U.S. patent number RE48,599 [Application Number 16/366,676] was granted by the patent office on 2021-06-22 for compositions comprising tlia-ig fusion protein for the regulation of t regulatory cells, and methods for their use.
This patent grant is currently assigned to University of Miami. The grantee listed for this patent is University of Miami. Invention is credited to Samia Q. Khan, Eckhard R. Podack, Taylor H. Schreiber.
United States Patent |
RE48,599 |
Podack , et al. |
June 22, 2021 |
Compositions comprising TLIA-Ig fusion protein for the regulation
of T regulatory cells, and methods for their use
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: |
1000005361099 |
Appl.
No.: |
16/366,676 |
Filed: |
March 27, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14151497 |
Jan 9, 2014 |
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61843558 |
Jul 8, 2013 |
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61842127 |
Jul 2, 2013 |
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61753634 |
Jan 17, 2013 |
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61750672 |
Jan 9, 2013 |
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Reissue of: |
14185295 |
Feb 20, 2014 |
9603925 |
Mar 28, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
38/20 (20130101); A61K 38/2013 (20130101); A61K
31/436 (20130101); A61K 39/3955 (20130101); A61K
31/7088 (20130101); C07K 14/55 (20130101); A61K
39/395 (20130101); C07K 2319/30 (20130101); C07K
14/52 (20130101); C07K 16/246 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); A61K 31/7088 (20060101); C07K
14/52 (20060101); C07K 14/525 (20060101); A61K
38/20 (20060101); C07K 14/55 (20060101); A61K
31/436 (20060101); C07K 16/24 (20060101) |
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|
Primary Examiner: Orwig; Kevin S
Attorney, Agent or Firm: Morgan, Lewis & Bockius LLP
Government Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under 5P01CA109094
awarded by the National Institutes of Health. The government has
certain rights in the invention.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
14/151,497, filed Jan. 9, 2014, which 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.
Claims
What is claimed is:
1. A composition comprising (i) 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 Tumor Necrosis Factor Receptor
Superfamily, Member 25 (TNFRSF25); and (b) a second polypeptide
comprising an immunoglobulin (Ig) polypeptide; and (ii) one or both
of an effective amount of interleukin (IL)-2 and an effective
amount of an mTOR inhibitor.
2. The composition of claim 1, wherein, when administered to a
human in need thereof, the composition reduces the frequency of
naive CD4 T cells in the human.
3. The composition of claim 1, wherein the first polypeptide
comprises (a) the 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.
4. The composition of claim 1, wherein the fusion protein is a
homomultimer, and wherein the homomultimer is a dimer of
trimers.
5. The composition of claim 1, wherein the Ig polypeptide comprises
one or more of a hinge region, a CH2 domain, and a CH3 domain of an
IgG polypeptide.
6. The composition claim 1, wherein the Ig polypeptide comprises
(a) the 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.
7. The composition of claim 1, wherein the fusion protein comprises
(a) the 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.
8. The composition of claim 1, wherein the in vivo efficacy of the
composition is higher than the in vivo efficacy of the first
polypeptide when it is not coupled with an Ig polypeptide.
9. The composition of claim 1, wherein the effective amount of IL-2
is an amount that, in combination with the TL1A-Ig fusion protein,
is sufficient to achieve a synergistic effect on the expansion of
Treg cells when administered to a subject.
10. The composition of claim 1, wherein the effective amount of
IL-2 is a dose of IL-2 that would induce suboptimal, or fail to
induce, expansion of Treg cells if administered alone to a human
patient.
11. The composition of claim 10, wherein the dose of IL-2 is less
than 1 million units per square meter per day.
12. The composition of claim 11, wherein the dose of IL-2 is an
amount in the range of about 30,000 to about 300,000 units per
square meter per day.
13. The composition of claim 12, wherein the dose of IL-2 is about
300,000 units per square meter per day.
14. The composition of claim 12, wherein the dose of IL-2 is about
30,000 units per square meter per day.
15. The composition of claim 1, wherein 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.
16. The composition of claim 15, wherein the mTOR inhibitor is
rapamycin.
17. The composition of claim 16, wherein the effective amount of
rapamycin is a dosage between about 25 .mu.g/kg and about 500
.mu.g/kg.
18. The composition of claim 1, wherein the composition comprises
the fusion protein and the IL-2.
19. The composition of claim 1, wherein the composition comprises
the fusion protein and the mTOR inhibitor.
.Iadd.20. A method of increasing the frequency of FoxP3+CD4+ Treg
cells out of total CD4+ cells in a patient, the method comprising:
(a) administering a fusion protein to the patient, the fusion
protein comprising, from N- to C-terminal: i) a human IgG domain,
and ii) a human TL1A extracellular domain that specifically binds
to Tumor Necrosis Factor Receptor Superfamily Member 25 (TNFRSF25);
and (b) administering to the patient an effective amount of
IL-2..Iaddend.
.Iadd.21. The method of claim 20, wherein an effective amount of
IL-2 is an amount that, in combination with the fusion protein, is
sufficient to achieve a synergistic effect on the expansion of Treg
cells when administered to a subject..Iaddend.
.Iadd.22. The method of claim 20, wherein an effective amount of
IL-2 is a dose of IL-2 that induces suboptimal, or fails to induce,
expansion of Treg cells if administered alone to a human
patient..Iaddend.
.Iadd.23. The method of claim 22, wherein the dose of IL-2 is less
than 1 million units per square meter per day..Iaddend.
.Iadd.24. The method of claim 23, wherein the dose of IL-2 is an
amount in the range of about 30,000 to about 300,000 units per
square meter per day..Iaddend.
.Iadd.25. The method of claim 23, wherein the dose of IL-2 is about
300,000 units per square meter per day..Iaddend.
.Iadd.26. The method of claim 23, wherein the dose of IL-2 is about
30,000 units per square meter per day..Iaddend.
.Iadd.27. A method of decreasing the frequency and/or expansion of
effector T cells in a patient, the method comprising: (a)
administering to the patient a fusion protein, the fusion protein
comprising, from N- to C-terminal: i) a human IgG domain, and ii) a
human TL1A extracellular domain that specifically binds to Tumor
Necrosis Factor Receptor Superfamily Member 25 (TNFRSF25); and (b)
administering to the patient an effective amount of an mTOR
inhibitor..Iaddend.
.Iadd.28. The method of claim 27, wherein 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..Iaddend.
.Iadd.29. A method of claim 28, wherein the mTOR inhibitor is
rapamycin..Iaddend.
.Iadd.30. A method of claim 29, wherein the effective amount of
rapamycin is between about 25 .mu.g/kg and about 500
.mu.g/kg..Iaddend.
Description
TECHNICAL FIELD
This disclosure relates to the fields of molecular biology and
immunology.
BACKGROUND
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
As discussed above, safe and effective therapies for so 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.
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).
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.
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.
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.
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.
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.
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.
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.
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 comprises 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.
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.
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.
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
FIG. 1 is a schematic outline of the murine adoptive transfer
model.
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.
FIGS. 3A-3D are line graphs quantifying the proportion of
FoxP3+Ki67+ mLN pTreg (FIGS. 3A and 3B) and in mLN tTreg (FIGS. 3C
and 3D) cells out of total CD4+ cells in groups of mice that
received 1% ovalbumnin (ova) in drinking water continuously (FIGS.
3A and 3C) 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) (FIGS. 3B and 3D). 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.
FIGS. 4A-4D are line graphs quantifying the proportion of splenic
FoxP3+Ki67+ pTreg (FIGS. 4A and 4B) and in splenic tTreg (FIGS. 4C
and 4D) cells out of total CD4+ cells in groups of mice that
received 1% ova in drinking water continuously (FIGS. 4A and 4C) 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) (FIGS. 4B and 4D). 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.
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.
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).
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 (FIGS. 7A-7D), mLN (FIGS. 8A-8D), and small intestine
(FIGS. 9A-9D) 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.
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.
FIGS. 11A and 11B are line graphs quantifying the absolute number
of leukocytes (10.sup.3/.mu.l) (FIG. 11A) and total
polymorphonuclear (PMN) cells (10.sup.3/.mu.l) (FIG. 11B)
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.
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.
FIGS. 13A and 13B are bar graphs quantifying the serum
concentration of IFN-.gamma. (ng/ml) (FIG. 13A) and TGFI.beta.
(ng/ml) (FIG. 13B) 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.
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.
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).
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).
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
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.
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 TL 1 A) or any fragment thereof that retains
specific binding to the TL 1 A 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.
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.
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.
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.
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 4C 12), 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.
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:
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.
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).
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.
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.
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.
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."
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%.
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.
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.
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.
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.
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).
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.
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).
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.
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.
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).
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").
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.
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.
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.
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.
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.
TNFRSF25 Agonists
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.
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).
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.
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_001204344.1/NP_001191273.1,
NM_005118.3/NP_005109.2, NM_001039664.1/NP 001034753.1,
NM_148970.1/NP_683871.1, NM 148967.1/NP_683868.1,
NM_148966.1/NP_683867.1, NM_148965.1/NP_683866.1, and
NM_003790.2/NP_003781.1, each of which is incorporated by reference
(including the referenced sequences).
Rhesus macaque TL1A nucleic acid and amino acid sequences can be
inferred from the Macaca mulatta chromosome 15, Mmul_051212, whole
genome shotgun sequence (GenBank.RTM. Accession No. NC_007872.1).
The mRNA sequence having GenBank.RTM. Accession No. NM_001194132.1
(SEQ ID NO: 3). An exemplary rhesus macaque amino acid sequence has
GenBank.RTM. Accession No. NP_001181061, which is incorporated by
reference (including the referenced sequence). (SEQ ID NO: 4).
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.
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.
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. 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.
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.
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).
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.
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)
aaaggacaggagtttgcaccttcacatcagcaagtttatgcacctcttag
agcagacggagataagccaagggcacacctgacagttgtgacacaaactc
ccacacagcactttaaaaatcagttcccagctctgcactgggaacatgaa
ctaggcctggccttcaccaagaaccgaatgaactataccaacaaattcct
gctgatcccagagtcgggagactacttcatttactcccaggtcacattcc
gtgggatgacctctgagtgcagtgaaatcagacaagcaggccgaccaaac
aagccagactccatcactgtggtcatcaccaaggtaacagacagctaccc
tgagccaacccagctcctcatggggaccaagtctgtgtgcgaagtaggta
gcaactggttccagcccatctacctcggacccatgttctccttgcaagaa
ggggacaagctaatggtgaacgtcagtgacatctccttggtggattacac
aaaagaagataaaaccttctttggagccttcttactatag; and (SEQ ID NO: 6)
kgqefapshqqvyaplradgdkprahltvvtqtptqhfknqfpalhwehe
lglaftknrmnytnkfllipesgdyfiysqvtfrgmtsecseirpgrpnk
pdsitvvitkvtdsypeptqllmgtksvcevgsnwfqpiy1gpmfslqeg
dklmvnvedislvdytkedktffgafll.
Furthermore, in some embodiments the nucleic acid of the rhesus
macaque Ig sequence (IgG1 hinge-CH2-CH3 sequence) is:
ataaaaacatgtggtggtggcagcaaacctcccacgtgcccaccgtgccca
gcacctgaactcctggggggac cgtcagtcttcctcttccccccaaaaccc
aaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtagacgt
gagccaggaagaccccgatgtcaagttcaactggtacgtaaacggcgcggaggt
gcatcatgcccagacgaagccacggga gacgcagtacaacagcacatatc
gtgtggtcagcgtcctcaccgtcacgcaccaggactggctgaacggcaaggagtacacgt
gcaaggtctccaacaaagccctcccggtccccatccagaaaaccatct
ccaaagacaaagggcagccccgagagcctcagg tgtacaccct
gcccccgtcccgggaggagctgaccaagaaccaggtcagcctgacctgcctgg
tcaaaggcttctacccca gcgacatcgtcgtggagtgggagaacagcgggcagccggag
aacacctacaagaccaccccgcccgtgctggactccg acggctcctactt
cctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtct
tctcatgctccgtgat 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:
iktcgggskpptcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvd
vsqedpdvkfnwyvngaevh haqtkpretqynstyrvvsvltvthq
dwlngkeytckvsnkalpvpiqktiskdkgqprepqvytlppsreeltknqvsltcl
vkgfypsdivvewensgqentykttppvldsdgsyflyskltvdksr
wqqgnvfscsvmhealhnhytq (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.
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)
atggagacagacacactcctgctatgggtactgctgctctgggttccagg ttccactggtgac
ataaaaacatgtggtggtggcagcaaacctc
ccacgtgcccaccgtgcccagcacctgaactcctggggggaccgtcagtc
ttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccc
tgaggtcacatgcgtggtggtagacgtgagccaggaagaccccgatgtca
agttcaactggtacgtaaacggcgcggaggtgcatcatgcccagacgaag
ccacgggagacgcagtacaacagcacatatcgtgtggtcagcgtcctcac
cgtcacgcaccaggactggctgaacggcaaggagtacacgtgcaaggtct
ccaacaaagccctcccggtccccatccagaaaaccatctccaaagacaaa
gggcagccccgagagcctcaggtgtacaccctgcccccgtcccgggagga
gctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctacc
ccagcgacatcgtcgtggagtgggagaacagcgggcagccggagaacacc
tacaagaccaccccgcccgtgctggactccgacggctcctacttcctcta
cagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttct
catgctccgtgatgcatgaggctctgcacaaccactacacgcag
aaaggacaggagtttgcaccttcacatcagcaagtttatgcacctcttag
agcagacggagataagccaagggcacacctgacagttgtgacacaaactc
ccacacagcactttaaaaatcagtteccagctctgcactgagaacatgaa
ctaggcctggccttcaccaagaaccgaatgaactataccaacaaattcct
gctgatcccagagtcgggagactacttcatttactcccaggtcacattcc
gtgggatgacctctgagtgcagtgaaatcagacaagcaggccgaccaaac
aagccagactccatcactgtggtcatcaccaaggtaacagacagctaccc
tgagccaacccagctcctcatggggaccaagtctgtgtgcgaagtaggta
gcaactggttccagcccatctacctcggacccatgttctccttgcaagaa
ggggacaagctaatggtgaacgtcagtgacatctccttggtggattacac
aaaagaagataaaaccttctttggagccttcttactatag; and (SEQ ID NO: 10:
metdtlllwvlllwvpgstqd iktcgggskpptcppcpapellggps
vflfppkpkdtlmisrtpevtcyvvdvsgedpdvkfnwyvngaevhhagt
kpretqynstyrvvsyltvthqdwlngkeytckvsnkalpvpiqktiskd
kgqprepgvytlppereeltkngveltclvkgfypsdivvewensggpen
tykttppvldsdgsyflyskltvdksrwqqgnvfscsvmhealhnhytq
kgqefapshqqvyaplradgdkprahltvvtqtptqhfknqfpalhwe
helqlaftknrmnytnkfllipesgdyfiysqvtfrgmtsecseirqaqr
pnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwfqpiylgpmfsl
qegdklmvnvsdislvdytkedktffgafll.
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).
In some embodiments, the human TL1A polypeptide comprises or
consists of amino acid residues 68-252 from the extracellular
domain of human TL1A.
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)
cgggcccagggagaggcctgtgtgcagttccaggctctaaaaggacagga
gtttgcaccttcacatcagcaagtttatgcacctcttagagcagacggag
ataagccaagggcacacctgacagttgtgagacaaactcccacacagcac
tttaaaaatcagttcccagctctgcactgggaacatgaactaggcctggc
cttcaccaagaaccgaatgaactataccaacaaattcctgctgatcccag
agtcgggagactacttcatttactcccaggtcacattccgtgggatgacc
tctgagtgcagtgaaatcagacaagcaggccgaccaaacaagccagactc
catcactgtggtcatcaccaaggtaacagacagctaccctgagccaaccc
agctcctcatggggaccaagtctgtgtgcgaagtaggtagcaactggttc
cagcccatctacctcggagccatgttctccttgcaagaaggggacaagct
aatggtgaacgtcagtgacatctctttggtggattacacaaaagaagata
aaaccttctttggagccttcttactatag (encoding the extra- cellular domain
of human TL1A); and (SEQ ID NO: 12)
raqgeacvqfgalkgqefapshqqvyaplradgdkprahltvvrqtptqh
fknqfpalhwehelglaftknrmnytnkfllipesgdyfiysqvtfrgmt
secseirqagrpnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwf
qpiylgamfslqegdklmvnvsdislvdytkedktffgafll (the extracellular
domain of human TL1A).
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)
tgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggg
gggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga
tctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa
gaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataa
tgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtgg
tcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtac
aagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccat
ctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc
catcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtc
aaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggca
gccggagaacaactacaagaccacgcctcccgtgctggactccgacggct
ccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag
gggaacgtatctcatgctccgtgatgcatgaggctctgcacaaccactac
acgcagaagagcctctccctgtctccgggtaaa; and (SEQ ID NO: 14)
cdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshe
dpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkey
kckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclv
kgfypsdiavewesngqpennykttppvldsdgsfflyskltvdkerwqg
gnvfscsvmhealhnhytqkslslspgk (the hinge, CH2 and CH3 region of
human IgG).
In some embodiments the nucleic acid and amino acid sequences of
the human TL1A fusion protein are:
TABLE-US-00005 (SEQ ID NO: 15)
tgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggg
gggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga
tctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa
gaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataa
tgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtgg
tcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtac
aagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccat
ctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc
catcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtc
aaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggca
gccggagaacaactacaagaccacgcctcccgtgctggactccgacggct
ccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag
gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccacta
cacgcagaagagcctctccctgtctccgggtaaa cgggcccagg
gagaggcctgtgtgcagttccaggctctaaaaggacaggagtttgcacct
tcacatcagcaagtttatgcacctettagagcagacggagataagccaag
ggcacacctgacagttgtgagacaaactcccacacagcactttaaaaatc
agtteccagctctgcactgggaacatgaactaggcctggccttcaccaag
aaccgaatgaactataccaacaaattcctgctgatcccagagtcgggaga
ctacttcatttactcccaggtcacattccgtgggatgacctctgagtgca
gtgaaatcagacaagcaggccgaccaaacaagccagactccatcactgtg
gtcatcaccaaggtaacagacagctaccctgagccaacccagctcctcat
ggggaccaagtctgtgtgcgaagtaggtagcaactggttccagcccatct
acctcggagccatgttctecttgcaagaaggggacaagctaatggtgaac
gtcagtgacatctctttggtggattacacaaaagaagataaaaccttctt
tggagccttcttactatag; and (SEQ ID NO: 16)
cdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshe
dpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkey
kckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclv
kgfypsdiavewesngqpennykttppvldsdgsfflyskltvdkerwqq
gnvfscsvmhealhnhytqkslslspgk raqgeacvqfgalkgqefap
shqgvyaplradgdkprahltvvrgtptghfknqfpalhwehelglaftk
nrmnytnkfllipesgdyfiysqvtfrgmtsecseirqagrpnkpdsitv
vitkvtdsypeptqllmgtksvcevqsnwfqpiylqamfslqegdklmvn
vsdislvdytkedktffgafll.
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).
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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. Nos. 5,218,088; 5,175,269; 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)).
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.
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.
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.
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.
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 .beta.-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.
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 (Ampr), tetracycline resistance gene
(Tc.sup.r) and the kanamycin resistance gene (Kan).
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.
In some embodiments preferred plasmids for transformation of E.
coli cells include the pET expression vectors (e.g., pET-11 a,
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-IllompAplasmids (see U.S. Pat. No. 4,575,013; see also Duffaud
et al., Meth. Enz. 153:492-507, 1987), such as pIN-IIIompA2.
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.
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
.lamda.c1857 repressor, and the like.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
"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).
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, 989; Kunkle, PNAS 82:488,
1985).
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.
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,
T19-14.times., 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.
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.
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.
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.
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.
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).
mTOR Inhibitors
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.
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.
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.
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.
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.
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)).
Interleukins
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.
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).
Compositions and Pharmaceutical Compositions
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.
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.
In some embodiments, provided herein are compositions comprising a
TL1A fusion protein as described herein and an effective amount of
IL-2.
In some embodiments, provided herein are compositions comprising an
agonistic TNFRSF25 antibody as described herein and an effective
amount of IL-2.
In some embodiments, provided herein are compositions comprising a
small molecule agonist of TNFRSF25 as described herein and an
effective amount of IL-2.
In some embodiments, provided herein are compositions comprising a
TL1A fusion protein as described herein and an effective amount of
an mTOR inhibitor.
In some embodiments, provided herein are compositions comprising an
agonistic TNFRSF25 antibody as described herein and an effective
amount of an mTOR inhibitor.
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.
In some embodiments, provided herein are compositions comprising a
TL1A fusion protein as described herein and an effective amount of
rapamycin.
In some embodiments, provided herein are compositions comprising an
agonistic TNFRSF25 antibody as described herein and an effective
amount of rapamycin.
In some embodiments, provided herein are compositions comprising a
small molecule agonist of TNFRSF25 as described herein and an
effective amount of rapamycin.
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.
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.]
Administration and Dosage
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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).
Uses of TL1A Fusion Proteins and Other TNFRSF25 Agonists
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.
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.
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.
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.
Exemplary effective amounts of TNFRSF25 agonists, interleukins, and
mTOR inhibitors for use in the methods disclosed herein are
described above.
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).
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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).
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).
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.
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
The following are the materials and methods used in the Examples
set forth below.
Mice and Adoptive Transfer Model
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/yc-/- (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).
Cloning of Rhesus Macaque and Human TL1A-Ig
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).
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-CH.sub.2--CH3 domain
of human IgG1, and cloned into the pVITRO2-hygro (InvivoGen, San
Diego, Calif.).
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)
aaaggacaggagtttgcaccttcacatcagcaagtttatgcacctcttag
agcagacggagataagccaagggcacacctgacagttgtgacacaaactc
ccacacagcactttaaaaatcagttcccagctctgcactgggaacatgaa
ctaggcctggccttcaccaagaaccgaatgaactataccaacaaattcct
gctgatcccagagtcgggagactacttcatttactcccaggtcacattcc
gtgggatgacctctgagtgcagtgaaatcagacaagcaggccgaccaaac
aagccagactccatcactgtggtcatcaccaaggtaacagacagctaccc
tgagccaacccagctcctcatggggaccaagtctgtgtgcgaagtaggta
gcaactggttccagcccatctacctcggacccatgttctccttgcaagaa
ggggacaagctaatggtgaacgtcagtgacatctccttggtggattacac
aaaagaagataaaaccttctttggagccttcttactatag; and Amino Acid Sequence
(SEQ ID NO: 6) kgqefapshqqvyaplradgdkprahltvvtqtptqhfknqfpalhwehe
lglaftknrmnytnkfllipesgdyfiysqvtfrgmtsecseirpgrpnk
pdsitvvitkvtdsypeptqllmgtksvcevgsnwfqpiy1gpmfslqeg
dklmvnvedislvdytkedktffgafll.
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)
ataaaaacatgtggtggtggcagcaaacctcccacgtgcccaccgtgccc
agcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaac
ccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtg
gtagacgtgagccaggaagaccccgatgtcaagttcaactggtacgtaaa
cggcgcggaggtgcatcatgcccagacgaagccacgggagacgcagtaca
acagcacatatcgtgtggtcagcgtcctcaccgtcacgcaccaggactgg
ctgaacggcaaggagtacacgtgcaaggtctccaacaaagccctcccggt
ccccatccagaaaaccatctccaaagacaaagggcagccccgagagcctc
aggtgtacaccctgcccccgtcccgggaggagctgaccaagaaccaggtc
agcctgacctgcctggtcaaaggcttctaccccagcgacatcgtcgtgga
gtgggagaacagcgggcagccggagaacacctacaagaccaccccgcccg
tgctggactccgacggctcctacttcctctacagcaagctcaccgtggac
aagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatga
ggctctgcacaaccactacacgcag; and Amino Acid Sequence: (SEQ ID NO: 8)
iktcgggskpptcppcpapellggpsvflfppkpkdtlmisrtpevtcvv
vdvsqedpdvkfnwyvngaevhhaqtkpretqynstyrvvsyltythqdw
lngkeytekvsnkalpvpigktiskdkgqprepqvytlppereeltknqv
sltelvkgfypsdivvewensggpentykttppvldsdgsyflyskltvd
kerwqqgnvfscsvmhealhnhytg.
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)
atggagacagacacactcctgctatgggtactgctgctctgggttccagg ttccactggtgac
ataaaaacatgtggtggtggcagcaaacctc
ccacgtgcccaccgtgcccagcacctgaactcctggggggaccgtcagtc
ttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccc
tgaggtcacatgcgtggtggtagacgtgagccaggaagaccccgatgtca
agttcaactggtacgtaaacggcgcggaggtgcatcatgcccagacgaag
ccacgggagacgcagtacaacagcacatatcgtgtggtcagcgtcctcac
cgtcacgcaccaggactggctgaacggcaaggagtacacgtgcaaggtct
ccaacaaagccctcccggtccccatccagaaaaccatctccaaagacaaa
gggcagccccgagagcctcaggtgtacaccctgcccccgtcccgggagga
gctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctacc
ccagcgacatcgtcgtggagtgggagaacagcgggcagccggagaacacc
tacaagaccaccccgcccgtgctggactccgacggctcctacttcctcta
cagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttct
catgctccgtgatgcatgaggctctgcacaaccactacacgcag
aaaggacaggagtttgcaccttcacatcagcaagtttatgcacctcttag
agcagacggagataagccaagggcacacctgacagttgtgacacaaactc
ccacacagcactttaaaaatcagtteccagctctgcactgagaacatgaa
ctaggcctggccttcaccaagaaccgaatgaactataccaacaaattcct
gctgatcccagagtcgggagactacttcatttactcccaggtcacattcc
gtgggatgacctctgagtgcagtgaaatcagacaagcaggccgaccaaac
aagccagactccatcactgtggtcatcaccaaggtaacagacagctaccc
tgagccaacccagctcctcatggggaccaagtctgtgtgcgaagtaggta
gcaactggttccagcccatctacctcggacccatgttctccttgcaagaa
ggggacaagctaatggtgaacgtcagtgacatctccttggtggattacac
aaaagaagataaaaccttctttggagccttcttactatag; and Amino Acid Sequence:
(SEQ ID NO: 10) metdtlllwvlllwvpgstqd iktcgggskpptcppcpapellggps
vflfppkpkdtlmisrtpevtcyvvdvsgedpdvkfnwyvngaevhhagt
kpretqynstyrvvsyltvthqdwlngkeytckvsnkalpvpiqktiskd
kgqprepgvytlppereeltkngveltclvkgfypsdivvewensggpen
tykttppvldsdgsyflyskltvdksrwqqgnvfscsvmhealhnhytq
kgqefapshqqvyaplradgdkprahltvvtqtptqhfknqfpalhwe
helqlaftknrmnytnkfllipesgdyfiysqvtfrgmtsecseirqaqr
pnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwfqpiylgpmfsl
qegdklmvnvsdislvdytkedktffgafll.
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. 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)
cgggcccagggagaggcctgtgtgcagttccaggctctaaaaggacagga
gtttgcaccttcacatcagcaagtttatgcacctcttagagcagacggag
ataagccaagggcacacctgacagttgtgagacaaactcccacacagcac
tttaaaaatcagttcccagctctgcactgggaacatgaactaggcctggc
cttcaccaagaaccgaatgaactataccaacaaattcctgctgatcccag
agtcgggagactacttcatttactcccaggtcacattccgtgggatgacc
tctgagtgcagtgaaatcagacaagcaggccgaccaaacaagccagactc
catcactgtggtcatcaccaaggtaacagacagctaccctgagccaaccc
agctcctcatggggaccaagtctgtgtgcgaagtaggtagcaactggttc
cagcccatctacctcggagccatgttctccttgcaagaaggggacaagct
aatggtgaacgtcagtgacatctctttggtggattacacaaaagaagata
aaaccttctttggagccttcttactatag; and Amino Acid Sequence: (SEQ ID NO:
12) raqgeacvqfgalkgqefapshqqvyaplradgdkprahltvvrqtptqh
fknqfpalhwehelglaftknrmnytnkfllipesgdyfiysqvtfrgmt
secseirqagrpnkpdsitvvitkvtdsypeptqllmgtksvcevgsnwf
qpiylgamfslqegdklmvnvsdislvdytkedktffgafll.
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)
tgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggg
gggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga
tctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa
gaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataa
tgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtgg
tcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtac
aagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccat
ctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc
catcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtc
aaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggca
gccggagaacaactacaagaccacgcctcccgtgctggactccgacggct
ccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag
gggaacgtatctcatgctccgtgatgcatgaggctctgcacaaccactac
acgcagaagagcctctccctgtctccgggtaaa; and Amino Acid Sequence: (SEQ ID
NO: 14) cdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshe
dpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkey
kckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclv
kgfypsdiavewesngqpennykttppvldsdgsfflyskltvdkerwqq
gnvfscsvmhealhnhytqkslslspgk.
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)
tgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggg
gggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga
tctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa
gaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataa
tgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtgg
tcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtac
aagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccat
ctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc
catcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtc
aaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggca
gccggagaacaactacaagaccacgcctcccgtgctggactccgacggct
ccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag
gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccacta
cacgcagaagagcctctccctgtctccgggtaaa cgggcccagg
gagaggcctgtgtgcagttccaggctctaaaaggacaggagtttgcacct
tcacatcagcaagtttatgcacctettagagcagacggagataagccaag
ggcacacctgacagttgtgagacaaactcccacacagcactttaaaaatc
agtteccagctctgcactgggaacatgaactaggcctggccttcaccaag
aaccgaatgaactataccaacaaattcctgctgatcccagagtcgggaga
ctacttcatttactcccaggtcacattccgtgggatgacctctgagtgca
gtgaaatcagacaagcaggccgaccaaacaagccagactccatcactgtg
gtcatcaccaaggtaacagacagctaccctgagccaacccagctcctcat
ggggaccaagtctgtgtgcgaagtaggtagcaactggttccagcccatct
acctcggagccatgttctecttgcaagaaggggacaagctaatggtgaac
gtcagtgacatctctttggtggattacacaaaagaagataaaaccttctt
tggagccttcttactatag; and Amino Acid Sequence (SEQ ID NO: 16)
cdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshe
dpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkey
kckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclv
kgfypsdiavewesngqpennykttppvldsdgsfflyskltvdkerwqq
gnvfscsvmhealhnhytqkslslspgk raqgeacvqfgalkgqefap
shqgvyaplradgdkprahltvvrgtptghfknqfpalhwehelglaftk
nrmnytnkfllipesgdyfiysqvtfrgmtsecseirqagrpnkpdsitv
vitkvtdsypeptqllmgtksvcevqsnwfqpiylqamfslqegdklmvn
vsdislvdytkedktffgafll.
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.
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.
Cell Culture
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.
Reagents, antibodies and flow cytometry
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.
Western Blot
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).
Caspase Detection Assay
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).
T Cell Proliferation Assay
These assays were performed as described in Khan et al. J. Immunol.
2013 Feb. 15; 190(4):1540-50.
Generation of Humanized Mice
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).
Safety and Activity of TL1A-Ig in Rhesus Macaque 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. LSR11 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 .mu.L 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 .mu.L 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.
Statistical Analysis
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
This Example demonstrates that Treg proliferation stimulated by
TNFRSF25 is dependent on cognate antigen.
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.
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.
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
proportionvof 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 (FIGS. 3A-3D) and spleen (FIGS.
4A-4D), 1% ova in drinking water was either continued (FIGS. 3A,
3C, 4A and 4C) or replaced with normal water to `washout` ovalbumin
(ova) for the indicated number of days (FIGS. 3B, 3D, 4B, and 4D).
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.
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 (FIGS. 3A-3D). 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 (FIGS. 3A-3D) and spleen
(FIGS. 4A-4D). 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 4C 12 stimulated proliferation,
indicating an antigen-specific response.
Example 3
Human TL1A-Ig Stimulates Proliferation of Human tTre2 in Humanized
Mice
This Example demonstrates that human TL1A-Ig fusion protein induced
strong proliferation of human Tregs systemically and in the mucosa
of humanized mice.
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).
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%).
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% (FIGS. 7A-7D). In lymph nodes, the
control mean Treg value was 0.91.+-.0.12 whereas for hTL1A-Ig it
was 1.94.+-.0.21 (FIGS. 8A-8D). 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% (FIGS. 9A-9D). 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%)
pheootype. 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.
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-Ig in Rhesus Macaques
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.
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.
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.
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 (FIGS. 13A-13B). 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.
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 (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.
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.
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.
Discussion
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
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.
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.
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.
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.
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.
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
4C 12/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-Ig and Rapamycin
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.
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.
As shown in FIG. 18 and FIGS. 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.
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-Ig Fusion
Protein
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.
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
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.
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
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.
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 LISTINGS
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 15Met Leu Pro Glu His Gly Ser Cys Arg Pro
Lys Ala Arg Ser Ser Ser 20 25 30Ala Arg Trp Ala Leu Thr Cys Cys Leu
Val Leu Leu Pro Phe Leu Ala 35 40 45Gly Leu Thr Thr Tyr Leu Leu Val
Ser Gln Leu Arg Ala Gln Gly Glu 50 55 60Ala Cys Val Gln Phe Gln Ala
Leu Lys Gly Gln Glu Phe Ala Pro Ser65 70 75 80His Gln Gln Val Tyr
Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg 85 90 95Ala His Leu Thr
Val Val Arg Gln Thr Pro Thr Gln His Phe Lys Asn 100 105 110Gln Phe
Pro Ala Leu His Trp Glu His Glu Leu Gly Leu Ala Phe Thr 115 120
125Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser
130 135 140Gly Asp Tyr Phe Ile Tyr Ser Gln Val Thr Phe Arg Gly Met
Thr Ser145 150 155 160Glu Cys Ser Glu Ile Arg Gln Ala Gly Arg Pro
Asn Lys Pro Asp Ser 165 170 175Ile Thr Val Val Ile Thr Lys Val Thr
Asp Ser Tyr Pro Glu Pro Thr 180 185 190Gln Leu Leu Met Gly Thr Lys
Ser Val Cys Glu Val Gly Ser Asn Trp 195 200 205Phe Gln Pro Ile Tyr
Leu Gly Ala Met Phe Ser Leu Gln Glu Gly Asp 210 215 220Lys Leu Met
Val Asn Val Ser Asp Ile Ser Leu Val Asp Tyr Thr Lys225 230 235
240Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 245
25031659DNARhesus 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 15Met Leu Pro Glu
His Gly Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser 20 25 30Ala Cys Trp
Ala Leu Thr Cys Cys Leu Val Leu Leu Pro Phe Leu Ala 35 40 45Gly Leu
Thr Thr Tyr Leu Leu Val Ser Gln Leu Arg Ala Gln Gly Glu 50 55 60Ala
Cys Val Gln Leu Gln Asp Leu Lys Gly Gln Glu Phe Ala Pro Ser65 70 75
80His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg
85 90 95Ala His Leu Thr Val Val Arg Gln Thr Pro Thr Gln His Leu Lys
Asn 100 105 110Gln Phe Pro Ala Leu His Trp Glu His Glu Leu Gly Leu
Ala Phe Thr 115 120 125Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu
Leu Ile Pro Glu Ser 130 135 140Gly Asp Tyr Phe Val Tyr Ser Gln Val
Thr Phe Arg Gly Met Thr Ser145 150 155 160Glu Cys Ser Glu Ile Arg
Gln Ala Gly Arg Pro Asn Lys Pro Asp Ser 165 170 175Ile Thr Val Val
Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu Pro Thr 180 185 190Gln Leu
Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly Ser Asn Trp 195 200
205Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu Gln Glu Gly Asp
210 215 220Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp Tyr
Thr Lys225 230 235 240Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu
245 2505540DNArhesus 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 15Arg Ala Asp Gly Asp Lys Pro Arg Ala
His Leu Thr Val Val Thr Gln 20 25 30Thr Pro Thr Gln His Phe Lys Asn
Gln Phe Pro Ala Leu His Trp Glu 35 40 45His Glu Leu Gly Leu Ala Phe
Thr Lys Asn Arg Met Asn Tyr Thr Asn 50 55 60Lys Phe Leu Leu Ile Pro
Glu Ser Gly Asp Tyr Phe Ile Tyr Ser Gln65 70 75 80Val Thr Phe Arg
Gly Met Thr Ser Glu Cys Ser Glu Ile Arg Gln Ala 85 90 95Gly Arg Pro
Asn Lys Pro Asp Ser Ile Thr Val Val Ile Thr Lys Val 100 105 110Thr
Asp Ser Tyr Pro Glu Pro Thr Gln Leu Leu Met Gly Thr Lys Ser 115 120
125Val Cys Glu Val Gly Ser Asn Trp Phe Gln Pro Ile Tyr Leu Gly Pro
130 135 140Met Phe Ser Leu Gln Glu Gly Asp Lys Leu Met Val Asn Val
Ser Asp145 150 155 160Ile Ser Leu Val Asp Tyr Thr Lys Glu Asp Lys
Thr Phe Phe Gly Ala 165 170 175Phe 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 15Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 20 25 30Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 35 40 45Val Val Val Asp Val Ser Gln
Glu Asp Pro Asp Val Lys Phe Asn Trp 50 55 60Tyr Val Asn Gly Ala Glu
Val His His Ala Gln Thr Lys Pro Arg Glu65 70 75 80Thr Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Thr 85 90 95His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Thr Cys Lys Val Ser Asn 100 105 110Lys
Ala Leu Pro Val Pro Ile Gln Lys Thr Ile Ser Lys Asp Lys Gly 115 120
125Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
130 135 140Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr145 150 155 160Pro Ser Asp Ile Val Val Glu Trp Glu Asn Ser
Gly Gln Pro Glu Asn 165 170 175Thr Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Tyr Phe 180 185 190Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215
220Gln22591290DNAArtificial 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 15Gly Ser Thr Gly Asp Leu Glu Ile Lys Thr Cys Gly Gly
Gly Ser Lys 20 25 30Pro Pro Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 35 40 45Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 50 55 60Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp65 70 75 80Pro Asp Val Lys Phe Asn Trp Tyr
Val Asn Gly Ala Glu Val His His 85 90 95Ala Gln Thr Lys Pro Arg Glu
Thr Gln Tyr Asn Ser Thr Tyr Arg Val 100 105 110Val Ser Val Leu Thr
Val Thr His Gln Asp Trp Leu Asn Gly Lys Glu 115 120 125Tyr Thr Cys
Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile Gln Lys 130 135 140Thr
Ile Ser Lys Asp Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr145 150
155 160Leu Pro Pro Ser Arg Glu Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr 165 170 175Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val
Glu Trp Glu 180 185 190Asn Ser Gly Gln Pro Glu Asn Thr Tyr Lys Thr
Thr Pro Pro Val Leu 195 200 205Asp Ser Asp Gly Ser Tyr Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 210 215 220Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu225 230 235 240Ala Leu His Asn
His Tyr Thr Gln Glu Phe Lys Gly Gln Glu Phe Ala 245 250 255Pro Ser
His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys 260 265
270Pro Arg Ala His Leu Thr Val Val Thr Gln Thr Pro Thr Gln His Phe
275 280 285Lys Asn Gln Phe Pro Ala Leu His Trp Glu His Glu Leu Gly
Leu Ala 290 295 300Phe Thr Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe
Leu Leu Ile Pro305 310 315 320Glu Ser Gly Asp Tyr Phe Ile Tyr Ser
Gln Val Thr Phe Arg Gly Met 325 330 335Thr Ser Glu Cys Ser Glu Ile
Arg Gln Ala Gly Arg Pro Asn Lys Pro 340 345 350Asp Ser Ile Thr Val
Val Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu 355 360 365Pro Thr Gln
Leu Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly Ser 370 375 380Asn
Trp Phe Gln Pro Ile Tyr Leu Gly Pro Met Phe Ser Leu Gln Glu385 390
395 400Gly Asp Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp
Tyr 405 410 415Thr Lys Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu
420 42511579DNAHomo 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 15Glu Phe Ala Pro Ser His Gln Gln Val Tyr Ala Pro Leu
Arg Ala Asp 20 25 30Gly Asp Lys Pro Arg Ala His Leu Thr Val Val Arg
Gln Thr Pro Thr 35 40 45Gln His Phe Lys Asn Gln Phe Pro Ala Leu His
Trp Glu His Glu Leu 50 55 60Gly Leu Ala Phe Thr Lys Asn Arg Met Asn
Tyr Thr Asn Lys Phe Leu65 70 75 80Leu Ile Pro Glu Ser Gly Asp Tyr
Phe Ile Tyr Ser Gln Val Thr Phe 85 90 95Arg Gly Met Thr Ser Glu Cys
Ser Glu Ile Arg Gln Ala Gly Arg Pro 100 105 110Asn Lys Pro Asp Ser
Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser 115 120 125Tyr Pro Glu
Pro Thr Gln Leu Leu Met Gly Thr Lys Ser Val Cys Glu 130 135 140Val
Gly Ser Asn Trp Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser145 150
155 160Leu Gln Glu Gly Asp Lys Leu Met Val Asn Val Ser Asp Ile Ser
Leu
165 170 175Val Asp Tyr Thr Lys Glu Asp Lys Thr Phe Phe Gly Ala Phe
Leu Leu 180 185 19013684DNAHomo 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 15Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 20 25 30Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 35 40 45His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120
125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro 165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr 180 185 190Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220Ser Pro Gly
Lys225151269DNAArtificial 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 15Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55
60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65
70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn 85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro 100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val 130 135 140Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200
205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220Ser Pro Gly Lys Glu Phe Arg Ala Gln Gly Glu Ala Cys Val
Gln Phe225 230 235 240Gln Ala Leu Lys Gly Gln Glu Phe Ala Pro Ser
His Gln Gln Val Tyr 245 250 255Ala Pro Leu Arg Ala Asp Gly Asp Lys
Pro Arg Ala His Leu Thr Val 260 265 270Val Arg Gln Thr Pro Thr Gln
His Phe Lys Asn Gln Phe Pro Ala Leu 275 280 285His Trp Glu His Glu
Leu Gly Leu Ala Phe Thr Lys Asn Arg Met Asn 290 295 300Tyr Thr Asn
Lys Phe Leu Leu Ile Pro Glu Ser Gly Asp Tyr Phe Ile305 310 315
320Tyr Ser Gln Val Thr Phe Arg Gly Met Thr Ser Glu Cys Ser Glu Ile
325 330 335Arg Gln Ala Gly Arg Pro Asn Lys Pro Asp Ser Ile Thr Val
Val Ile 340 345 350Thr Lys Val Thr Asp Ser Tyr Pro Glu Pro Thr Gln
Leu Leu Met Gly 355 360 365Thr Lys Ser Val Cys Glu Val Gly Ser Asn
Trp Phe Gln Pro Ile Tyr 370 375 380Leu Gly Ala Met Phe Ser Leu Gln
Glu Gly Asp Lys Leu Met Val Asn385 390 395 400Val Ser Asp Ile Ser
Leu Val Asp Tyr Thr Lys Glu Asp Lys Thr Phe 405 410 415Phe Gly Ala
Phe Leu Leu 420
* * * * *
References