgac ctctgagtgc
agtgaaatca gacaagcagg ccgaccaaac 300aagccagact ccatcactgt
ggtcatcacc aaggtaacag acagctaccc tgagccaacc 360cagctcctca
tggggaccaa gtctgtgtgc gaagtaggta gcaactggtt ccagcccatc
420tacctcggac ccatgttctc cttgcaagaa ggggacaagc taatggtgaa
cgtcagtgac 480atctccttgg tggattacac aaaagaagat aaaaccttct
ttggagcctt cttactatag 5406179PRTrhesus macaque 6Lys Gly Gln Glu Phe
Ala Pro Ser His Gln Gln Val Tyr Ala Pro Leu1 5 10 15 Arg Ala Asp
Gly Asp Lys Pro Arg Ala His Leu Thr Val Val Thr Gln 20 25 30 Thr
Pro Thr Gln His Phe Lys Asn Gln Phe Pro Ala Leu His Trp Glu 35 40
45 His Glu Leu Gly Leu Ala Phe Thr Lys Asn Arg Met Asn Tyr Thr Asn
50 55 60 Lys Phe Leu Leu Ile Pro Glu Ser Gly Asp Tyr Phe Ile Tyr
Ser Gln65 70 75 80 Val Thr Phe Arg Gly Met Thr Ser Glu Cys Ser Glu
Ile Arg Gln Ala 85 90 95 Gly Arg Pro Asn Lys Pro Asp Ser Ile Thr
Val Val Ile Thr Lys Val 100 105 110 Thr Asp Ser Tyr Pro Glu Pro Thr
Gln Leu Leu Met Gly Thr Lys Ser 115 120 125 Val Cys Glu Val Gly Ser
Asn Trp Phe Gln Pro Ile Tyr Leu Gly Pro 130 135 140 Met Phe Ser Leu
Gln Glu Gly Asp Lys Leu Met Val Asn Val Ser Asp145 150 155 160 Ile
Ser Leu Val Asp Tyr Thr Lys Glu Asp Lys Thr Phe Phe Gly Ala 165 170
175 Phe Leu Leu7675DNArhesus macaque 7ataaaaacat gtggtggtgg
cagcaaacct cccacgtgcc caccgtgccc agcacctgaa 60ctcctggggg gaccgtcagt
cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120tcccggaccc
ctgaggtcac atgcgtggtg gtagacgtga gccaggaaga ccccgatgtc
180aagttcaact ggtacgtaaa cggcgcggag gtgcatcatg cccagacgaa
gccacgggag 240acgcagtaca acagcacata tcgtgtggtc agcgtcctca
ccgtcacgca ccaggactgg 300ctgaacggca aggagtacac gtgcaaggtc
tccaacaaag ccctcccggt ccccatccag 360aaaaccatct ccaaagacaa
agggcagccc cgagagcctc aggtgtacac cctgcccccg 420tcccgggagg
agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctac
480cccagcgaca tcgtcgtgga gtgggagaac agcgggcagc cggagaacac
ctacaagacc 540accccgcccg tgctggactc cgacggctcc tacttcctct
acagcaagct caccgtggac 600aagagcaggt ggcagcaggg gaacgtcttc
tcatgctccg tgatgcatga ggctctgcac 660aaccactaca cgcag
6758225PRTrhesus macaque 8Ile Lys Thr Cys Gly Gly Gly Ser Lys Pro
Pro Thr Cys Pro Pro Cys1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 20 25 30 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45 Val Val Val Asp
Val Ser Gln Glu Asp Pro Asp Val Lys Phe Asn Trp 50 55 60 Tyr Val
Asn Gly Ala Glu Val His His Ala Gln Thr Lys Pro Arg Glu65 70 75 80
Thr Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Thr 85
90 95 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Thr Cys Lys Val Ser
Asn 100 105 110 Lys Ala Leu Pro Val Pro Ile Gln Lys Thr Ile Ser Lys
Asp Lys Gly 115 120 125 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu 130 135 140 Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr145 150 155 160 Pro Ser Asp Ile Val Val
Glu Trp Glu Asn Ser Gly Gln Pro Glu Asn 165 170 175 Thr Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 180 185 190 Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210
215 220 Gln225 91290DNAArtificial Sequencesynthetic oligonucleotide
9atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt
60gacctcgaga taaaaacatg tggtggtggc agcaaacctc ccacgtgccc accgtgccca
120gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc
caaggacacc 180ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg
tagacgtgag ccaggaagac 240cccgatgtca agttcaactg gtacgtaaac
ggcgcggagg tgcatcatgc ccagacgaag 300ccacgggaga cgcagtacaa
cagcacatat cgtgtggtca gcgtcctcac cgtcacgcac 360caggactggc
tgaacggcaa ggagtacacg tgcaaggtct ccaacaaagc cctcccggtc
420cccatccaga aaaccatctc caaagacaaa gggcagcccc gagagcctca
ggtgtacacc 480ctgcccccgt cccgggagga gctgaccaag aaccaggtca
gcctgacctg cctggtcaaa 540ggcttctacc ccagcgacat cgtcgtggag
tgggagaaca gcgggcagcc ggagaacacc 600tacaagacca ccccgcccgt
gctggactcc gacggctcct acttcctcta cagcaagctc 660accgtggaca
agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag
720gctctgcaca accactacac gcaggaattc aaaggacagg agtttgcacc
ttcacatcag 780caagtttatg cacctcttag agcagacgga gataagccaa
gggcacacct gacagttgtg 840acacaaactc ccacacagca ctttaaaaat
cagttcccag ctctgcactg ggaacatgaa 900ctaggcctgg ccttcaccaa
gaaccgaatg aactatacca acaaattcct gctgatccca 960gagtcgggag
actacttcat ttactcccag gtcacattcc gtgggatgac ctctgagtgc
1020agtgaaatca gacaagcagg ccgaccaaac aagccagact ccatcactgt
ggtcatcacc 1080aaggtaacag acagctaccc tgagccaacc cagctcctca
tggggaccaa gtctgtgtgc 1140gaagtaggta gcaactggtt ccagcccatc
tacctcggac ccatgttctc cttgcaagaa 1200ggggacaagc taatggtgaa
cgtcagtgac atctccttgg tggattacac aaaagaagat 1260aaaaccttct
ttggagcctt cttactatag 129010429PRTArtificial Sequencesynthetic
peptide 10Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp
Val Pro1 5 10 15 Gly Ser Thr Gly Asp Leu Glu Ile Lys Thr Cys Gly
Gly Gly Ser Lys 20 25 30 Pro Pro Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro 35 40 45 Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser 50 55 60 Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp65 70 75 80 Pro Asp Val Lys
Phe Asn Trp Tyr Val Asn Gly Ala Glu Val His His 85 90 95 Ala Gln
Thr Lys Pro Arg Glu Thr Gln Tyr Asn Ser Thr Tyr Arg Val 100 105 110
Val Ser Val Leu Thr Val Thr His Gln Asp Trp Leu Asn Gly Lys Glu 115
120 125 Tyr Thr Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile Gln
Lys 130 135 140 Thr Ile Ser Lys Asp Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr145 150 155 160 Leu Pro Pro Ser Arg Glu Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr 165 170 175 Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Val Val Glu Trp Glu 180 185 190 Asn Ser Gly Gln Pro Glu
Asn Thr Tyr Lys Thr Thr Pro Pro Val Leu 195 200 205 Asp Ser Asp Gly
Ser Tyr Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 210 215 220 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu225 230 235
240 Ala Leu His Asn His Tyr Thr Gln Glu Phe Lys Gly Gln Glu Phe Ala
245 250 255 Pro Ser His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly
Asp Lys 260 265 270 Pro Arg Ala His Leu Thr Val Val Thr Gln Thr Pro
Thr Gln His Phe 275 280 285 Lys Asn Gln Phe Pro Ala Leu His Trp Glu
His Glu Leu Gly Leu Ala 290 295 300 Phe Thr Lys Asn Arg Met Asn Tyr
Thr Asn Lys Phe Leu Leu Ile Pro305 310 315 320 Glu Ser Gly Asp Tyr
Phe Ile Tyr Ser Gln Val Thr Phe Arg Gly Met 325 330 335 Thr Ser Glu
Cys Ser Glu Ile Arg Gln Ala Gly Arg Pro Asn Lys Pro 340 345 350 Asp
Ser Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu 355 360
365 Pro Thr Gln Leu Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly Ser
370 375 380 Asn Trp Phe Gln Pro Ile Tyr Leu Gly Pro Met Phe Ser Leu
Gln Glu385 390 395 400 Gly Asp Lys Leu Met Val Asn Val Ser Asp Ile
Ser Leu Val Asp Tyr 405 410 415 Thr Lys Glu Asp Lys Thr Phe Phe Gly
Ala Phe Leu Leu 420 425 11579DNAHomo sapiens 11cgggcccagg
gagaggcctg tgtgcagttc caggctctaa aaggacagga gtttgcacct 60tcacatcagc
aagtttatgc acctcttaga gcagacggag ataagccaag ggcacacctg
120acagttgtga gacaaactcc cacacagcac tttaaaaatc agttcccagc
tctgcactgg 180gaacatgaac taggcctggc cttcaccaag aaccgaatga
actataccaa caaattcctg 240ctgatcccag agtcgggaga ctacttcatt
tactcccagg tcacattccg tgggatgacc 300tctgagtgca gtgaaatcag
acaagcaggc cgaccaaaca agccagactc catcactgtg 360gtcatcacca
aggtaacaga cagctaccct gagccaaccc agctcctcat ggggaccaag
420tctgtgtgcg aagtaggtag caactggttc cagcccatct acctcggagc
catgttctcc 480ttgcaagaag gggacaagct aatggtgaac gtcagtgaca
tctctttggt ggattacaca 540aaagaagata aaaccttctt tggagccttc ttactatag
57912192PRTHomo sapiens 12Arg Ala Gln Gly Glu Ala Cys Val Gln Phe
Gln Ala Leu Lys Gly Gln1 5 10 15 Glu Phe Ala Pro Ser His Gln Gln
Val Tyr Ala Pro Leu Arg Ala Asp 20 25 30 Gly Asp Lys Pro Arg Ala
His Leu Thr Val Val Arg Gln Thr Pro Thr 35 40 45 Gln His Phe Lys
Asn Gln Phe Pro Ala Leu His Trp Glu His Glu Leu 50 55 60 Gly Leu
Ala Phe Thr
Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu65 70 75 80 Leu Ile Pro
Glu Ser Gly Asp Tyr Phe Ile Tyr Ser Gln Val Thr Phe 85 90 95 Arg
Gly Met Thr Ser Glu Cys Ser Glu Ile Arg Gln Ala Gly Arg Pro 100 105
110 Asn Lys Pro Asp Ser Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser
115 120 125 Tyr Pro Glu Pro Thr Gln Leu Leu Met Gly Thr Lys Ser Val
Cys Glu 130 135 140 Val Gly Ser Asn Trp Phe Gln Pro Ile Tyr Leu Gly
Ala Met Phe Ser145 150 155 160 Leu Gln Glu Gly Asp Lys Leu Met Val
Asn Val Ser Asp Ile Ser Leu 165 170 175 Val Asp Tyr Thr Lys Glu Asp
Lys Thr Phe Phe Gly Ala Phe Leu Leu 180 185 190 13684DNAHomo
sapiens 13tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg
gggaccgtca 60gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac
ccctgaggtc 120acatgcgtgg tggtggacgt gagccacgaa gaccctgagg
tcaagttcaa ctggtacgtg 180gacggcgtgg aggtgcataa tgccaagaca
aagccgcggg aggagcagta caacagcacg 240taccgtgtgg tcagcgtcct
caccgtcctg caccaggact ggctgaatgg caaggagtac 300aagtgcaagg
tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc
360aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga
tgagctgacc 420aagaaccagg tcagcctgac ctgcctggtc aaaggcttct
atcccagcga catcgccgtg 480gagtgggaga gcaatgggca gccggagaac
aactacaaga ccacgcctcc cgtgctggac 540tccgacggct ccttcttcct
ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600gggaacgtct
tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag
660agcctctccc tgtctccggg taaa 68414228PRTHomo sapiens 14Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15 Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25
30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155
160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys225
151269DNAArtificial Sequencesynthetic oligonucleotide 15tgtgacaaaa
ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 60gtcttcctct
tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc
120acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa
ctggtacgtg 180gacggcgtgg aggtgcataa tgccaagaca aagccgcggg
aggagcagta caacagcacg 240taccgtgtgg tcagcgtcct caccgtcctg
caccaggact ggctgaatgg caaggagtac 300aagtgcaagg tctccaacaa
agccctccca gcccccatcg agaaaaccat ctccaaagcc 360aaagggcagc
cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc
420aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga
catcgccgtg 480gagtgggaga gcaatgggca gccggagaac aactacaaga
ccacgcctcc cgtgctggac 540tccgacggct ccttcttcct ctacagcaag
ctcaccgtgg acaagagcag gtggcagcag 600gggaacgtct tctcatgctc
cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660agcctctccc
tgtctccggg taaagaattc cgggcccagg gagaggcctg tgtgcagttc
720caggctctaa aaggacagga gtttgcacct tcacatcagc aagtttatgc
acctcttaga 780gcagacggag ataagccaag ggcacacctg acagttgtga
gacaaactcc cacacagcac 840tttaaaaatc agttcccagc tctgcactgg
gaacatgaac taggcctggc cttcaccaag 900aaccgaatga actataccaa
caaattcctg ctgatcccag agtcgggaga ctacttcatt 960tactcccagg
tcacattccg tgggatgacc tctgagtgca gtgaaatcag acaagcaggc
1020cgaccaaaca agccagactc catcactgtg gtcatcacca aggtaacaga
cagctaccct 1080gagccaaccc agctcctcat ggggaccaag tctgtgtgcg
aagtaggtag caactggttc 1140cagcccatct acctcggagc catgttctcc
ttgcaagaag gggacaagct aatggtgaac 1200gtcagtgaca tctctttggt
ggattacaca aaagaagata aaaccttctt tggagccttc 1260ttactatag
126916422PRTArtificial Sequencesynthetic peptide 16Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15 Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35
40 45 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165
170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys Glu Phe Arg Ala Gln
Gly Glu Ala Cys Val Gln Phe225 230 235 240 Gln Ala Leu Lys Gly Gln
Glu Phe Ala Pro Ser His Gln Gln Val Tyr 245 250 255 Ala Pro Leu Arg
Ala Asp Gly Asp Lys Pro Arg Ala His Leu Thr Val 260 265 270 Val Arg
Gln Thr Pro Thr Gln His Phe Lys Asn Gln Phe Pro Ala Leu 275 280 285
His Trp Glu His Glu Leu Gly Leu Ala Phe Thr Lys Asn Arg Met Asn 290
295 300 Tyr Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser Gly Asp Tyr Phe
Ile305 310 315 320 Tyr Ser Gln Val Thr Phe Arg Gly Met Thr Ser Glu
Cys Ser Glu Ile 325 330 335 Arg Gln Ala Gly Arg Pro Asn Lys Pro Asp
Ser Ile Thr Val Val Ile 340 345 350 Thr Lys Val Thr Asp Ser Tyr Pro
Glu Pro Thr Gln Leu Leu Met Gly 355 360 365 Thr Lys Ser Val Cys Glu
Val Gly Ser Asn Trp Phe Gln Pro Ile Tyr 370 375 380 Leu Gly Ala Met
Phe Ser Leu Gln Glu Gly Asp Lys Leu Met Val Asn385 390 395 400 Val
Ser Asp Ile Ser Leu Val Asp Tyr Thr Lys Glu Asp Lys Thr Phe 405 410
415 Phe Gly Ala Phe Leu Leu 420
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