U.S. patent application number 16/993569 was filed with the patent office on 2021-02-11 for single-chain trail-receptor agonist proteins.
The applicant listed for this patent is AbbVie Inc., Apogenix AG. Invention is credited to Fritz G. Buchanan, Christian Gieffers, Oliver Hill, Susan E. Lappe, Darren C. Phillips, Meinholf Thiemann.
Application Number | 20210040178 16/993569 |
Document ID | / |
Family ID | 1000005170044 |
Filed Date | 2021-02-11 |
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United States Patent
Application |
20210040178 |
Kind Code |
A1 |
Buchanan; Fritz G. ; et
al. |
February 11, 2021 |
Single-Chain Trail-Receptor Agonist Proteins
Abstract
Provided herein are specific TRAIL receptor agonist proteins,
nucleic acids encoding the same, and methods of treating a subject
having a TRAIL-associated disease or disorder. The TRAIL receptor
agonist proteins provided herein comprise three soluble TRAIL
domains and an Fc fragment. The TRAIL receptor agonist proteins are
substantially non-aggregating and suitable for therapeutic,
diagnostic and/or research applications.
Inventors: |
Buchanan; Fritz G.; (Salem,
WI) ; Gieffers; Christian; (Dossenheim, DE) ;
Hill; Oliver; (Neckarsteinach, DE) ; Lappe; Susan
E.; (Riverwoods, IL) ; Phillips; Darren C.;
(Glenview, IL) ; Thiemann; Meinholf; (Schriesheim,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Inc.
Apogenix AG |
North Chicago
Heidelberg |
IL |
US
DE |
|
|
Family ID: |
1000005170044 |
Appl. No.: |
16/993569 |
Filed: |
August 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15887509 |
Feb 2, 2018 |
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16993569 |
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14694358 |
Apr 23, 2015 |
9908927 |
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15887509 |
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61983152 |
Apr 23, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/30 20130101;
C07K 14/70575 20130101; C07K 2319/00 20130101; A61K 38/00
20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705 |
Claims
1. A TRAIL receptor agonist protein comprising a polypeptide having
the amino acid sequence set forth in SEQ ID NO: 19.
2. A TRAIL receptor agonist protein comprising two polypeptides
having the amino acid sequence set forth in SEQ ID NO: 19.
3. The TRAIL receptor agonist protein of claim 2, wherein the two
polypeptides are covalently linked through three interchain
disulfide bonds formed between cysteine residues 513, 519, and 522
of each polypeptide.
4. The TRAIL receptor agonist protein of claim 2, wherein one or
more of the asparagine residues at positions 168 and 337 of the
polypeptide(s) are N-glycosylated.
5. The TRAIL receptor agonist protein of claim 2, wherein the
asparagine residues at positions 168 and 337 of the polypeptide(s)
are both N-glycosylated.
6. The TRAIL receptor agonist protein of claim 5, wherein the
polypeptide(s) are further post-translationally modified.
7. The TRAIL receptor agonist protein of claim 6, wherein the
post-translational modification comprises modification of the
N-terminal glutamine to pyroglutamate.
8. A pharmaceutical composition comprising the TRAIL receptor
agonist protein of any one of claim 6 and one or more
pharmaceutically acceptable carriers, diluents, excipients, and/or
adjuvants.
9. A nucleic acid molecule encoding the TRAIL receptor agonist
protein of claim 1.
10. An expression vector comprising the nucleic acid molecule of
claim 9.
11. A cell comprising the nucleic acid molecule of claim 9.
12. The cell of claim 11, which is a eukaryotic cell.
13. The cell of claim 11, wherein the cell is a mammalian cell.
14. The cell of claim 11, wherein the cell is a Chinese Hamster
Ovary (CHO) cell.
15. A method of treating a subject having a TRAIL-associated
disease or disorder, the method comprising administering to the
subject an effective amount of the TRAIL receptor agonist protein
of any one of claim 6.
16. The method of claim 15, wherein the disease or disorder is
selected from the group consisting of: tumors, infectious diseases,
inflammatory diseases, metabolic diseases, autoimmune disorders,
degenerative diseases, apoptosis-associated diseases, and
transplant rejections.
17. The method of claim 16, wherein the tumors are solid
tumors.
18. The method of claim 16, wherein the tumors are lymphatic
tumors.
19. The method of claim 16, wherein the autoimmune disorders are
rheumatoid diseases, arthritic diseases, or rheumatoid and
arthritic diseases.
20. The method of claim 16, wherein the disease or disorder is
rheumatoid arthritis.
21. The method of claim 16, wherein the degenerative disease is a
neurodegenerative disease.
22. The method of claim 16, wherein the neurodegenerative disease
is multiple sclerosis.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/887,509, filed Feb. 2, 2018 which is a continuation of U.S.
application Ser. No. 14/694,358, filed Apr. 23, 2015, now U.S. Pat.
No. 9,908,927, which claims the benefit of priority to U.S.
Provisional Patent Application No. 61/983,152, filed Apr. 23, 2014.
All of which are incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention provides specific TRAIL receptor
agonist proteins comprising three soluble TRAIL domains and an Fc
fragment, nucleic acid molecules encoding the TRAIL receptor
agonist proteins, and uses thereof. The TRAIL receptor agonist
proteins are substantially non-aggregating and suitable for
therapeutic, diagnostic and/or research applications.
BACKGROUND OF THE INVENTION
[0003] It is known that trimerization of TNF superfamily (TNFSF)
cytokines is required for efficient receptor binding and
activation. Trimeric complexes of TNF superfamily cytokines,
however, are difficult to prepare from recombinant monomeric
units.
[0004] WO 01/49866 and WO 02/09055 disclose recombinant fusion
proteins comprising a TNF cytokine and a multimerization component,
particularly a protein from the C1q protein family or a collectin.
A disadvantage of these fusion proteins is, however, that the
trimerization domain usually has a large molecular weight and/or
that the trimerization is rather inefficient.
[0005] Schneider et al. (J Exp Med 187 (1989), 1205-1213) describe
that trimers of TNF cytokines are stabilized by N-terminally
positioned stabilization motifs. In CD95L, the stabilization of the
receptor binding domain trimer is presumably caused by N-terminal
amino acid domains which are located near the cytoplasmic
membrane.
[0006] Shiraishi et al. (Biochem Biophys Res Commun 322 (2004),
197-202) describe that the receptor binding domain of CD95L may be
stabilized by N-terminally positioned artificial .alpha.-helical
coiled-coil (leucine zipper) motifs. It was found, however, that
the orientation of the polypeptide chains to each other, e.g.
parallel or antiparallel orientation, can hardly be predicted.
Further, the optimal number of heptad-repeats in the coiled-coil
zipper motif are difficult to determine. In addition, coiled-coil
structures have the tendency to form macromolecular aggregates
after alteration of pH and/or ionic strength.
[0007] WO 01/25277 relates to single-chain oligomeric polypeptides
which bind to an extracellular ligand binding domain of a cellular
receptor, wherein the polypeptide comprises at least three receptor
binding sites of which at least one is capable of binding to a
ligand binding domain of the cellular receptor and at least one is
incapable of effectively binding to a ligand binding domain of the
cellular receptor, whereby the single-chain oligomeric polypeptides
are capable of binding to the receptor, but incapable of activating
the receptor. For example, the monomers are derived from cytokine
ligands of the TNF family, particularly from TNF-.alpha..
[0008] WO 2005/103077 discloses single-chain fusion polypeptides
comprising at least three monomers of a TNF family ligand member
and at least two peptide linkers that link the monomers of the TNF
ligand family members to one another. Recent experiments, however,
have shown that these single-chain fusion polypeptides show
undesired aggregation.
[0009] WO 2010/010051 discloses single-chain fusion polypeptides
comprising three soluble TNF family cytokine domains and at least
two peptide linkers. The described fusion polypeptides are
substantially non-aggregating.
[0010] Moreover, previous work, including that of Papadopoulos et
al. (Cancer Chemother Pharmacol, 2015, DOI
10.1007/s00280-015-2712-0), has demonstrated that TRAIL receptor
superclustering can result in toxicity.
[0011] Accordingly, there is a need in the art for novel TRAIL
receptor agonists that exhibit high biological activity, high
stability, low toxicity, and allow for efficient recombinant
manufacturing.
SUMMARY OF THE INVENTION
[0012] The present invention provides specific TRAIL receptor
agonist proteins that exhibit low proteolytic degradation, long
half-life, and low TRAIL receptor superclustering in vivo (along
with concomitant toxicity).
[0013] The TRAIL receptor agonist proteins of the instant invention
generally comprise: (i) a first soluble TRAIL cytokine domain; (ii)
a first peptide linker; (iii) a second soluble TRAIL domain; (iv) a
second peptide linker; (v) a third soluble TRAIL domain; and (vi)
an antibody Fc fragment.
[0014] In one aspect, the present invention provides a single-chain
fusion polypeptide comprising: (i) a first soluble TRAIL domain,
(ii) a first peptide linker, (iii) a second soluble TRAIL domain,
(iv) a second peptide linker, (v) a third soluble TRAIL domain, and
(vi) an antibody Fc fragment. In one embodiment, the antibody Fc
fragment (vi) is located N terminal to the first TRAIL domain (i)
and/or C-terminal to the third TRAIL domain (v). In another
embodiment the antibody Fc fragment is located C-terminally to the
third TRAIL domain (v). In one embodiment, the polypeptide is
substantially non aggregating. In another embodiment, the second
and/or third soluble TRAIL domain is an N-terminally shortened
domain which optionally comprises amino acid sequence
mutations.
[0015] In one embodiment, at least one of the soluble TRAIL
domains, particularly at least one of the soluble TRAIL domains
(iii) and (v), is a soluble TRAIL domain with an N-terminal
sequence which starts between amino acid GIn120 and Val122 of human
TRAIL and wherein Arg121 may be replaced by a neutral amino acid,
e.g., Ser or Gly. In another embodiment, at least one of the
soluble TRAIL domains, particularly at least one of the soluble
TRAIL domains (iii) and (v), is a soluble TRAIL domain with an
N-terminal sequence selected from (a) Arg121-Val122-Ala123 and (b)
(Gly/Ser)121-Val122-Ala123. In one embodiment, the soluble TRAIL
domain ends with amino acid Gly281 of human TRAIL and/or optionally
comprises a mutation at positions R130, G160, H168, R170, H177,
Y189, R191, Q193, E195, N199, K201, Y213, T214, S215, H264, 1266,
D267 or D269 or at two or more of said positions. In one
embodiment, the soluble TRAIL domain (i) consists of amino acids
GIn120-Gly281 of human TRAIL according to SEQ ID NO: 1 and the
soluble TRAIL domains (iii) and (v) consist of amino acids
Arg121-Gly281 of human TRAIL according to SEQ ID NO: 1.
[0016] In one embodiment, the first and second peptide linkers (ii)
and (iv) independently have a length of 3-8 amino acids,
particularly a length of 3, 4, 5, 6, 7, or 8 amino acids, and
preferably are glycine/serine linkers, optionally comprising an
asparagine residue which may be glycosylated. In one embodiment,
the first and the second peptide linkers (ii) and (iv) consist of
the amino acid sequence according to SEQ ID NO: 2. In another
embodiment, the polypeptide additionally comprises an N-terminal
signal peptide domain, e.g., of SEQ ID NO: 12, which may comprise a
protease cleavage site, and/or which additionally comprises a
C-terminal element which may comprise and/or connect to a
recognition/purification domain, e.g., a Strep-tag according to SEQ
ID NO: 13.
[0017] In one embodiment, the antibody Fc fragment (vi) is fused to
the soluble TRAIL domain (i) and/or (v) via a hinge-linker,
preferably of SEQ ID NO: 11. In another embodiment, the antibody Fc
fragment (vi) consists of the amino acid sequence as shown in SEQ
ID NO: 10 or 17. In one embodiment, the polypeptide comprises the
amino acid sequence of SEQ ID NO: 14, 15 or 18.
[0018] In another aspect, the present invention provides a TRAIL
receptor agonist protein comprising a polypeptide having the amino
acid sequence set forth in SEQ ID NO: 19, 20 or 21.
[0019] In another aspect, the present invention provides a TRAIL
receptor agonist protein comprising a polypeptide having the amino
acid sequence set forth in SEQ ID NO: 26, 27, 28, 29, or 30.
[0020] In another aspect, the present invention provides a TRAIL
receptor agonist protein comprising two polypeptides having the
amino acid sequence set forth in SEQ ID NO:
[0021] 19. In one embodiment, the two polypeptides are covalently
linked through three interchain disulfide bonds formed between
cysteine residues 513, 519, and 522 of each polypeptide.
[0022] In one embodiment, one or more of the asparagine residues at
positions 168 and 337 of the polypeptide(s) are N-glycosylated. In
another embodiment, the asparagine residues at positions 168 and
337 of the polypeptide(s) are both N-glycosylated.
[0023] In another embodiment, the polypeptide(s) are further
post-translationally modified. In another embodiment, the
post-translational modification comprises the N-terminal glutamine
modified to pyroglutamate.
[0024] In another aspect, the present invention provides a
pharmaceutical composition comprising a TRAIL receptor agonist
protein disclosed herein and one or more pharmaceutically
acceptable carriers, diluents, excipients, and/or adjuvants.
[0025] In another aspect, the present invention provides a nucleic
acid molecule encoding the TRAIL receptor agonist protein. In
another embodiment, the present invention provides an expression
vector comprising the nucleic acid molecule. In another embodiment,
the present invention provides a cell comprising the nucleic acid
molecule. In a further embodiment, the cell is a eukaryotic cell.
In another embodiment, the cell is a mammalian cell. In another
embodiment, the cell is a Chinese Hamster Ovary (CHO) cell. In
other embodiments, the cell is selected from the group consisting
of CHO-DBX11, CHO-DG44, CHO-S, and CHO-K1 cells. In other
embodiments, the cell is selected from the group consisting of
Vero, BHK, HeLa, COS, MDCK, HEK-293, NIH-3T3, W138, BT483, Hs578T,
HTB2, BT20, T47D, NSO, CRL7030, HsS78Bst, PER.C6, SP2/0-Agl4, and
hybridoma cells.
[0026] In another aspect, the present invention provides a method
of treating a subject having a TRAIL-associated disease or
disorder, the method comprising administering to the subject an
effective amount of the TRAIL receptor agonist protein. In one
embodiment, the TRAIL receptor agonist protein is administered
alone. In another embodiment, the TRAIL receptor agonist protein is
administered before, concurrently, or after the administration of a
second agent. In another embodiment, the disease or disorder is
selected from the group consisting of: tumors, infectious diseases,
inflammatory diseases, metabolic diseases, autoimmune disorders,
degenerative diseases, apoptosis-associated diseases, and
transplant rejections. In one embodiment, the tumors are solid
tumors. In one embodiment, the tumors arise from the group of
cancers consisting of sarcoma, esophageal cancer, and gastric
cancer. In another embodiment, the tumors arise from Ewing's
sarcoma or fibrosarcoma, In another embodiment, the tumors arise
from the group of cancers consisting of Non-Small Cell Lung
Carcinoma (NSCLC), pancreatic cancer, colorectal cancer, breast
cancer, ovarian cancer, head and neck cancers, and Small Cell Lung
Cancer (SCLC). In another embodiment, the tumors are lymphatic
tumors. In one embodiment, the tumors are hematologic tumors. In
another embodiment, the tumors arise from non-Hodgkin's lymphoma,
leukemia, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), B cell lymphoma, Burkitt's lymphoma, chronic
myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), or
hairy cell leukemia. In another embodiment, the autoimmune
disorders are rheumatoid diseases, arthritic diseases, or
rheumatoid and arthritic diseases. In a further embodiment, the
disease or disorder is rheumatoid arthritis. In another embodiment,
the degenerative disease is a neurodegenerative disease. In a
further embodiment, the neurodegenerative disease is multiple
sclerosis.
[0027] In one embodiment, the second agent is a chemotherapeutic,
radiotherapeutic, or biological agent. In one embodiment, the
second agent is selected from the group consisting of Duvelisib,
Ibrutinib, Navitoclax, and Venetoclax, In another embodiment, the
second agent is an apoptotic agent. In one embodiment, the
apoptotic second agent is selected from the group consisting of
Bortezomib, Azacitidine, Dasatinib, and Gefitinib. In a particular
embodiment, the pharmaceutical compositions disclosed herein are
administered to a patient by intravenous or subcutaneous
administration. In other embodiments, the disclosed pharmaceutical
compositions are administered to a patient byoral, parenteral,
intramuscular, intrarticular, intrabronchial, intraabdominal,
intracapsular, intracartilaginous, intracavitary, intracelial,
intracerebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,
buccal, sublingual, intranasal, or transdermal administration.
[0028] In one embodiment, the TRAIL receptor agonist protein is
administered as a single bolus. In another embodiment, TRAIL
receptor agonist protein may be administered over several divided
doses. The TRAIL receptor agonist protein can be administered at
about 0.1-100 mg/kg. In one embodiment, the TRAIL receptor agonist
protein can be administered at a dosage selected from the group
consisting of: about 0.1-0.5, 0.1-1, 0.1-10, 0.1-20, 0.1-50,
0.1-75, 1-10, 1-15, 1-7.5, 1.25-15, 1.25-7.5, 2.5-7.5, 2.5-15,
5-15, 5-7.5, 1-20, 1-50, 7-75, 1-100, 5-10, 5-15, 5-20, 5-25, 5-50,
5-75, 10-20, 10-50, 10-75, and 10-100 mg/kg. In other embodiments,
the TRAIL receptor agonist protein is present in pharmaceutical
compositions at about 0.1-100 mg/ml. In one embodiment, the TRAIL
receptor agonist protein is present in pharmaceutical compositions
at an amount selected from the group consisting of: about 0.1-0.5,
0.1-1, 0.1-10, 0.1-20, 0.1-50, 0.1-75, 1-10, 1-20, 1-50, 1-75,
1-100, 5-10, 5-15, 5-20, 5-25, 5-50, 5-75, 10-20, 10-50, 10-75, or
10-100 mg/ml. In other embodiments, a therapeutically effective
amount of TRAIL receptor agonist protein is administered to a
subject. In another embodiment, a prophylactically effective amount
of TRAIL receptor agonist protein is administered to a subject.
DESCRIPTION OF THE FIGURES
[0029] FIG. 1 Domain structure of a single-chain fusion polypeptide
comprising three TRAIL domains. I., II., III. Soluble TRAIL
domains.
[0030] FIG. 2 Schematic picture representing the general structure
of TRAIL. [0031] .box-solid. .box-solid. .box-solid. Cell membrane,
N-terminus located within the cell, [0032] 1. anti-parallel
.beta.-fold of receptor-binding domain (RBD), [0033] 2. interface
of RBD and cell membrane, [0034] 3. protease cleavage site.
[0035] FIG. 3 Schematic picture representing the structure of the
native TRAIL trimer. Cylindric structures represent RBDs. N-termini
connect RBDs with the cell membrane.
[0036] FIG. 4 Schematic picture representing the structure of three
soluble domains comprising the receptor-binding domain of a TRAIL.
I., II., III. soluble TRAIL domains.
[0037] FIG. 5 Trimerization of the soluble domains comprising the
RBD of TRAIL, characterized in that the N- and C-termini of the
three soluble domains form a surface.
[0038] FIG. 6 Schematic picture representing the structure of the
single-chain TRAIL comprising all or a part of the stalk-region
illustrating the requirement of longer linkers to compensate for
the distance to the N-terminus of the next soluble domain.
[0039] FIG. 7 scFv-TRAIL fusion protein known from the art.
[0040] FIG. 8 Fc-TRAIL fusion protein known from the art.
[0041] FIG. 9A Single-chain fusion polypeptide comprising an
additional Fab antibody fragment.
[0042] FIG. 9B Single-chain fusion polypeptide comprising an
additional scFv antibody fragment.
[0043] FIG. 10 Dimerization of two N-terminally fused scFc fusion
polypeptides via disulfide bridges.
[0044] FIG. 11 Dimerization of two C-terminally fused scFc fusion
polypeptides via disulfide bridges.
[0045] FIG. 12 Dimerization of single-chain fusion polypeptides via
a linker.
[0046] FIG. 13 Single-chain fusion polypeptide comprising an
additional Fab antibody fragment further fused to a second fusion
polypeptide or to a scFv fusion polypeptide.
[0047] FIG. 14 Dimerization of two scFab fusion polypeptides via
disulfide bridges.
[0048] FIG. 15 N-terminally fused scFc fusion polypeptides further
comprising a Fv and/or Fab antibody fragment.
[0049] FIG. 16 C-terminally fused scFc fusion polypeptides further
comprising a Fv and/or Fab antibody fragment.
[0050] FIG. 17A The exemplary TRAIL receptor agonist protein as
shown with the N-terminal signal peptide domain is set forth in SEQ
ID NO: 14. The mature protein (which does not include the
N-terminal signal peptide domain) is set forth in SEQ ID NO:
19.
[0051] FIG. 17B Schematic picture representing the overall
structure and annotated sequence of an exemplary TRAIL receptor
agonist protein.
[0052] FIG. 18 Assay setup of the ELISA for the quantitation of the
TRAIL-receptor agonists containing an FC-domain.
[0053] FIG. 19 A TRAIL receptor agonist protein comprising two
polypeptides having the amino acid sequence set forth in SEQ ID NO:
19 induces cell death in human tumor cell lines in vitro. SKM-1,
Colo205 or Jurkat cells were treated with increasing concentrations
the TRAIL receptor agonist protein for 24 hours and cell viability
assessed.
[0054] FIGS. 20(A-C) A TRAIL receptor agonist protein comprising
two polypeptides having the amino acid sequence set forth in SEQ ID
NO: 19 synergizes with anti-tumorigenic agents in vitro. SU-DHL-4
cells were incubated with increasing concentrations of the TRAIL
receptor agonist protein in the presence or absence of the
indicated concentrations of venetoclax (FIG. 20A) or navitoclax
(FIG. 20B) for 24 hours. Alternatively, (FIG. 20C) NCI-H596 cells
were treated with increasing concentrations of the TRAIL receptor
agonist protein in the presence or absence of the indicated
concentrations of docetaxel (DTX) for 72 hours. Cell viability was
assessed and synergy determined by Bliss sum.
[0055] FIG. 21 Effect of TRAIL receptor agonist protein comprising
two polypeptides having the amino acid sequence set forth in SEQ ID
NO: 19 on tumor growth in the Colo205 colorectal carcinoma
xenograft model.
[0056] FIG. 22 Effect of TRAIL receptor agonist protein comprising
two polypeptides having the amino acid sequence set forth in SEQ ID
NO: 19 on tumor growth in the SKM-1 acute myeloid leukemia
xenograft model.
[0057] FIG. 23 Effect of TRAIL receptor agonist protein comprising
two polypeptides having the amino acid sequence set forth in SEQ ID
NO: 19 on tumor growth in the H460LM non-small cell lung xenograft
model.
[0058] FIGS. 24(A-G) Effect of TRAIL receptor agonist protein
comprising two polypeptides having the amino acid sequence set
forth in SEQ ID NO: 19 on tumor growth in PDX models. Diamonds,
TRAIL receptor agonist protein-treated; Squares, untreated. Tumor
volumes are shown for (A) CTG-0069, (B) CTG-0167, (C) CTG-0293, (D)
CTG-0785, (E) CTG-0714, (F) CTG-0136, and (G) CTG-0485.
DETAILED DESCRIPTION OF THE INVENTION
[0059] According to the present invention, it was found that fusing
a single-chain TRAIL receptor-binding domain to an Fc domain
results in a hexavalent TRAIL receptor agonist providing high
biological activity combined with good stability. Accordingly, a
single-chain fusion polypeptide comprising at least three soluble
TRAIL domains connected by two peptide linkers and N-terminally
and/or C-terminally an antibody Fc fragment, is provided.
[0060] Preferably, the single-chain fusion polypeptide is
non-aggregating. The term "non-aggregating" refers to a monomer
content of the preparation of 50%, preferably 70% and more
preferably 90%. The ratio of monomer content to aggregate content
may be determined by examining the amount of aggregate formation
using size-exclusion chromatography (SEC). The stability concerning
aggregation may be determined by SEC after defined time periods,
e.g. from a few to several days, to weeks and months under
different storage conditions, e.g. at 4.degree. C. or 25.degree. C.
For the fusion protein, in order to be classified as substantially
non-aggregating, it is preferred that the monomer content is as
defined above after a time period of several days, e.g. 10 days,
more preferably after several weeks, e.g. 2, 3 or 4 weeks, and most
preferably after several months, e.g. 2 or 3 months of storage at
4.degree. C., or 25.degree. C.
[0061] The single-chain fusion polypeptide may comprise additional
domains which may be located at the N- and/or C-termini thereof.
Examples for additional fusion domains are e.g. an N-terminal
signal peptide domain which may comprise a protease cleave site or
a C-terminal element which may comprise and/or connect to a
recognition/purification domain. According to a preferred
embodiment, the fusion polypeptide comprises a Strep-tag at its
C-terminus that is fused via a linker. An exemplary Strep-tag
including a short serine linker is shown in SEQ ID NO: 13.
[0062] The TRAIL receptor agonist protein of the present invention
comprises three soluble domains derived from TRAIL. Preferably,
those soluble domains are derived from a mammalian, particularly
human TRAIL including allelic variants and/or derivatives thereof.
The soluble domains comprise the extracellular portion of TRAIL
including the receptor binding domain without membrane located
domains. Like other proteins of the TNF superfamily, TRAIL is
anchored to the membrane via an N-terminal portion of 15-30 amino
acids, the so-called stalk-region. The stalk region contributes to
trimerization and provides a certain distance to the cell membrane.
However, the stalk region is not part of the receptor binding
domain (RBD).
[0063] Importantly, the RBD is characterized by a particular
localization of its N- and C-terminal amino acids. Said amino acids
are immediately adjacent and are located centrally to the axis of
the trimer. The first N-terminal amino acids of the RBD form an
anti-parallel beta-strand with the C-terminal amino acids of the
RBD (FIGS. 2 and 3).
[0064] Thus, the anti-parallel beta-strand of the RBD forms an
interface with the cell membrane, which is connected to and
anchored within the cell membrane via the amino acids of the stalk
region. It is highly preferred that the soluble TRAIL domains of
the TRAIL receptor agonist protein comprise a receptor binding
domain of the TRAIL lacking any amino acids from the stalk region
(FIGS. 4 and 5). Otherwise, a long linker connecting the C-terminus
of one of the soluble domains with the N-terminus of the next
soluble domain would be required to compensate for the N-terminal
stalk-region of the next soluble domain (FIG. 6), which might
result in instability and/or formation of aggregates.
[0065] A further advantage of such soluble domains is that the N-
and C-terminal amino acids of the RBD are not accessible for any
anti-drug antibodies. Preferably, the single-chain fusion
polypeptide is capable of forming an ordered trimeric structure
comprising at least one functional binding site for the respective
TRAIL receptor.
[0066] The TRAIL receptor agonist protein comprises three
functional TRAIL receptor binding sites, i.e. amino acid sequences
capable of forming a complex with a TRAIL receptor. Thus, the
soluble domains are capable of binding to the corresponding TRAIL
receptor. In one embodiment, at least one of the soluble domains is
capable of receptor activation, whereby apoptotic and/or
proliferative activity may be affected. In a further embodiment,
one or more of the soluble domains are selected as not being
capable of receptor activation.
[0067] The soluble TRAIL domain may be derived from human TRAIL as
shown in SEQ ID NO: 1. Preferably, the soluble TRAIL domains are
derived from human TRAIL, particularly starting from amino acids
120-122 and comprise particularly amino acids 120-281, 121-281 or
122-281 of SEQ ID NO: 1. Optionally, amino acid Arg121 of SEQ ID
NO: 1 may be replaced by a non-charged amino acid, e.g. Ser or
Gly.
TABLE-US-00001 TABLE 1 Sequence of Human TRAIL Protein SEQ ID NO
Sequence 1 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNE
LKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKW
QLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVA
AHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFL
SNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQ
MVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGG
IFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG
[0068] As indicated above, the soluble TRAIL domains may comprise
the wild-type sequences as indicated in SEQ ID NO: 1. It should be
noted, however, that it is possible to introduce mutations in one
or more of these soluble domains, e.g. mutations which alter (e.g.
increase or decrease) the binding properties of the soluble
domains. In one embodiment, soluble domains may be selected which
cannot bind to the corresponding cytokine receptor.
[0069] In a further preferred embodiment of the invention, the
soluble TRAIL domain (i) comprises a mutant of TRAIL or a receptor
binding domain thereof which binds and/or activates TRAIL-receptor
1 (TRAILR1) and/or TRAIL-receptor 2 (TRAILR2). The binding and/or
activity of the mutant may be, e.g., determined by the assays as
described in van der Sloot et al. (PNAS, 2006, 103:8634-8639),
Kelley et al. (J. Biol. Chem., 2005, 280:2205-2215), or MacFarlane
et al. (Cancer Res., 2005, 65: 11265-11270).
[0070] The mutant may be generated by any technique and is known by
the skilled person, e.g., the techniques described in van der Sloot
et al. (PNAS, 2006, 103:8634-8639), Kelley et al. (J. Biol. Chem.,
2005, 280:2205-2215), or MacFarlane et al. (Cancer Res., 2005, 65:
11265-11270) and may comprise any type of structural mutations,
e.g., substitution, deletion, duplication and/or insertion of an
amino acid. A preferred embodiment is the generation of
substitutions. The substitution may affect at least one amino acid
of TRAIL or a receptor binding domain thereof as described herein.
In a preferred embodiment, the substitution may affect at least one
of the amino acids of TRAIL, e.g., human TRAIL (e.g., SEQ ID NO:
1). Preferred substitutions in this regard affect at least one of
the following amino acids of human TRAIL of SEQ ID NO: 1: R130,
G160, Y189, R191, Q193, E195, N199, K201, Y213, T214, S215, H264,
1266, D267, D269. Preferred amino acid substitutions of human TRAIL
of SEQ ID NO:1 are at least one of the following substitutions:
R130E, G160M, Y189A, Y189Q, R191K, Q193S, Q193R, E195R, N199V,
N199R, K201R, Y213W, T214R, S215D, H264R, I266L, D267Q, D269H,
D269R, or D269K.
[0071] The amino acid substitution(s) may affect the binding and/or
activity of TRAIL, e.g., human TRAIL, to or on either the TRAILR1
or the TRAILR2. Alternatively, the amino acid substitution(s) may
affect the binding and/or activity of TRAIL, e.g., human TRAIL, to
or on both, the TRAILR1 and the TRAILR2. The binding and/or
activity of the TRAILR1 and/or TRAILR2 may be affected positively,
i.e., stronger, more selective or more specific binding and/or more
activation of the receptor. Alternatively, the binding and/or
activity of the TRAILR1 and/or TRAILR2 may be affected negatively,
i.e., weaker, less selective or less specific binding and/or less
or no activation of the receptor.
[0072] Examples of mutants of TRAIL with amino acid substitution(s)
of the invention that affect binding and/or activation of both
TRAILR1 and TRAILR2 may be found, e.g., in Table 1 of MacFarlane et
al. (cf. above) and may comprise a human TRAIL mutant with the
following two amino acid substitutions of SEQ ID NO: 1 Y213W and
S215D or with the following single amino acid substitution:
Y189A.
[0073] Examples of mutants of TRAIL with amino acid substitution(s)
of the invention that affect binding and/or activation of TRAILR1
may be found, e.g., in Table 1 of MacFarlane et al. (cf. above) and
may comprise a human TRAIL mutant with the following four amino
acid substitutions of SEQ ID NO: 1 N 199V, K201R, Y213W and S215D
or with the following five amino acid substitutions: Q193S, N199V,
K201R, Y213W and S215D, or may be found in Table 2 of Kelley et al.
(cf. above) and may comprise a human TRAIL mutant with the
following six amino acid substitutions: Y213W, S215D, Y189A, Q193S,
N199V, and K201R, or with Y213W, S215D, Y189A, Q193S, N199R, and
K201R.
[0074] Examples of mutants of TRAIL with amino acid substitution(s)
of the invention that affect binding and/or activation of TRAILR2
may be found, e.g., in Table 1 of MacFarlane et al. (cf. above) or
in Table 2 of Kelley et al. (cf. above) and may comprise a human
TRAIL mutant with the following six amino acid substitutions of SEQ
ID NO: 1: Y189Q, R191 K, Q193R, H264R, I266L, and D267Q, or may be
found in Table 2 of van der Sloot et al. (cf. above) and may
comprise a human TRAIL mutant with the following single amino acid
substitution: D269H, or with the following two amino acid
substitutions: D269H and E195R or D269H and T214R.
[0075] Thus one preferred embodiment is a TRAIL receptor agonist
protein as described herein wherein at least one of the soluble
domains comprises a mutant of TRAIL or of a receptor binding domain
thereof which binds and/or activates TRAILR1 and/or TRAILR2.
[0076] Further examples of mutants of TRAIL, which show reduced
TRAIL induced receptor aggregation are H168 (S, T, Q), R170 (E, S,
T, Q) and H177 (S, T).
[0077] One preferred embodiment of a TRAIL receptor agonist protein
comprising a mutant of TRAIL or of a receptor binding domain as
described herein is a TRAIL receptor agonist protein wherein
component (i) comprises at least one amino acid substitution,
particularly as indicated below.
[0078] Such an amino acid substitution affects at least one of the
following amino acid positions of human TRAIL (SEQ ID NO: 1): R130,
G160, H168, R170, H177, Y189, R191, Q193, E195, N199, K201, Y213,
T214, S215, H264, 1266, D267, D269.
[0079] Such an amino acid substitution is at least one of the
following: R130E, G160M, H168 (S, T, Q), R170 (E, S, T, Q), H177
(SJ)1 Y189A, Y189Q, R191K, Q193S, Q193R, E195R, N199V, N199R,
K201R, Y213W, T214R, S215D, H264R, I266L, D267Q, D269H, D269R, or
D269K.
[0080] A preferred TRAIL-R2 selective domain comprises amino acid
substitutions Y189Q, R191K, Q193R, H264R, I266L and D267Q.
[0081] A preferred TRAIL-R1 selective domain comprises amino acid
substitutions Y189A, Q193S, N199V, K201R, Y213W and S215D.
[0082] The single-chain fusion molecule of the present invention
comprises three soluble TRAIL domains, namely components (i), (iii)
and (v). The stability of a single-chain TRAIL fusion polypeptide
against aggregation is enhanced, if the second and/or third soluble
TRAIL domain is an N-terminally shortened domain which optionally
comprises amino acid sequence mutations. Thus, preferably, both the
second and the third soluble TRAIL domain are N-terminally
shortened domains which optionally comprise amino acid sequence
mutations in the N-terminal regions, preferably within the first
five amino acids of the N-terminus of the soluble TRAIL domain.
These mutations may comprise replacement of charged, e.g. acidic or
basic amino acids, by neutral amino acids, particularly serine or
glycine.
[0083] In contrast thereto, the selection of the first soluble
TRAIL domain is not as critical. Here, a soluble domain having a
full-length N-terminal sequence may be used. It should be noted,
however, that also the first soluble TRAIL domain may have an
N-terminally shortened and optionally mutated sequence.
[0084] In a further preferred embodiment of the present invention,
the soluble TRAIL domains (i), (iii) and (v) are soluble human
TRAIL domains. The first soluble TRAIL domain (i) may be selected
from native, shortened and/or mutated sequences. Thus, the first
soluble TRAIL domain (i) has an N-terminal sequence which may start
between amino acid Glu116 and Val122 of human TRAIL, and wherein
Arg121 may be replaced by a neutral amino acid, e.g. by Ser or Gly.
The second and third soluble TRAIL domains (iii) and (v) have a
shortened N-terminal sequence which preferably starts between amino
acid GIn120 and Val122 of human TRAIL and wherein Arg121 may be
replaced by another amino acid, e.g. Ser or Gly.
[0085] Preferably, the N-terminal sequence of the soluble TRAIL
domains (iii) and (v) is selected from:
(a) Arg121-Val122-Ala123 and
(b) (Gly/Ser) 121.
[0086] The soluble TRAIL domain preferably ends with amino acid
Gly281 of human TRAIL. In certain embodiments, the TRAIL domain may
comprise internal mutations as described above.
[0087] Components (ii) and (iv) of the TRAIL receptor agonist
protein are peptide linker elements located between components (i)
and (iii) or (iii) and (v), respectively. The flexible linker
elements have a length of 3-8 amino acids, particularly a length of
3, 4, 5, 6, 7, or 8 amino acids. The linker elements are preferably
glycine/serine linkers, i.e. peptide linkers substantially
consisting of the amino acids glycine and serine. In cases in which
the soluble cytokine domain terminates with S or G (C-terminus),
e.g. human TRAIL, the linker starts after S or G. In cases in which
the soluble cytokine domain starts with S or G (N-terminus), the
linker ends before this S or G.
[0088] It should be noted that linker (ii) and linker (iv) do not
need to be of the same length. In order to decrease potential
immunogenicity, it may be preferred to use shorter linkers. In
addition it turned out that shorter linkers lead to single chain
molecules with reduced tendency to form aggregates. Whereas linkers
that are substantially longer than the ones disclosed here may
exhibit unfavorable aggregations properties.
[0089] If desired, the linker may comprise an asparagine residue
which may form a glycosylate site Asn-Xaa-Ser. In certain
embodiments, one of the linkers, e.g. linker (ii) or linker (iv)
comprises a glycosylation site. In other embodiments, both linkers
(iv) comprise glycosylation sites. In order to increase the
solubility of the sc TRAIL proteins and/or in order to reduce the
potential immunogenicity, it may be preferred that linker (ii) or
linker (iv) or both comprise a glycosylation site.
[0090] Preferred linker sequences are selected from GSGSGSGS (SEQ
ID NO: 3), GSGSGNGS (SEQ ID NO: 2), GGSGSGSG (SEQ ID NO: 4), GGSGSG
(SEQ ID NO: 5), GGSG (SEQ ID NO: 6), GGSGNGSG (SEQ ID NO: 7),
GGNGSGSG (SEQ ID NO: 8), GGNGSG (SEQ ID NO: 9), and GSGS (SEQ ID
NO: 23).
[0091] According to a most preferred embodiment, the linker
sequences are each GSGSGNGS according to SEQ ID NO: 2. Example
linker sequences are shown in Table 2.
TABLE-US-00002 TABLE 2 Example Linker Sequences SEQ ID NO Sequence
2 GSGSGNGS 3 GSGSGSGS 4 GGSGSGSG 5 GGSGSG 6 GGSG 7 GGSGNGSG 8
GGNGSGSG 9 GGNGSG 22 GSGSGS 23 GSGS 24 GSG
[0092] The TRAIL receptor agonist protein additionally comprises an
antibody Fc fragment domain which may be located N-terminal to the
first TRAIL domain (i) and/or C-terminal to the third TRAIL domain
(v). Preferably, the antibody Fc fragment domain comprises or
consists of an amino acid sequence as shown in SEQ ID NO: 10.
Alternatively, the Fc fragment domain comprises or consists of an
amino acid sequence as shown in SEQ ID NO: 17. Example Fc fragment
domains are shown in Table 3.
TABLE-US-00003 TABLE 3 Example Fc Fragment Domains SEQ ID NO
Sequence 10 PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 17
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
[0093] The total number of glycosites and the individual position
of the carbohydrates in three dimensions impacts the in-vivo
stability of TRAIL receptor agonist proteins. Further, carbohydrate
recognition depends on local density of the terminal saccharides,
the branching of the carbohydrate tree and the relative position of
the carbohydrates matter.
[0094] Depletion of CH2-domain carbohydrates is necessary in order
to avoid Fc-receptor based crosslinking in vivo and potential
TRAIL-receptor superclustering-based toxicity. Further, partially
degraded carbohydrates reduce the in vivo half-life of TRAIL
receptor agonist proteins through lectin-driven mechanisms. By
reducing the total number of glycosylation sites on the molecule,
the resulting compound is less accessible to these mechanisms,
increasing half-life. Accordingly, in one embodiment, the overall
number of glycosites on the TRAIL receptor agonist proteins of the
instant invention was reduced through the depletion of CH2
glycosites, resulting in TRAIL receptor agonist proteins comprising
N297S equivalent mutations (according to the EU numbering system)
creating aglycosl-CH2 domains.
[0095] CH2-glycosites present on the inner surface areas normally
shield the subdomain from proteases during "open Fc-conformation
transits" wherein hinge-interchain disulfide bonds are reduced and
the covalent interchain linkage is disrupted. This enables
CH2-dissociation and exposure of the inner surface area towards
proteases. TRAIL receptor agonist proteins comprising an N297S
equivalent mutation (according to the EU numbering system) creating
an aglycosl-CH2 are therefore likely to be less proteolytically
stable that equivalent structures with wild-type CH2 glycosylation.
This would impact the compound's stability during USP/DSP/storage,
where host cell proteases are present and have long-term access to
the structure. Accordingly, in certain embodiments, the TRAIL
receptor agonist lacks CH2 glycosites, but comprises glycosites in
the linker sequences of each polypeptide chain (e.g., GSGSGNGS
according to SEQ ID NO: 2). In certain exemplary embodiments, the
TRAIL receptor agonist comprises two glycosites per polypeptide
chain, for a total of four glycosites, According to a preferred
embodiment of the invention, the antibody Fc fragment domain is
fused via a hinge-linker element. The hinge-linker element has a
length of 10-30 amino acids, particularly a length of 15-25 amino
acids, e.g. 22 amino acids. The hinge-linker element preferably
comprises the hinge-region sequence of an immunoglobulin, herein
referred to as "Ig hinge-region". The term "Ig hinge-region" means
any polypeptide comprising an amino acid sequence that shares
sequence identity or similarity with a portion of a naturally
occurring Ig hinge-region sequence which includes the cysteine
residues at which the disulfide bonds link the two heavy chains of
the immunoglobulin.
[0096] Derivatives and analogues of the hinge-region can be
obtained by mutations. A derivative or analogue as referred to
herein is a polypeptide comprising an amino acid sequence that
shares sequence identity or similarity with the full length
sequence of the wild type (or naturally occurring protein) except
that it has one or more amino acid sequence differences
attributable to a deletion, insertion and/or substitution.
According to the present invention, however, the term
"hinge-linker" is not limited to those linkers comprising an Ig
hinge-region or a derivative thereof, but any linkers long enough
to allow the domains attached by the hinge-linker element to attain
a biologically active confirmation.
[0097] The number of molecules with open Fc-conformation in an
individual TRAIL receptor agonist protein depends on the number of
interchain-disulfide bonds present in the hinge region.
Accordingly, in one embodiment a third cysteine was introduced into
the hinge region of the TRAIL receptor agonist proteins of the
instant invention in order to ameliorate the effect of depleting
the CH2-glycosites.
[0098] Further, the TRAIL receptor agonist proteins of the
invention additionally comprise mutation of the upper-hinge lysine
to a glycine to reduce proteolytic processing at this site.
[0099] A particularly preferred hinge-linker element comprises or
consists of the amino acid sequence as shown in SEQ ID NO: 11
(Table 4).
[0100] The TRAIL receptor agonist protein may additionally comprise
an N-terminal signal peptide domain, which allows processing, e.g.
extracellular secretion, in a suitable host cell. Preferably, the
N-terminal signal peptide domain comprises a protease cleavage
site, e.g. a signal peptidase cleavage site and thus may be removed
after or during expression to obtain the mature protein. A
particularly preferred N-terminal signal peptide domain comprises
the amino acid sequence as shown in SEQ ID NO: 12 (Table 4).
[0101] Further, the TRAIL receptor agonist protein may additionally
comprise a C-terminal element, having a length of e.g. 1-50,
preferably 10-30 amino acids which may include or connect to a
recognition/purification domain, e.g. a FLAG domain, a Strep-tag
or
[0102] Strep-tag II domain and/or a poly-His domain. According to a
particularly preferred embodiment, the fusion polypeptide comprises
a Strep-tag fused to the C-terminus via a short serine linker as
shown in SEQ ID NO: 13 (Table 4).
[0103] An exemplary hinge-linker element, N-terminal signal peptide
domain, and short serine linker are shown in Table 4.
TABLE-US-00004 TABLE 4 Exemplary domains and linkers SEQ ID NO
Sequence 11 GPGSSSSSSSGSCDKTHTCPPC 12 METDTLLVFVLLVWVPAGNG 13
SSSSSSAWSHPQFEK 25 GPGSSSSSSGSCDKTHTCPPC
[0104] According to a particularly preferred embodiment of the
invention, the fusion polypeptide comprises three soluble TRAIL
domains fused by peptide linker elements of SEQ ID NO: 2. The first
soluble TRAIL domain (i) consists of amino acids 120-281 of human
TRAIL according to SEQ ID NO: 1 and the soluble TRAIL domains (iii)
and (v) consist of amino acids 121-281 of human TRAIL according to
SEQ ID NO: 1. Additionally, the fusion polypeptide comprises an
antibody Fc fragment domain according to SEQ ID NO: 10 that is
fused C-terminally to the soluble TRAIL domain (v) via a
hinge-linker according to SEQ ID NO: 11. The inventors surprisingly
found that this particular fusion polypeptide provides improved
biological activity and is particularly stable. The amino acid
sequence of an exemplary embodiment of a TRAIL receptor agonist
protein of the invention is set forth in SEQ ID NO: 19.
[0105] Further, the fusion polypeptide may comprise an N-terminal
signal peptide domain e.g. according to SEQ ID NO: 12. A specific
example of a TRAIL receptor agonist protein of the invention is
shown in SEQ ID NO: 14.
[0106] According to another preferred embodiment, the fusion
polypeptide may additionally comprise a C-terminal Strep-tag that
is fused to the polypeptide of the invention via a short serine
linker as shown in SEQ ID NO: 13. According to this aspect of the
invention, the Fc fragment preferably consists of the amino acid
sequence as shown in SEQ ID NO: 10 or 17. Further, the Fc fragment
may consist of a shorter Fc fragment, for example including amino
acids 1-217 of SEQ ID NO: 10. Particularly preferred examples of
fusion polypeptides comprising a C-terminal Strep-tag are shown in
SEQ ID NOs: 15 and 18.
[0107] The exemplary TRAIL receptor agonist proteins as shown in
SEQ ID NOs: 14, 15 and 18 each comprise an N-terminal signal
peptide domain. The signal peptide domain includes amino acids
1-20. In each case, the mature protein starts with amino acid 21.
Mature exemplary TRAIL receptor agonist proteins of the instant
invention are set forth in SEQ ID NO: 19, 20, 21, 26, 27, 28, 29,
and 30. Exemplary TRAIL receptor agonist proteins described above
are shown in Table 5.
[0108] The TRAIL receptor agonist as set forth in SEQ ID NO: 19 has
a reduced total number of glycosylation sites (the N297S mutation
in the CH2 region providing an aglycosylated CH2 domain), an
increased number of inter-chain disulfide bonds in the hinge
region, and the mutation of an upper-hinge lysine to a glycine.
These alterations provide a decrease in potential degradation and
TRAIL receptor superclustering (along with concomitant toxicity)
while increasing the half-life of the molecule. In some
embodiments, the N-terminal glutamine is modified to pyroglutamate
(Liu et al. 2011, J. Biol. Chem. 286:11211-11217).
TABLE-US-00005 TABLE 5 Exemplary TRAIL Receptor Agonist Proteins
SEQ ID NO Sequence 14 METDTLLVFVLLVWVPAGNGQRVAAHITGTRGRSNTL
SSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGE
LVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYI
YKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGI
FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSG
SGNGSRVAAHITGTRGRSNTLSSPNSKNEKALGRKIN
SWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTY
FRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSA
RNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNE
HLIDMDHEASFFGAFLVGGSGSGNGSRVAAHITGTRG
RSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLH
LRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQ
MVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSI
YQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFL
VGGPGSSSSSSSGSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 15
METDTLLVFVLLVVVVPAGNGQRVAAHITGTRGRSNT
LSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNG
ELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQY
IYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGG
IFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGS
GSGNGSRVAAHITGTRGRSNTLSSPNSKNEKALGRKI
NSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQT
YFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKS
ARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTN
EHLIDMDHEASFFGAFLVGGSGSGNGSRVAAHITGTR
GRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNL
HLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDK
QMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYS
IYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAF
LVGGPGSSSSSSSGSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVW
YVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGSSSSSSAWSHPQFEK 18
METDTLLVFVLLVWVPAGNGQRVAAHITGTRGRSNTL
SSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGE
LVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYI
YKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGI
FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSG
SGNGSRVAAHITGTRGRSNTLSSPNSKNEKALGRKIN
SWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTY
FRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSA
RNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNE
HLIDMDHEASFFGAFLVGGSGSGNGSRVAAHITGTRG
RSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLH
LRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQ
MVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSI
YQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFL
VGGPGSSSSSSSGSCDKTHTCPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 19
QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWES
SRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQ
EEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSC
WSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLID
MDHEASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNT
LSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNG
ELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQY
IYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGG
IFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGS
GSGNGSRVAAHITGTRGRSNTLSSPNSKNEKALGRKI
NSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQT
YFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKS
ARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTN
EHLIDMDHEASFFGAFLVGGPGSSSSSSSGSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYSS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 20
QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWES
SRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQ
EEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSC
WSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLID
MDHEASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNT
LSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNG
ELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQY
IYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGG
IFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGS
GSGNGSRVAAHITGTRGRSNTLSSPNSKNEKALGRKI
NSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQT
YFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKS
ARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTN
EHLIDMDHEASFFGAFLVGGPGSSSSSSSGSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGS SSSSSAWSHPQFEK 21
QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWES
SRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQ
EEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSC
WSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLID
MDHEASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNT
LSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNG
ELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQY
IYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGG
IFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGS
GSGNGSRVAAHITGTRGRSNTLSSPNSKNEKALGRKI
NSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQT
YFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKS
ARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTN
EHLIDMDHEASFFGAFLVGGPGSSSSSSSGSCDKTHT
CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 26
QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWES
SRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQ
EEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSC
WSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLID
MDHEASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNT
LSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNG
ELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQY
IYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGG
IFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGS
GSGNGSRVAAHITGTRGRSNTLSSPNSKNEKALGRKI
NSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQT
YFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKS
ARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTN
EHLIDMDHEASFFGAFLVGGPGSSSSSSGSDKTHTCP
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSSSS SSAWSHPQFEK 27
QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWES
SRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQ
EEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSC
WSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLID
MDHEASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNT
LSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNG
ELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQY
IYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGG
IFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGS
GSRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWE
SSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRF
QEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNS
CWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLI
DMDHEASFFGAFLVGGPGSSSSSSSGSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSSSSS SAWSHPQFEK 28
QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWES
SRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQ
EEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSC
WSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLID
MDHEASFFGAFLVGGSGSGSRVAAHITGTRGRSNTLS
SPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGEL
VIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIY
KYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIF
ELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGS
GSRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWE
SSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRF
QEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNS
CWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLI
DMDHEASFFGAFLVGGPGSSSSSSSGSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSSSSS SAWSHPQFEK 29
QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWES
SRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQ
EEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSC
WSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLID
MDHEASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNT
LSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNG
ELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQY
IYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGG
IFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGS
GSRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWE
SSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRF
QEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNS
CWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLI
DMDHEASFFGAFLVGGPGSSSSSSSGSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 30
QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWES
SRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQ
EEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSC
WSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLID
MDHEASFFGAFLVGGSGSGSRVAAHITGTRGRSNTLS
SPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGEL
VIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIY
KYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIF
ELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGS
GSRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWE
SSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRF
QEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNS
CWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLI
DMDHEASFFGAFLVGGPGSSSSSSSGSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0109] A further aspect of the present invention relates to a
nucleic acid molecule encoding a TRAIL receptor agonist protein as
described herein. The nucleic acid molecule may be a DNA molecule,
e.g. a double-stranded or single-stranded DNA molecule, or an RNA
molecule. The nucleic acid molecule may encode the TRAIL receptor
agonist protein or a precursor thereof, e.g. a pro- or pre-proform
of the TRAIL receptor agonist protein which may comprise a signal
sequence or other heterologous amino acid portions for secretion or
purification which are preferably located at the N- and/or
C-terminus of the TRAIL receptor agonist protein. The heterologous
amino acid portions may be linked to the first and/or second domain
via a protease cleavage site, e.g. a Factor X3, thrombin or IgA
protease cleavage site. A specific example of a nucleic acid
sequence of the invention is shown in Table 6 as SEQ ID NO: 16.
This nucleic acid molecule encodes the fusion polypeptide of SEQ ID
NO: 14.
TABLE-US-00006 TABLE 6 Nucleic Acid Sequence of Exemplary TRAIL
Receptor Agonist Protein SEQ ID NO Sequence 16
gatatcggtaccgccaccatggaaaccgacaccctgctggtgt
tcgtgctgctcgtgtgggtgccagccggcaatggacagagagt
ggccgctcatatcaccggcacccggggcagatctaacaccctg
tccagccccaactccaagaacgagaaggccctgggccggaaga
tcaactcctgggagtcctccagatccggccactcctttctgtc
caacctgcacctgagaaacggcgagctggtcatccacgagaag
ggcttctactacatctactcccagacctacttcaggtttcagg
aagagatcaaagagaacacaaagaacgacaagcagatggtgca
gtatatctacaagtacacctcctaccccgaccccatcctgctg
atgaagtccgcccggaactcctgctggtccaaggatgctgagt
acggcctgtacagcatctaccagggcggcatcttcgagctgaa
agagaacgaccggatcttcgtgtccgtgaccaacgagcacctg
atcgacatggaccacgaggccagctttttcggcgcctttctcg
tgggcggatccggaagcggaaacggcagtagagtggctgccca
cattaccggaaccagaggccggtccaacaccctgagcagccct
aacagcaaaaatgagaaagctctcgggcgcaagatcaacagct
gggaatctagcagaagcggccacagctttctgagcaatctgca
tctgcggaacggcgaactcgtgattcatgagaaggggttttat
tatatctatagccagacatactttcgattccaggaggaaatca
aggaaaacaccaaaaatgataaacagatggtccagtacattta
taagtataccagctaccctgatcctatcctcctcatgaagtct
gccagaaactcttgttggagcaaggacgccgagtatggactgt
actctatctatcagggggggatctttgaactcaaagaaaacga
tcgcatctttgtcagcgtcaccaatgagcatctcattgatatg
gatcatgaagctagtttcttcggggcattcctcgtgggaggct
ccggctctggcaacggatctagagtcgccgcacacatcacagg
gaccagaggcagaagcaataccctgtcctccccaaatagtaaa
aacgaaaaggcactcggccgcaaaattaattcctgggagagca
gcagatccgggcacagttttctgtctaatctccatctgaggaa
tggggagctggtgattcacgaaaaaggattttactacatttac
agtcagacttactttcgttttcaggaagagattaaggaaaata
ccaaaaacgacaagcagatggtccagtacatctataaatacac
ctcttatcctgacccaattctgctcatgaagagtgcccgcaac
agctgctggtctaaagacgccgaatacgggctgtattccattt
accaggggggaatttttgagctgaaggaaaatgatcggatttt
tgtctctgtcacaaacgaacacctcatcgatatggatcacgaa
gcctctttctttggcgccttcctggtcggaggccctggctcga
gttccagctcctcttctggctcctgcgacaagacccacacctg
tcccccttgtcctgcccctgaactgctgggcggaccttccgtg
ttcctgttccccccaaagcccaaggacaccctgatgatctccc
ggacccccgaagtgacctgcgtggtggtggatgtgtctcacga
ggaccctgaagtgaagttcaattggtacgtggacggcgtggaa
gtgcacaacgccaagaccaagcccagagaggaacagtactcct
ccacctaccgggtggtgtctgtgctgaccgtgctgcaccagga
ctggctgaacggcaaagagtacaagtgcaaggtgtccaacaag
gccctgcctgcccccatcgaaaagaccatctccaaggccaagg
gccagccccgggaaccccaggtgtacacactgccccctagccg
ggaagagatgaccaagaaccaggtgtccctgacctgcctggtc
aagggcttttacccctccgacattgccgtggaatgggagtcca
acggccagcctgagaacaactacaagaccaccccccctgtgct
ggactccgacggctcattcttcctgtactccaagctgacagtg
gacaagtcccggtggcagcagggcaacgtgttctcctgctccg
tgatgcacgaggccctgcacaaccactacacccagaagtccct
gtccctgagccccggcaaatgatagaagcttgatatc
[0110] The nucleic acid molecule may be operatively linked to an
expression control sequence, e.g. an expression control sequence
which allows expression of the nucleic acid molecule in a desired
host cell. The nucleic acid molecule may be located on a vector,
e.g. a plasmid, a bacteriophage, a viral vector, a chromosomal
integration vector, etc. Examples of suitable expression control
sequences and vectors are described for example by Sambrook et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, and Ausubel et al. (1989), Current Protocols in Molecular
Biology, John Wiley & Sons or more recent editions thereof.
[0111] Various expression vector/host cell systems may be used to
express the nucleic acid sequences encoding the TRAIL receptor
agonist proteins of the present invention. Suitable host cells
include, but are not limited to, prokaryotic cells such as
bacteria, e.g. E. coli, eukaryotic host cells such as yeast cells,
insect cells, plant cells or animal cells, preferably mammalian
cells and, more preferably, human cells. Further, the invention
relates to a non-human organism transformed or transfected with a
nucleic acid molecule as described above. Such transgenic organisms
may be generated by known methods of genetic transfer including
homologous recombination.
[0112] A further aspect of the present invention relates to a
pharmaceutical or diagnostic composition comprising as the active
agent at least one TRAIL receptor agonist protein, a respective
nucleic acid encoding therefore, or a transformed or transfected
cell, all as described herein.
[0113] The term "TRAIL-associated disease or disorder" as used
herein is any disease or disorder which may be ameliorated by
addition of a TRAIL receptor agonist. At least one TRAIL receptor
agonist protein, respective nucleic acid encoding therefore, or
transformed or transfected cell, all as described herein may be
used in therapy, e.g., in the prophylaxis and/or treatment of
disorders caused by, associated with and/or accompanied by
dysfunction of TRAIL, particularly proliferative disorders, such as
tumors, e.g. solid or lymphatic tumors; infectious diseases;
inflammatory diseases; metabolic diseases; autoimmune disorders,
e.g. rheumatoid and/or arthritic diseases; degenerative diseases,
e.g. neurodegenerative diseases such as multiple sclerosis;
apoptosis-associated diseases or transplant rejections.
[0114] The term "dysfunction of TRAIL" as used herein is to be
understood as any function or expression of TRAIL that deviates
from the normal function or expression of TRAIL, e.g.,
overexpression of the TRAIL gene or protein, reduced or abolished
expression of the TRAIL gene or protein compared to the normal
physiological expression level of TRAIL, increased activity of
TRAIL, reduced or abolished activity of TRAIL, increased binding of
TRAIL to any binding partners, e.g., to a receptor, particularly a
TRAIL receptor or another cytokine molecule, reduced or abolished
binding to any binding partner, e.g. to a receptor, particularly a
TRAIL receptor or another cytokine molecule, compared to the normal
physiological activity or binding of TRAIL.
[0115] In various embodiments, a method is provided for diagnosing
and/or treating a human subject suffering from a disorder which can
be diagnosed and/or treated by targeting TRAIL receptors comprising
administering to the human subject a TRAIL receptor agonist protein
disclosed herein such that the effect on the activity of the
target, or targets, in the human subject is agonistic, one or more
symptoms is alleviated, and/or treatment is achieved. The TRAIL
receptor agonist proteins provided herein can be used to diagnose
and/or treat humans suffering from primary and metastatic cancers,
including carcinomas of breast, colon, rectum, lung (e.g., small
cell lung cancer "SCLC" and non-small cell lung cancer "NSCLC"),
oropharynx, hypopharynx, esophagus, stomach, pancreas, liver,
gallbladder and bile ducts, small intestine, urinary tract
(including kidney, bladder and urothelium), female genital tract
(including cervix, uterus, and ovaries as well as choriocarcinoma
and gestational trophoblastic disease), male genital tract
(including prostate, seminal vesicles, testes and germ cell
tumors), endocrine glands (including the thyroid, adrenal, and
pituitary glands), and skin, as well as hemangiomas, melanomas,
sarcomas (including those arising from bone and soft tissues as
well as Kaposi's sarcoma), tumors of the brain, nerves, eyes, and
meninges (including astrocytomas, gliomas, glioblastomas,
retinoblastomas, neuromas, neuroblastomas, Schwannomas, and
meningiomas), tumors arising from hematopoietic malignancies, acute
leukemia, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), B cell lymphoma, Burkitt's lymphoma, chronic
myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL),
hairy cell leukemia, Hodgkin's and non-Hodgkin's lymphomas, DLBCL,
follicular lymphomas, hematopoietic malignancies, Kaposi's sarcoma,
malignant lymphoma, malignant histiocytosis, malignant melanoma,
multiple myeloma, paraneoplastic syndrome/hypercalcemia of
malignancy, or solid tumors.
[0116] A pharmaceutical composition comprising a TRAIL receptor
agonist protein disclosed herein and a pharmaceutically acceptable
carrier is provided. In some embodiments, the pharmaceutical
composition comprises at least one additional therapeutic agent for
treating a disorder. For example, the additional agent may be a
therapeutic agent, a chemotherapeutic agent; an imaging agent, a
cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor
(including but not limited to a KDR and a TIE-2 inhibitor), a
co-stimulation molecule modulator or an immune checkpoint inhibitor
(including but not limited to anti-B7.1, anti-B7.2, anti-B7.3,
anti-B7.4, anti-CD28, anti-B7RP1, CTLA4-Ig, anti-CTLA-4, anti-PD-1,
anti-PD-L1, anti-PD-L2, anti-ICOS, anti-LAG-3, anti-Tim3,
anti-VISTA, anti-HVEM, anti-BTLA, LIGHT fusion protein, anti-CD137,
anti-CD137L, anti-OX40, anti-OX4OL, anti-CD70, anti-CD27,
anti-GAL9, anti-A2AR, anti-KIR, anti-IDO-1, anti-CD20), a dendritic
cell/antigen-presenting cell modulator (including but not limited
to anti-CD40 antibody, anti-CD40 L, anti-DC-SIGN, anti-Dectin-1,
anti-CD301, anti-CD303, anti-CD123, anti-CD207, anti-DNGR1,
anti-CD205, anti-DCIR, anti-CD206, anti-ILT7), a modulator for
Toll-like receptors (including but not limited to anti-TLR-1,
anti-TLR-2, anti-TLR-3, anti-TLR-4, anti-TLR-4, anti-TLR-5,
anti-TLR-6, anti-TLR-7, anti-TLR-8, anti-TLR-9), an adhesion
molecule blocker (including but not limited to an anti-LFA-1
antibody, an anti-E/L selectin antibody, a small molecule
inhibitor), an anti-cytokine antibody or functional fragment
thereof (including but not limited to an anti-IL-18, an anti-TNF,
or an anti-IL-6/cytokine receptor antibody), a bispecific
redirected T cell or NK cell cytotoxicity (including but not
limited to a BiTE.RTM.), a chimeric T cell receptor (CAR-T) based
therapy, a T cell receptor (TCR)-based therapy, a therapeutic
cancer vaccine, methotrexate, cyclosporin, rapamycin, FK506, a
detectable label or reporter, a TNF antagonist, an anti-rheumatic,
a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug
(NSAID), an analgesic, an anesthetic, a sedative, a local
anesthetic, a neuromuscular blocker, an antimicrobial, an
antipsoriatic, a corticosteriod, an anabolic steroid, an
erythropoietin, an immunization, an immunoglobulin, an
immunosuppressive, a growth hormone, a hormone replacement drug, a
radiopharmaceutical, an antidepressant, an antipsychotic, a
stimulant, an asthma medication, a beta agonist, an inhaled
steroid, an epinephrine or analog, a cytokine, or a cytokine
antagonist.
[0117] In an embodiment, a method of treating a cancer or in the
prevention or inhibition of metastases from the tumors described
herein, the TRAIL receptor agonist protein(s) can be used alone or
in combination with one or more additional agents, e.g., a
chemotherapeutic, radiotherapy, or biological agent. In some
embodiments, the agent can include the following: 13-cis-Retinoic
Acid; 2-CdA; 2-Chlorodeoxyadenosine; 5-Azacitidine; 5-Fluorouracil;
5-FU; 6-Mercaptopurine; 6-MP; 6-TG; 6-Thioguanine; Abraxane;
Accutane.RTM.; Actinomycin-D; Adriamycin.RTM.; Adrucil.RTM.;
Afinitor.RTM.; Agrylin.RTM.; Ala-Cod.RTM.; Aldesleukin;
Alemtuzumab; ALIMTA; Alitretinoin; Alkaban-AQ.RTM.; Alkeran.RTM.;
All-transretinoic Acid; Alpha Interferon; Altretamine;
Amethopterin; Amifostine; Aminoglutethimide; Anagrelide;
Anandron.RTM.; Anastrozole; Arabinosylcytosine; Ara-C Aranesp.RTM.;
Aredia.RTM.; Arimidex.RTM.; Aromasin.RTM.; Arranon.RTM.; Arsenic
Trioxide; Arzerra.TM.; Asparaginase; ATRA; Avastin.RTM.;
Azacitidine; BCG; BCNU; Bendamustine; Bevacizumab; Bexarotene;
BEXXAR.RTM.; Bicalutamide; BiCNU; Blenoxane.RTM.; Bleomycin;
Bortezomib; Busulfan; Busulfex.RTM.; C225; Calcium Leucovorin;
Campath.RTM.; Camptosar.RTM.; Camptothecin-11; Capecitabine
Carac.TM.; Carboplatin; Carmustine; Carmustine Wafer; Casodex.RTM.;
CC-5013; CCI-779; CCNU; CDDP; CeeNU; Cerubidine.RTM.; Cetuximab;
Chlorambucil; Cisplatin; Citrovorum Factor; Cladribine; Cortisone;
Cosmegen.RTM.; CPT-11; Cyclophosphamide; Cytadren.RTM.; Cytarabine;
Cytarabine Liposomal; Cytosar-U.RTM.; Cytoxan.RTM.; Dacarbazine;
Dacogen; Dactinomycin; Darbepoetin Alfa; Dasatinib; Daunomycin;
Daunorubicin; Daunorubicin Hydrochloride; Daunorubicin Liposomal;
DaunoXome.RTM.; Decadron; Decitabine; Delta-Cortef.RTM.;
Deltasone.RTM.; Denileukin; Diftitox; DepoCyt.TM.; Dexamethasone;
Dexamethasone Acetate; Dexamethasone Sodium Phosphate; Dexasone;
Dexrazoxane; DHAD; DIC; Diodex; Docetaxel; Doxil.RTM.; Doxorubicin;
Doxorubicin Liposomal; Droxia.TM.; DTIC; DTIC-Dome.RTM.;
Duralone.RTM.; Duvelisib; Efudex.RTM.; Eligard.TM.; Ellence.TM.;
Eloxatin.TM.; Elspar.RTM.; Emcyt.RTM.; Epirubicin; Epoetin Alfa;
Erbitux; Erlotinib; Erwinia L-asparaginase; Estramustine; Ethyol
Etopophos.RTM.; Etoposide; Etoposide Phosphate; Eulexin.RTM.;
Everolimus; Evista.RTM.; Exemestane; Fareston.RTM.; Faslodex.RTM.;
Femara.RTM.; Filgrastim; Floxuridine; Fludara.RTM.; Fludarabine;
Fluoroplex.RTM.; Fluorouracil; Fluorouracil (cream);
Fluoxymesterone; Flutamide; Folinic Acid; FUDR.RTM.; Fulvestrant;
Gefitinib; Gemcitabine; Gemtuzumab ozogamicin; Gemzar; Gleevec.TM.;
Gliadel.RTM.; Wafer; GM-CSF; Goserelin; Granulocyte-Colony
Stimulating Factor (G-CSF); Granulocyte Macrophage Colony
Stimulating Factor (G-MCSF); Halotestin.RTM.; Herceptin.RTM.;
Hexadrol; Hexalen.RTM.; Hexamethylmelamine; HMM; Hycamtin.RTM.;
Hydrea.RTM.; Hydrocort Acetate.RTM.; Hydrocortisone; Hydrocortisone
Sodium Phosphate; Hydrocortisone Sodium Succinate; Hydrocortone
Phosphate; Hydroxyurea; Ibrutinib; Ibritumomab; Ibritumomab
Tiuxetan; Idamycin.RTM.; Idarubicin Ifex.RTM.; Interferon-alpha;
Interferon-alpha-2b (PEG Conjugate); Ifosfamide; Interleukin-11
(IL-11); Interleukin-2 (IL-2); Imatinib mesylate; Imidazole
Carboxamide; Intron A.RTM.; ipilimumab, Iressa.RTM.; Irinotecan;
Isotretinoin; Ixabepilone; Ixempra.TM.; KADCYCLA.RTM.; Kidrolase
(t) Lanacort.RTM.; Lapatinib; L-asparaginase; LCR; Lenalidomide;
Letrozole; Leucovorin; Leukeran; Leukine.TM.; Leuprolide;
Leurocristine; Leustatin.TM.; Lirilumab; Liposomal Ara-C; Liquid
Pred.RTM.; Lomustine; L-PAM; L-Sarcolysin; Lupron.RTM.; Lupron
Depot.RTM.; Matulane.RTM.; Maxidex; Mechlorethamine;
Mechlorethamine Hydrochloride; Medralone.RTM.; Medrol.RTM.;
Megace.RTM.; Megestrol; Megestrol Acetate; MEK inhibitors;
Melphalan; Mercaptopurine; Mesna; Mesnex.TM.; Methotrexate;
Methotrexate Sodium; Methylprednisolone; Meticorten.RTM.;
Mitomycin; Mitomycin-C; Mitoxantrone M-Prednisol.RTM.; MTC; MTX;
Mustargen.RTM.; Mustine; Mutamycin.RTM.; Myleran.RTM.; Mylocel.TM.;
Mylotarg.RTM.; Navitoclax; Navelbine.RTM.; Nelarabine; Neosar.RTM.;
Neulasta.TM.; Neumega.RTM.; Neupogen.RTM.; Nexavar.RTM.;
Nilandron.RTM.; Nilotinib; Nilutamide; Nipent.RTM.; Nitrogen
Mustard Novaldex.RTM.; Nivolumab; Novantrone.RTM.; Nplate;
Octreotide; Octreotide acetate; Ofatumumab; Oncospar.RTM.;
Oncovin.RTM.; Ontak.RTM.; Onxal.TM.; Oprelvekin; Orapred.RTM.;
Orasone.RTM.; Oxaliplatin; Paclitaxel; Paclitaxel Protein-bound;
Pamidronate; Panitumumab; Panretin.RTM.; Paraplatin.RTM.;
Pazopanib; Pediapred.RTM.; PEG Interferon; Pegaspargase;
Pegfilgrastim; PEG-INTRON.TM.; PEG-L-asparaginase; PEMETREXED;
Pembrolizumab; Pentostatin; Pertuzumab; Phenylalanine Mustard;
Pidilizumab; Platinol.RTM.; Platinol-AQ.RTM.; Prednisolone;
Prednisone; Prelone.RTM.; Procarbazine; PROCRIT.RTM.;
Proleukin.RTM.; Prolifeprospan 20 with Carmustine Implant;
Purinethol.RTM.; BRAF inhibitors; Raloxifene; Revlimid.RTM.;
Rheumatrex.RTM.; Rituxan.RTM.; Rituximab; Roferon-A.RTM.;
Romiplostim; Rubex.RTM.; Rubidomycin hydrochloride;
Sandostatin.RTM.; Sandostatin LAR.RTM.; Sargramostim;
Solu-Cortef.RTM.; Solu-Medrol.RTM.; Sorafenib; SPRYCEL.TM.;
STI-571; STIVAGRA.TM., Streptozocin; SU11248; Sunitinib;
Sutent.RTM.; Tamoxifen Tarceva.RTM.; Targretin.RTM.; Tasigna.RTM.;
Taxol.RTM.; Taxotere.RTM.; Temodar.RTM.; Temozolomide Temsirolimus;
Teniposide; TESPA; Thalidomide; Thalomid.RTM.; TheraCys.RTM.;
Thioguanine; Thioguanine Tabloid.RTM.; Thiophosphoamide;
Thioplex.RTM.; Thiotepa; TICE.RTM.; Toposar.RTM.; Topotecan;
Toremifene; Torisel.RTM.; Tositumomab; Trastuzumab; Treanda.RTM.;
Tremelimumab; Tretinoin; Trexall.TM.; Trisenox.RTM.; TSPA;
TYKERB.RTM.; Urelumab; VCR; Vectibix.TM. Velban.RTM.; Velcade.RTM.;
Venetoclax; VePesid.RTM.; Vesanoid.RTM.; Viadur.TM.; Vidaza.RTM.;
Vinblastine; Vinblastine Sulfate; Vincasar Pfs.RTM.; Vincristine;
Vinorelbine; Vinorelbine tartrate; VLB; VM-26; Vorinostat;
Votrient; VP-16; Vumon.RTM.; Xeloda.RTM.; Zanosar.RTM.; Zevalin.TM.
Zinecard.RTM.; Zoladex.RTM.; Zoledronic acid; Zolinza; or
Zometa.RTM., and/or any other agent not specifically listed here
that target similar pathways.
[0118] When two or more substances or principles are to be used as
part of a combined treatment regimen, they can be administered via
the same route of administration or via different routes of
administration, at essentially the same time or at different times
(e.g. essentially simultaneously, consecutively, or according to an
alternating regime). When the substances or principles are to be
administered simultaneously via the same route of administration,
they may be administered as different pharmaceutical formulations
or compositions or part of a combined pharmaceutical formulation or
composition, as will be clear to the skilled person.
[0119] Also, when two or more active substances or principles are
to be used as part of a combined treatment regimen, each of the
substances or principles may be administered in the same amount and
according to the same regimen as used when the compound or
principle is used on its own, and such combined use may or may not
lead to a synergistic effect. However, when the combined use of the
two or more active substances or principles leads to a synergistic
effect, it may also be possible to reduce the amount of one, more
than one, or all of the substances or principles to be
administered, while still achieving the desired therapeutic action.
This may, e.g., be useful for avoiding, limiting or reducing any
unwanted side-effects that are associated with the use of one or
more of the substances or principles when they are used in their
usual amounts, while still obtaining the desired pharmaceutical or
therapeutic effect.
[0120] The effectiveness of the treatment regimen used according to
the invention may be determined and/or followed in any manner known
per se for the disease or disorder involved, as will be clear to
the clinician. The clinician will also be able, where appropriate
and on a case-by-case basis, to change or modify a particular
treatment regimen, so as to achieve the desired therapeutic effect,
to avoid, limit or reduce unwanted side-effects, and/or to achieve
an appropriate balance between achieving the desired therapeutic
effect on the one hand and avoiding, limiting or reducing undesired
side effects on the other hand.
[0121] Generally, the treatment regimen will be followed until the
desired therapeutic effect is achieved and/or for as long as the
desired therapeutic effect is to be maintained. Again, this can be
determined by the clinician.
[0122] In various embodiments, pharmaceutical compositions
comprising one or more TRAIL receptor agonist proteins, either
alone or in combination with prophylactic agents, therapeutic
agents, and/or pharmaceutically acceptable carriers are provided
herein. In various embodiments, nonlimiting examples of the uses of
the pharmaceutical compositions disclosed herein include
diagnosing, detecting, and/or monitoring a disorder, preventing,
treating, managing, and/or ameliorating a disorder or one or more
symptoms thereof, and/or in research. The formulation of
pharmaceutical compositions, either alone or in combination with
prophylactic agents, therapeutic agents, and/or pharmaceutically
acceptable carriers, are known to one skilled in the art (US Patent
Publication No. 20090311253 A1).
[0123] In various embodiments, a pharmaceutical formulation can
comprise one or more amino acid, one or more polysaccharide and/or
polysorbate, and a TRAIL receptor agonist protein present at a
concentration of between about 0.1 and 100 mg/ml, inclusive of
endpoints (e.g., 0.1-10, 1-10, 0.01-50, 1-50, 1-100, 10-100,
25-100, 25-50, or 50-100 mg/ml), where the formulation is at a pH
between about 5.0 and 7.0, inclusive of endpoints (e.g., a pH of
about 5.0-6.0, 5.5-6.0, 5.0-6.5, 5.5-6.5, or 6.0-7.0). In an
embodiment, at least one amino acid in the formulation is histidine
and is present at a concentration of about 10-20 mM, 10-15 mM,
15-20 mM, or about 15 mM. In an embodiment, at least one
polysaccharide in the formulation is sucrose and is present at a
concentration of about 0-8.0% weight/volume (w/v). In an
embodiment, the polysorbate in the formulation is polysorbate 80
and is at a concentration of about 0-0.06% w/v. In an embodiment,
at least one amino acid in the formulation is arginine and is
present at a concentration of about 0-1.5% w/v (e.g., 0.5-1.5,
1.0-1.5, or 0.5-1.0 w/v). In an embodiment, the TRAIL receptor
agonist protein is present in the formulation at a concentration of
about 0.1-100 mg/ml, (e.g., about 1-100 mg/ml, or about 1-15 mg/ml,
or about 1-7.5 mg/ml, or about 2.5-7.5 mg/ml, or about 5-7.5 mg/ml,
or about 25-100 mg/ml, or about 20-60 mg/ml, or about 25-50 mg/ml,
or about 25 mg/ml, or about 50 mg/ml, or about 0.1-60 mg/ml, or
about 0.1-25 mg/ml, or about 1.0-60 mg/ml, or about 0.5-60 mg/ml,
or about 0.1-2.0 mg/ml, or about 0.5-2.0 mg/ml, or about 1-5 mg/ml,
or about 1-7.5 mg/ml, or about 1-15 mg/ml, or about 0.5 mg/ml, or
about 1.0 mg/m).
[0124] As used herein, the phrase "effective amount" means an
amount of TRAIL agonist protein that results in a detectable
improvement (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more from baseline) in
one or more parameters associated with a dysfunction of TRAIL or
with a TRAIL-associated disease or disorder.
[0125] In various embodiments, the pharmaceutical formulation is an
aqueous formulation, a lyophilized formulation, or a lyophilized
and rehydrated formulation. In an embodiment, the hydrating
solution is dextrose and/or saline (e.g., dextrose at a
concentration of about 5% w/v and/or the saline at a concentration
of about 0.9% w/v). In an embodiment, the pharmaceutical
formulation comprises about 15 mM histidine, about 0.03% (w/v)
polysorbate 80, about 4% (w/v) sucrose, and about 0.1-25 mg/ml of
the TRAIL receptor agonist protein, or about 1-15 mg/ml of TRAIL
receptor agonist protein, and is at a pH of about 6. In an
embodiment, the formulation further comprises at least one
additional agent.
[0126] In various embodiments, a formulation is used containing
about 25 mg/ml TRAIL receptor agonist protein, about 15 mM
histidine, 0.03% polysorbate 80 (weight/volume, w/v), 4.0% sucrose
(w/v), and a pH of about 6.0. In some embodiments, the formulation
does not comprise arginine. In some embodiments, the formulation
exhibits unexpectedly improved freeze-thaw stability, liquid
formulation stability, and/or lyophilized formulation stability, as
compared to other formulations comprising other components or
concentrations.
[0127] Methods of administering a therapeutic agent provided herein
include, but are not limited to, oral administration, parenteral
administration (e.g., intradermal, intramuscular, intraperitoneal,
intravenous and subcutaneous), epidural administration,
intratumoral administration, mucosal administration (e.g.,
intranasal and oral routes) and pulmonary administration (e.g.,
aerosolized compounds administered with an inhaler or nebulizer).
The formulation of pharmaceutical compositions for specific routes
of administration, and the materials and techniques necessary for
the various methods of administration are available and known to
one skilled in the art (US Patent Publication No. 20090311253
A1).
[0128] In various embodiments, dosage regimens may be adjusted to
provide for an optimum desired response (e.g., a therapeutic or
prophylactic response). For example, a single bolus may be
administered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated
by the exigencies of the therapeutic situation. In some
embodiments, parenteral compositions are formulated in dosage unit
form for ease of administration and uniformity of dosage. The term
"dosage unit form" refers to physically discrete units suited as
unitary dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier.
[0129] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a TRAIL receptor agonist
protein provided herein is about 0.1-100 mg/kg, (e.g., about
0.1-0.5, 0.1-1, 0.1-10, 0.1-20, 0.1-50, 0.1-75, 1-10, 1-15, 1-7.5,
1.25-15, 1.25-7.5, 2.5-7.5, 2.5-15, 5-15, 5-7.5, 1-20, 1-50, 7-75,
1-100, 5-10, 5-15, 5-20, 5-25, 5-50, 5-75, 10-20, 10-50, 10-75, or
10-100 mg/kg, or any concentration in between). In some
embodiments, the TRAIL receptor agonist protein is present in a
pharmaceutical composition at a therapeutically effective
concentration, e.g., a concentration of about 0.1-100 mg/ml (e.g.,
about 0.1-0.5, 0.1-1, 0.1-10, 0.1-20, 0.1-50, 0.1-75, 1-10, 1-20,
1-50, 1-75, 1-100, 5-10, 5-15, 5-20, 5-25, 5-50, 5-75, 10-20,
10-50, 10-75, or 10-100 mg/ml, or any concentration in between).
Note that dosage values may vary with the type and/or severity of
the condition to be alleviated. It is to be further understood that
for any particular subject, specific dosage regimens may be
adjusted over time according to the individual need and/or the
professional judgment of the person administering or supervising
the administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed composition.
Examples
1. Manufacture of a TRAIL Receptor Agonist Protein (Sc TRAIL
Wt)
1.1 Polypeptide Structure
[0130] A) Amino acids Met1-Gly20 [0131] Ig-Kappa-signal peptide,
assumed signal peptidase cleavage site after amino acid Gly 20. B)
Amino acids GIn21-Gly182 [0132] First soluble cytokine domain of
the human TRAIL ligand (TRAIL, amino acid 120-281 of SEQ ID NO: 1).
C) Amino acids Gly 183-Ser 190 [0133] First peptide linker element
of SEQ ID NO: 2. D) Amino acids Arg191-Gly351 [0134] Second soluble
cytokine domain of the human TRAIL ligand (TRAIL, amino acids
121-281 of SEQ ID NO: 1). E) Amino acids Gly352-Ser359. [0135]
Second peptide linker element of SEQ ID NO: 2. F) Amino acids
Arg360-Gly520 [0136] Third soluble cytokine domain of the human
TRAIL ligand (TRAIL, amino acids 121-281 of SEQ ID NO: 1). G) Amino
acids Gly521-Cys542 [0137] Hinge-linker element of SEQ ID NO: 11.
H) Amino acids Pro543-Lys760 [0138] Antibody Fc fragment domain of
SEQ ID NO: 10.
[0139] The above TRAIL receptor agonist protein is shown in SEQ ID
NO: 14.
[0140] The indicated linkers may be replaced by other preferred
linkers, e.g. as shown in SEQ ID NOs: 3-9.
[0141] It should be noted that the first and second peptide linkers
do not need to be identical.
[0142] The signal peptide sequence (A) may be replaced by any other
suitable, e.g. mammalian signal peptide sequence.
1.2 Gene Cassette Encoding the Polypeptide
[0143] The synthetic gene may be optimized in view of its codon
usage for the expression in suitable host cells, e.g. insect cells
or mammalian cells. A preferred nucleic acid sequence is shown in
SEQ ID NO: 16.
2. Expression and Purification
Cloning, Expression and Purification of Fusion Polypeptides
[0144] The aforementioned fusion proteins were expressed
recombinantly in two different eukaryotic host cells:
[0145] For initial analysis of aforementioned TRAIL receptor
agonist fusion proteins, Hek293T cells grown in DMEM+GlutaMAX
(GibCo) supplemented with 10% FBS, 100 units/ml Penicillin and 100
[mu]g/ml Streptomycin were transiently transfected with a plasmid
containing an expression cassette for a fusion polypeptide and an
appropriate selection marker, e.g. a functional expression cassette
comprising a blasticidine, puromycin or hygromycin resistance gene.
In those cases, where a plurality of polypeptide chains is
necessary to achieve the final product, the expression cassettes
were either combined on one plasmid or positioned on different
plasmids during the transfection. Cell culture supernatant
containing recombinant fusion polypeptide was harvested three days
post transfection and clarified by centrifugation at 300.times.g
followed by filtration through a 0.22 .mu.m sterile filter.
[0146] For larger scale expression of TRAIL receptor agonist fusion
proteins to be used in vivo, synthetic DNA cassettes encoding the
aforementioned proteins were inserted into eukaryotic expression
vectors comprising appropriate selection markers (e.g. a functional
expression cassette comprising a blasticidin, puromycin or
hygromycin resistance gene) and genetic elements suitable to
enhance the number of transcriptionally active insertion sites
within the host cells genome. The sequence verified expression
vectors were introduced by electroporation into suspension adapted
Chinese Hamster Ovary cells (CHO-S, Invitrogen). Appropriate
selection pressure was applied three days post-transfection to the
transfected cells. Surviving cells carrying the vector derived
resistance gene(s) were recovered by subsequent cultivation under
selection pressure. Upon stable growth of the selected cell pools
in chemically defined medium (PowerCHO2-CD, Lonza) at 37.degree. C.
and 7% CO2 atmosphere in an orbital shaker incubator (100 rpm, 50
mm shaking throw), the individual supernatants were analysed by
ELISA-assays detecting the aforementioned proteins and the cell
pools with the highest specific productivity were expanded in shake
flasks prior to protein production (orbital shaker, 100 rpm,
shaking throw 50 mm).
[0147] For lab-scale protein production, individual cell pools were
cultured for 7-12 days in chemically defined medium (PowerCHO2-CD,
Lonza) at 37.degree. C. and 7% CO2 atmosphere in a Wave bioreactor
20/50 EHT (GE-Healthcare). The basal medium was PowerCHO2-CD
supplemented with 4 mM Glutamax. Wave culture started with a viable
cell concentration of 0.3 to 0.4.times.10e6 cells/ml and the
following settings (for a five- or ten liter bag): shaking
frequency 18 rpm, shaking ankle 7.degree., gas current 0.2-0.3
L/min, 7% CO2, 36.5.degree. C. During the Wave run, the cell
culture were fed twice with PowerFeed A (Lonza), usually on day 2
(20% feed) and day 5 (30% feed). After the second feed, shaking
frequency was increased to 22 rpm, as well as the shaking ankle to
8.degree..
[0148] The bioreactor was usually harvested in between day 7 to day
12 when the cell viability dropped below 80%. First, the culture
supernatant was clarified using a manual depth filtration system
(Millipore Millistak Pod, MCOHC 0.054 m.sup.2). For Strep-tagged
proteins, Avidin was added to a final concentration of 0.5 mg/L.
Finally, the culture supernatant containing the TRAIL receptor
agonist fusion protein was sterile filtered using a bottle top
filter (0.22 .mu.m, PES, Corning) and stored at 2-8.degree. C.
until further processing.
[0149] For affinity purification Streptactin Sepharose was packed
to a column (gel bed 1 ml), equilibrated with 15 ml buffer W (100
mM Tris-HCl, 150 mM NaCl, pH 8.0) or PBS pH 7.4 and the cell
culture supernatant was applied to the column with a flow rate of 4
ml/min. Subsequently, the column was washed with 15 ml buffer W and
bound polypeptide was eluted stepwise by addition of 7.times.1 ml
buffer E (100 mM Tris HCl, 150 mM NaCl, 2.5 mM Desthiobiotin, pH
8.0). Alternately, PBS pH 7.4 containing 2.5 mM Desthiobiotin can
be used for this step.
[0150] Alternately to the Streptactin Sepharose based method, the
affinity purification was performed employing a column with
immobilized Protein-A as affinity ligand and a Akta chromatography
system (GE-Healthcare). A solid phase material with high affinity
for the FC-domain of the fusion protein was chosen: MABSelect
Sure.TM. (GE Healthcare). Briefly, the clarified cell culture
supernatant was loaded on a HiTrap MabSelectSure column (CV=5 ml)
equilibrated in wash-buffer-1 (20 mM Pi, 95 mM NaCl, pH7.2) not
exceeding a load of 10 mg fusion protein per ml column-bed. The
column was washed with ten column-volumes (10CV) of aforementioned
equilibration buffer followed by four column-volumes (4CV) of
wash-buffer-2 (20 mM Pi, 95 mM NaCl, pH 8.0) to deplete host-cell
protein and host-cell DNA. The column was then eluted with elution
buffer (20 mM Pi, 95 mM NaCl, pH 3.5) and the eluate was collected
in up to ten fractions with each fraction having a volume equal to
column-bed volume (5 ml). Each fraction was neutralized with an
equal volume of aforementioned wash-buffer-2. The linear velocity
was set to 150 cm/h and kept constant during the aforementioned
affinity chromatography method.
[0151] The protein amount of the eluate fractions was quantitated
and peak fractions were concentrated by ultrafiltration and further
purified by size exclusion chromatography (SEC).
[0152] SEC was performed on Superdex 200 10/300 GL or HiLoad 26/60
columns using an Akta chromatography system (GE-Healthcare). The
columns were equilibrated with phosphate buffered saline and the
concentrated, affinity-purified polypeptide was loaded onto the SEC
column with the sample volume not exceeding 2% (v/v) of the
column-volume. In the case of Superdex200 10/300 GL columns (GE
Healthcare), a flow rate of 0.5 ml per minute was applied. In the
case of HiLoad 26/60 Superdex200 columns, a flow rate of 2.5 ml per
minute was applied. The elution profile of the polypeptide was
monitored by absorbance at 280 nm
[0153] For determination of the apparent molecular weight of
purified fusion polypeptide under native conditions a Superdex 200
column was loaded with standard proteins of known molecular weight.
Based on the elution volume of the standard proteins a calibration
curve was plotted and the apparent molecular weight of purified
fusion polypeptide was determined. The FC-domain comprising TRAIL
receptor agonist fusion proteins typically eluted from the
Supoerdex200 columns with an apparent molecular weight for the
homodimer of approx. 160-180 kDa.
3. Apoptosis Assay
[0154] A cellular assay with a Jurkat A3 permanent T-cell line was
used to determine the apoptosis inducing activity of the
TRAIL-receptor agonist fusion proteins. Jurkat cells were grown in
flasks with RPMI 1640-medium+GlutaMAX (GibCo) supplemented with 10%
FBS, 100 units/ml Penicillin and 100 .mu.g/ml Streptomycin. Prior
to the assay, 100,000 cells were seeded per well into a 96-well
microtiterplate. The addition of different concentrations of fusion
peptides to the wells was followed by a 3 hour incubation at
37.degree. C. Cells were lysed by adding lysis buffer (250 mM
HEPES, 50 mM MgCl2, 10 mM EGTA, 5% Triton-X-100, 100 mM DTT, 10 mM
AEBSF, pH 7.5) and plates were put on ice for 30 minutes to 2
hours. Apoptosis is paralleled by an increased activity of
caspases, e.g. Caspase-3. Hence, cleavage of the specific caspase
substrate Ac-DEVD-AFC (Biomol) was used to determine the extent of
apoptosis. In fact, Caspase activity correlates with the percentage
of apoptotic cells determined morphologically after staining the
cells with propidium iodide and Hoechst-33342. For the caspase
activity assay, 20 .mu.l cell lysate was transferred to a black
96-well microtiterplate. After the addition of 80 .mu.l buffer
containing 50 mM HEPES, 1% Sucrose, 0.1% CHAPS, 50 .mu.M
Ac-DEVD-AFC, and 25 mM DTT, pH 7.5, the plate was transferred to a
Tecan Infinite 500 microtiterplate reader and the increase in
fluorescence intensity was monitored (excitation wavelength 400 nm,
emission wavelength 505 nm).
3.1 Cell Death Assay
[0155] For the determination of cell death in HT1080 fibrosarcoma
cells 15,000 cells were plated in 96-well plates overnight in RPM1
1640-medium+GlutaMAX (GibCo) supplemented with 10% FBS (Biochrom).
Cells were coincubated with cycloheximide (Sigma) at a final
concentration of 2.5 g/ml. Cell death was quantified by staining
with buffer KV (0.5% crystal violet, 20% methanol). After staining,
the wells were washed with water and air-dried. The dye was eluted
with methanol and optical density at 595 nm was measured with an
ELISA reader.
4. Stability/Aggregation Test
4.1 Principle of the Aggregation Analysis (Definition for Soluble
Protein)
[0156] The content of monomers (defined trimeric assembly of TRAIL
receptor binding modules) and aggregates is determined by
analytical SEC as described in Example 2. For this particular
purpose the analysis is performed in buffers containing
physiological salt concentrations at physiological pH (e.g. 0.9%
NaCl, pH 7.4; PBS pH 7.4). A typical aggregation analysis is done
on a Superdex200 column (GE Healthcare). This column separates
proteins in the range between 10 to 800 kDa.
[0157] For determination of the apparent molecular weight of
purified fusion polypeptide under native conditions a Superdex 200
column is loaded with standard proteins of known molecular weight.
Based on the elution volume of the standard proteins a calibration
curve is plotted and the apparent molecular weight of purified
fusion polypeptide is calculated based on the elution volume.
[0158] SEC analysis of soluble, non-aggregated proteins, e.g.
trimeric TRAIL, typically shows a distinct single protein peak at a
defined elution volume. This elution volume corresponds to the
apparent native molecular weight of the particular protein and
approximately complies to the theoretical molecular weight
calculated on the basis of the primary amino acid sequence.
[0159] If protein aggregation occurs the SEC analysis shows
additional protein peaks with lower retention volumes. For TRAIL,
the aggregation of soluble proteins occurs in a characteristic
manner. The proteins tend to form oligomers of the "trimers",
forming nonamers (3.times.3) and 27mers (3.times.9). These
oligomers serve as aggregation seeds and a high content of
oligomers potentially leads to aggregation of the protein.
Oligomers of large molecular weight and aggregates elute in the
void volume of the Superdex200 column and cannot be analysed by SEC
with respect to their native molecular weight.
[0160] Due to the induction of (complete) aggregation, purified
preparations of TRAIL-SF fusion proteins should preferably contain
only defined trimeric proteins and only a very low amount of
oligomerised protein. The degree of aggregation/oligomerisation of
a particular TRAIL-SF protein preparation is determined on basis of
the SEC analysis by calculating the peak areas of the OD280 diagram
for the defined trimeric and the oligomer/aggregate fraction,
respectively. Based on the total peak area the percentage of
defined trimeric protein is calculated as follows:
(% Trimer content=[Peak area trimer]/[Total peak
area].times.100)
[0161] The definition for soluble protein as used in this text,
describes a protein preparation of purified TRAIL protein in a
buffer of physiological salt concentrations at physiological pH
that contains a defined soluble protein (trimeric assembly of TRAIL
domains) content of >90% within a typical protein concentration
range from 0.2 to 10.0 mg/ml.
5. Half-Life Determination
[0162] Molecules A-D are each made up of two polypeptides
covalently linked by interchain disulfide bonds. The number of
glycosites and hinge cysteines (resulting in interchain disulfide
bonds between proteins) were tested in order to determine the
effect that altering these characteristics has on the half-life of
these compounds.
[0163] Female NMRI mice were treated with 1.2 mg/kg bw and/or with
4 mg/kg bw of the specified compounds as a single intravenous bolus
injection. Whole blood was collected before application (predose),
and up to 168 hours after test item administration. Serum was
prepared and samples were stored at -80.degree. C. until
determination of serum concentrations. Pharmacokinetic parameters
were calculated using the mean serum concentrations and the
pharmacokinetic evaluation program PK Solutions Version 2.0 for
non-compartmental pharmacokinetic data analysis (Summit Research
Services, Montrose, Colo.). PK Solutions is an automated,
Excel-based application, which computes pharmacokinetic parameters
from concentration-time data obtained from analysis of e.g.
biological samples following intravenous or extra-vascular routes
of administration. PK Solutions calculates results without
presuming any specific compartmental model.
[0164] Quantitation of the test items in serum was performed with
an ELISA-assay detecting the individual TRAIL-receptor agonists
shown in Table 7 independent of a Strep-Tag being part of the
molecules. The general layout is shown in FIG. 18. The results are
summarized in Table 7.
[0165] Molecule A (made up of two polypeptides of SEQ ID NO:26) has
two hinge cysteines (forming two interchain disulfide bonds) and an
N residue at position 297 of the Fc region (according to the EU
index), resulting in wild-type CH2 glycosylation. Molecule A also
has glycosites at positions 168 and 337. Molecule B (made up of two
polypeptides of SEQ ID NO: 19) has three hinge cysteines (forming
three interchain disulfide bonds) (at positions 513, 519, and 522)
and an N297S mutation at position 297 of the Fc region (according
to the EU index), resulting in aglycosylation of the CH2 domain.
Molecule B also has glycosites at positions 168 and 337. Molecule C
(made up of two polypeptides of SEQ ID NO: 27) has three hinge
cysteines (forming three interchain disulfide bonds) and an N297S
mutation at position 297 of the Fc region (according to the EU
index), resulting in aglycosylation of the CH2 domain. Further,
there is a glycosite at position 168 (linker 1), but not at
position 337 (linker 2). Molecule D (made up of two polypeptides of
SEQ ID NO:28) has three hinge cysteines (forming three interchain
disulfide bonds) and an N297S mutation at position 297 of the Fc
region (according to the EU index), resulting in aglycosylation of
the CH2 domain. Further, the glycosites on both linker 1 and linker
2 (positions 168 and 337, respectively) have been depleted in
Molecule D.
[0166] The in vivo stability (as judged by compound half-life) of
Molecule B (both linkers glycosylated, CH2 glycosites depleted, and
the addition of a third hinge cysteine) was enhanced when compared
to Molecule A. Further, the depletion of all glycosites in from the
compound (Molecule D) resulted in reduced in vivo stability and low
productivity during transient expression. Molecule C (first linker
glycosylated, second linker aglycosylated, CH2 glycosites depleted)
demonstrated an intermediate in vivo stability when compared to
Molecules B and D (see results in Table 7).
TABLE-US-00007 TABLE 7 Results of Compound Half-life Testing in
NMRI-mice Terminal Number of Terminal Half- Half-life glycosylation
Number of life 4 mg/kg 1.2 mg/kg Molecule sites hinge cysteines
i.v. (hour) i.v. (hour) A 6 2 23.1 17.7 B 4 3 33.94 28.28 C 2 3
21.03 -- D 0 3 8.81 --
[0167] These experimental results demonstrate that combining linker
glycosylation (in both linkers 1 and 2) with a third interchain
disulfide bond (through the addition of a hinge cysteine) and the
deglycosylation of the CH2 domain in the Fc region results in
greater in vivo stability in the molecules of the instant
invention.
6. In Vitro Demonstration of Efficacy
6.1 TRAIL Receptor Agonist Protein of SEQ ID NO: 19 Inhibits Human
Hematologic and Solid Tumor Cell Survival In Vitro
[0168] Tumor cells were seeded at 10,000 cells/well in 96-well
plates in the recommended media containing 10% FBS and treated with
a TRAIL receptor agonist protein made up of two polypeptides having
the amino acid sequence set forth in SEQ ID NO: 19 for 24 hours at
37.degree. C. in a humidified, 5% CO2 atmosphere. Cell viability
was subsequently assessed using CellTiter-Glo.RTM. reagent as
described by the manufacturer's instructions (Promega; Madison,
Wis.). IC.sub.50 values were determined by non-linear regression
analysis of the concentration response data normalized to
non-treated control cells. Examples of resulting concentration
response curves for Colo205, Jurkat and SKM-1 cells that
demonstrate a loss in cell viability in response to TRAIL receptor
agonist protein made up of two polypeptides having the amino acid
sequence set forth in SEQ ID NO: 19 treatment are shown in FIG. 19.
Table 8 shows the results of hematologic (A; (n=40; Non-Hodgkin's
Lymphoma, NHL; Acute Myeloid Lymphoma, AML; Acute Lymphoblastic
Leukemia, ALL) and solid tumor (B; (n=44; Non-Small Cell Lung
Carcinoma, NSCLC; Pancreatic; Colorectal Cancer, CRC; Breast
Cancer, BrCa; Ovarian, Fibrosarcoma; Head and Neck, H&N; Small
Cell Lung Cancer, SCLC) cell lines treated with a TRAIL receptor
agonist protein made up of two polypeptides having the amino acid
sequence set forth in SEQ ID NO: 19 for 24 hours and viability
assessed by CellTiter-Glo.RTM.. Resulting IC.sub.50s for TRAIL
receptor agonist protein made up of two polypeptides having the
amino acid sequence set forth in SEQ ID NO: 19-mediated effects on
tumor cell viability are presented.
TABLE-US-00008 TABLE 8 Potency of TRAIL receptor agonist protein of
SEQ ID NO: 19 in human tumor cancer cell lines in vitro Tumor cell
SEQ ID NO: 19 line Tumor Type IC.sub.50 (ng/ml) A SU-DHL-8 NHL 1.36
NUDHL-1 NHL 6.50 OCl-Ly8 NHL 7.49 ULA NHL 8.44 OCl-Ly2 NHL 18.98
OCl-LY19 NHL 26.34 WSU-NHL NHL 31.60 OCl-Ly7 NHL 63.76 SU-DHL-5 NHL
82.07 OCl-Ly18 NHL 196.20 OCl-Ly1 NHL 416.95 SU-DHL-16 NHL 545.55
SU-DHL-2 NHL 1000.00 WSU-DLCL2 NHL 1000.00 Toledo NHL 1000.00
OCl-LY3 NHL 1000.00 RL NHL 1000.00 SU-DHL-4 NHL 1000.00 U2932 NHL
1000.00 HT NHL 1000.00 RC-K8 NHL 1000.00 SKM-1 AML 0.95 PL-21 AML
10.67 EOL-1 AML 18.31 HL-60 AML 76.62 OCl-AML2 AML 124.32 UKE-1 AML
205.35 MV4-11 AML 312.55 SET-2 AML 384.80 MOLM-13 AML 722.10
OCl-AML5 AML 1032.60 Kasumi-1 AML 1000.00 KG-1 AML 1000.00 OCl-AML3
AML 1000.00 SHI-1 AML 1000.00 SKNO-1 AML 1000.00 TF-1 AML 1000.00
THP-1 AML 1000.00 HEL AML 1000.00 Jurkat ALL 3.08 B NCl-H847 NSCLC
14.53 NCl-H647 NSCLC 24.75 NCl-H2444 NSCLC 27.75 NCl-H2170 NSCLC
30.16 NCl-H460 NSCLC 36.85 NCl-H838 NSCLC 44.48 NCl-H1792 NSCLC
61.09 NCl-H2347 NSCLC 81.06 NCl-H1373 NSCLC 125.15 NCl-H522 NSCLC
259.87 NCl-H2110 NSCLC 314.20 NCl-H596 NSCLC 397.80 HCC4006 NSCLC
407.24 NCl-H2122 NSCLC 480.55 NCl-H1299 NSCLC 716.00 NCl-H1975
NSCLC 741.50 HCC827 NSCLC 2824.50 NCl-H727 NSCLC 3178.00 NCl-H1944
NSCLC 4068.75 NCl-H1299 NSCLC 4214.87 Calu-6 NSCLC 4757.00
NCl-H1693 NSCLC 5000.00 HCC2935 NSCLC 5000.00 A549 NSCLC 5000.00
NCl-H1395 NSCLC 5000.00 NCl-H2172 NSCLC 5000.00 Calu-1 NSCLC
5000.00 NCl-H441 NSCLC 5000.00 NCl-H23 NSCLC 5000.00 NCl-H661 NSCLC
5000.00 NCl-H1650-GFP NSCLC >3 BxPC3 Pancreatic 16.00 Capan-1
Pancreatic 393.00 MIA PaCa-2 Pancreatic 158.00 PANC-1 Pancreatic
>1000 SW48 CRC 6.10 Colo205 CRC 1.30 SW480 CRC 132.00 HCT
116-GFP CRC 337.00 HCC38 BrCa 3.00 HCC1569 BrCa 219.00 MCF7 BrCa
>3000 MDA-MB-231 BRCa 235.00 HeyA8-GFP Ovarian 141.00 Fadu-GFP
H&N >3 HT-1080 Fibrosarcoma 377.00 NCl-H211 SCLC 72.58
6.2 TRAIL Receptor Agonist Protein of SEQ ID NO: 19 Synergizes with
Anti-Tumorigenic Agents to Induce Tumor Cell Death
[0169] Tumor cells were seeded at 10,000 cells/well in 96-well
plates in the recommended media containing 10% FBS and co-treated
with a TRAIL receptor agonist protein made up of two polypeptides
having the amino acid sequence set forth in SEQ ID NO: 19 and
venetoclax (ABT-199), navitoclax (ABT-263) or docetaxel (DTX) for
24 hrs at 37.degree. C. in a humidified, 5% CO2 atmosphere. Cell
viability was subsequently assessed using CellTiter-Glo.RTM.
reagent as described by the manufacturer's instructions. The Bliss
independence model (Wong et al., 2012; Mol. Cancer Ther.
11:1026-1035; Bernebaum, 1981 Adv. Cancer Res. 35:269-335; Borisy
et al., 2003 Proc. Natl. Acad. Sci. USA 100:7977-7982) was employed
to assess combination activity, with negative integers indicating
antagonism, a value of zero indicating additive activity, and
positive integers indicating synergy. Bliss scores were calculated
for each combination in the dose matrix and totaled to give a
"Bliss sum" value. An example of synergistic tumor cell death
induced by co-treating human tumor cells with a TRAIL receptor
agonist protein made up of two polypeptides having the amino acid
sequence set forth in SEQ ID NO:19 and venetoclax, navitoclax or
DTX, with the associated Bliss sum is shown in FIGS. 20(A-C). Bliss
sums determined for these combinations in a number of tumor cell
lines are depicted in Table 9.
TABLE-US-00009 TABLE 9 Bliss synergy assessment of cell killing by
TRAIL receptor agonist protein of SEQ ID NO: 19 in combination with
DTX in NSCLC cell lines (A) and venetoclax or navitoclax in NHL
& AML cell lines (B) in vitro. Bliss Sum Bliss Sum Bliss Sum
(SEQ ID (SEQ ID (SEQ ID Tumor Cell NO: 19 + Tumor Cell NO: 19 + NO:
19 + line DTX) line venitoclax) navitoclax) LG0552 748.2 WSU-DLCL2
1292 560 NCl-H522 549.1 SU-DHL-4 898 617 NCl-H647 452 OCl-AML3
831.9 456.4 NCl-H727 429.4 OCl-AML5 777.8 174.5 NCl-H1373 387.1
U2932 736.2 636.1 NCl-H596 261 PL-21 600.8 244.9 HCC2935 224.2 ULA
343.8 79.4 NCl-H2347 154 OCl-Ly18 309.8 8.3 NCl-H2444 135 MV4; 11
301.1 351 A549 118.1 RL 286.1 446.7 NCl-H23 70.6 MOLM-13 270.6
264.8 NCl-H847 64.2 SKM-1 222.9 88.1 HCC4006 15.75 OCl-Ly1 218.8
69.1 NCl-H2170 -97.1 SU-DHL-16 217.5 142.5 LG0567 -105.2 OCl-AMI2
160.2 145.5 HCC2935 -183 OCl-Ly8 154.9 177 HCC827 -292.8 THP-1
152.7 43.1 NCl-H661 -344.5 OCl-Ly3 146.5 -242.2 NCl-H441 -362
OCl-Ly2 145 127.2 NCl-H1395 -512 OCl-Ly19 114.9 37.3 NCl-H1944 -565
SKNO-1 104.7 -138.9 NCl-H1693 -584 UKE-1 80.5 28.9 Calu-6 -628.7
WSU-NHK 79.8 84 LG0481 -803 EOL-1 69.7 -6.3 NCl-H2172 -1404
SU-DHL-2 53.5 -31.8 Toledo 51.4 -68.2 HEL 21.5 -92.4 NuDHL-1 -28.4
18.7 TF-1 -100.6 -50.2 RC-K8 -131 -68 HT -173 12.1 HL-60 -176
-112.7 SHI-1 -208.4 -122.6 SU-DHL-8 -210.6 -37 SU-DHL-5 -233.8
-280.6 SET-2 -248.4 71.2 KG-1 -260 -20.3 Kasumi-1 -356.4 -241.2
7. TRAIL Receptor Agonist Protein of SEQ ID NO:19 Treatment
Inhibits Tumor Growth In Vivo
[0170] The effect of a TRAIL receptor agonist protein made up of
two polypeptides having the amino acid sequence set forth in SEQ ID
NO:19 on tumor growth was evaluated in subcutaneous Colo205
(colorectal), SKM-1 (acute myeloid leukemia), and H460LM (non-small
cell lung) xenograft tumors implanted in SCID female mice (Charles
Rivers Laboratories; Wilmington, Mass.). Briefly, human cancer
cells were inoculated subcutaneously into the right hind flank of
female SCID mice on study day 0. Administration of TRAIL receptor
agonist protein of SEQ ID NO:19 (0.3, 1, or 3 mkd dosed IV, QDx5 or
IP, Q2Dx5 as indicated) was initiated at the time of size match.
Tumor volume was measured for the duration of the experiment until
the mean tumor volume in each group reached an endpoint of >2000
mm.sup.3 for Colo205 and SKM-1 or >2500 mm.sup.3 for H460LM.
Results are shown in FIGS. 21-23. Administration of a TRAIL
receptor agonist protein made up of two polypeptides having the
amino acid sequence set forth in SEQ ID NO:19 induced significant
tumor growth inhibition in the Colo205, SKM-1, and H460LM xenograft
tumor models.
[0171] The effect of a TRAIL receptor agonist protein made up of
two polypeptides having the amino acid sequence set forth in SEQ ID
NO:19 on tumor growth was also evaluated in patient-derived
xenograft models CTG-0069 (colorectal), CTG-0167 (NSCLC), CTG-0293
(pancreatic), CTG-0714 (sarcoma), CTG-0136 (esophageal), CTG-485
(gastric), and CTG-0785 (Ewing's sarcoma) implanted in NSG female
mice (Champions Oncology; Hackensack, N.J.). Briefly, tumor
fragments were propagated subcutaneously into the right hind flank
of female NSG mice on study day 0. Administration of a TRAIL
receptor agonist protein made up of two polypeptides having the
amino acid sequence set forth in SEQ ID NO: 19 (3 mkd dosed IP,
Q2Dx5) was initiated at the time of size match. Tumor volume was
measured for the duration of the experiment until the mean tumor
volume in each group reached an endpoint of >2000 mm.sup.3 or 60
days. Results are shown in FIGS. 24(A-G). Administration of a TRAIL
receptor agonist protein made up of two polypeptides having the
amino acid sequence set forth in SEQ ID NO: 19 induced significant
tumor growth inhibition in the CTG-0069 (colorectal), CTG-0167
(NSCLC), CTG-0293 (pancreatic), CTG-0714 (sarcoma), CTG-0136
(esophageal), CTG-485 (gastric), and CTG-0785 (Ewing's sarcoma) PDX
models.
Sequence CWU 1
1
311281PRTHomo sapienssource/note="human TRAIL" 1Met Ala Met Met Glu
Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys1 5 10 15Val Leu Ile Val
Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30Val Thr Tyr
Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45Tyr Ser
Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60Trp
Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val65 70 75
80Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser
85 90 95Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser
Pro 100 105 110Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His
Ile Thr Gly 115 120 125Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro
Asn Ser Lys Asn Glu 130 135 140Lys Ala Leu Gly Arg Lys Ile Asn Ser
Trp Glu Ser Ser Arg Ser Gly145 150 155 160His Ser Phe Leu Ser Asn
Leu His Leu Arg Asn Gly Glu Leu Val Ile 165 170 175His Glu Lys Gly
Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190Gln Glu
Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 195 200
205Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys
210 215 220Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly
Leu Tyr225 230 235 240Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys
Glu Asn Asp Arg Ile 245 250 255Phe Val Ser Val Thr Asn Glu His Leu
Ile Asp Met Asp His Glu Ala 260 265 270Ser Phe Phe Gly Ala Phe Leu
Val Gly 275 28028PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic linker peptide" 2Gly Ser Gly Ser Gly
Asn Gly Ser1 538PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic linker peptide" 3Gly Ser Gly Ser Gly
Ser Gly Ser1 548PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic linker peptide" 4Gly Gly Ser Gly Ser
Gly Ser Gly1 556PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic linker peptide" 5Gly Gly Ser Gly Ser
Gly1 564PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic linker peptide" 6Gly Gly Ser
Gly178PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic linker peptide" 7Gly Gly Ser Gly Asn Gly Ser
Gly1 588PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic linker peptide" 8Gly Gly Asn Gly Ser
Gly Ser Gly1 596PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic linker peptide" 9Gly Gly Asn Gly Ser
Gly1 510218PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic Fc fragment polypeptide" 10Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro1 5 10 15Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 20 25
30Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
35 40 45Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 50 55 60Glu Gln Tyr Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu65 70 75 80His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 85 90 95Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu 115 120 125Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 130 135 140Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn145 150 155 160Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 165 170
175Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
180 185 190Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 195 200 205Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210
2151122PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic hinge-linker peptide" 11Gly Pro Gly Ser Ser Ser
Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr1 5 10 15His Thr Cys Pro Pro
Cys 201220PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic signal peptide" 12Met Glu Thr Asp Thr
Leu Leu Val Phe Val Leu Leu Val Trp Val Pro1 5 10 15Ala Gly Asn Gly
201315PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic strep-tag + serine linker peptide" 13Ser Ser Ser
Ser Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys1 5 10
1514760PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic single-chain fusion polypeptide" 14Met Glu Thr
Asp Thr Leu Leu Val Phe Val Leu Leu Val Trp Val Pro1 5 10 15Ala Gly
Asn Gly Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly 20 25 30Arg
Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu 35 40
45Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe
50 55 60Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu
Lys65 70 75 80Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe
Gln Glu Glu 85 90 95Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val
Gln Tyr Ile Tyr 100 105 110Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu
Leu Met Lys Ser Ala Arg 115 120 125Asn Ser Cys Trp Ser Lys Asp Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr 130 135 140Gln Gly Gly Ile Phe Glu
Leu Lys Glu Asn Asp Arg Ile Phe Val Ser145 150 155 160Val Thr Asn
Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe 165 170 175Gly
Ala Phe Leu Val Gly Gly Ser Gly Ser Gly Asn Gly Ser Arg Val 180 185
190Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser
195 200 205Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn
Ser Trp 210 215 220Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn
Leu His Leu Arg225 230 235 240Asn Gly Glu Leu Val Ile His Glu Lys
Gly Phe Tyr Tyr Ile Tyr Ser 245 250 255Gln Thr Tyr Phe Arg Phe Gln
Glu Glu Ile Lys Glu Asn Thr Lys Asn 260 265 270Asp Lys Gln Met Val
Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp 275 280 285Pro Ile Leu
Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp 290 295 300Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu305 310
315 320Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu
Ile 325 330 335Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu
Val Gly Gly 340 345 350Ser Gly Ser Gly Asn Gly Ser Arg Val Ala Ala
His Ile Thr Gly Thr 355 360 365Arg Gly Arg Ser Asn Thr Leu Ser Ser
Pro Asn Ser Lys Asn Glu Lys 370 375 380Ala Leu Gly Arg Lys Ile Asn
Ser Trp Glu Ser Ser Arg Ser Gly His385 390 395 400Ser Phe Leu Ser
Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His 405 410 415Glu Lys
Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln 420 425
430Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr
435 440 445Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met
Lys Ser 450 455 460Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr
Gly Leu Tyr Ser465 470 475 480Ile Tyr Gln Gly Gly Ile Phe Glu Leu
Lys Glu Asn Asp Arg Ile Phe 485 490 495Val Ser Val Thr Asn Glu His
Leu Ile Asp Met Asp His Glu Ala Ser 500 505 510Phe Phe Gly Ala Phe
Leu Val Gly Gly Pro Gly Ser Ser Ser Ser Ser 515 520 525Ser Ser Gly
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 530 535 540Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro545 550
555 560Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val 565 570 575Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val 580 585 590Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln 595 600 605Tyr Ser Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln 610 615 620Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala625 630 635 640Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 645 650 655Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 660 665
670Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
675 680 685Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr 690 695 700Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr705 710 715 720Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe 725 730 735Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys 740 745 750Ser Leu Ser Leu Ser
Pro Gly Lys 755 76015774PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
single-chain fusion polypeptide" 15Met Glu Thr Asp Thr Leu Leu Val
Phe Val Leu Leu Val Trp Val Pro1 5 10 15Ala Gly Asn Gly Gln Arg Val
Ala Ala His Ile Thr Gly Thr Arg Gly 20 25 30Arg Ser Asn Thr Leu Ser
Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu 35 40 45Gly Arg Lys Ile Asn
Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe 50 55 60Leu Ser Asn Leu
His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys65 70 75 80Gly Phe
Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu 85 90 95Ile
Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr 100 105
110Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg
115 120 125Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser
Ile Tyr 130 135 140Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg
Ile Phe Val Ser145 150 155 160Val Thr Asn Glu His Leu Ile Asp Met
Asp His Glu Ala Ser Phe Phe 165 170 175Gly Ala Phe Leu Val Gly Gly
Ser Gly Ser Gly Asn Gly Ser Arg Val 180 185 190Ala Ala His Ile Thr
Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser 195 200 205Pro Asn Ser
Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp 210 215 220Glu
Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg225 230
235 240Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr
Ser 245 250 255Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn
Thr Lys Asn 260 265 270Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr
Thr Ser Tyr Pro Asp 275 280 285Pro Ile Leu Leu Met Lys Ser Ala Arg
Asn Ser Cys Trp Ser Lys Asp 290 295 300Ala Glu Tyr Gly Leu Tyr Ser
Ile Tyr Gln Gly Gly Ile Phe Glu Leu305 310 315 320Lys Glu Asn Asp
Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile 325 330 335Asp Met
Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly 340 345
350Ser Gly Ser Gly Asn Gly Ser Arg Val Ala Ala His Ile Thr Gly Thr
355 360 365Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn
Glu Lys 370 375 380Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser
Arg Ser Gly His385 390 395 400Ser Phe Leu Ser Asn Leu His Leu Arg
Asn Gly Glu Leu Val Ile His 405 410 415Glu Lys Gly Phe Tyr Tyr Ile
Tyr Ser Gln Thr Tyr Phe Arg Phe Gln 420 425 430Glu Glu Ile Lys Glu
Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr 435 440 445Ile Tyr Lys
Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser 450 455 460Ala
Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser465 470
475 480Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile
Phe 485 490 495Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His
Glu Ala Ser 500 505 510Phe Phe Gly Ala Phe Leu Val Gly Gly Pro Gly
Ser Ser Ser Ser Ser 515 520 525Ser Ser Gly Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala 530 535 540Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro545 550 555 560Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 565 570 575Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 580 585
590Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
595 600 605Tyr Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln 610 615 620Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala625 630 635 640Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro 645 650 655Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr 660 665 670Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 675 680 685Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 690 695 700Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr705 710
715 720Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe 725 730 735Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys 740 745 750Ser Leu Ser Leu Ser Pro Gly Ser Ser Ser Ser
Ser Ser Ala Trp Ser 755 760 765His Pro Gln Phe Glu Lys
770162316DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic DNA polynucleotide sequence encoding
a single-chain fusion polypeptide" 16gatatcggta ccgccaccat
ggaaaccgac accctgctgg tgttcgtgct gctcgtgtgg 60gtgccagccg gcaatggaca
gagagtggcc gctcatatca ccggcacccg gggcagatct 120aacaccctgt
ccagccccaa ctccaagaac gagaaggccc tgggccggaa gatcaactcc
180tgggagtcct ccagatccgg ccactccttt ctgtccaacc tgcacctgag
aaacggcgag 240ctggtcatcc acgagaaggg cttctactac atctactccc
agacctactt caggtttcag 300gaagagatca aagagaacac aaagaacgac
aagcagatgg tgcagtatat ctacaagtac 360acctcctacc ccgaccccat
cctgctgatg aagtccgccc ggaactcctg ctggtccaag 420gatgctgagt
acggcctgta cagcatctac cagggcggca tcttcgagct gaaagagaac
480gaccggatct tcgtgtccgt gaccaacgag cacctgatcg acatggacca
cgaggccagc 540tttttcggcg cctttctcgt gggcggatcc ggaagcggaa
acggcagtag agtggctgcc 600cacattaccg gaaccagagg ccggtccaac
accctgagca gccctaacag caaaaatgag 660aaagctctcg ggcgcaagat
caacagctgg gaatctagca gaagcggcca cagctttctg 720agcaatctgc
atctgcggaa cggcgaactc gtgattcatg agaaggggtt ttattatatc
780tatagccaga catactttcg attccaggag gaaatcaagg aaaacaccaa
aaatgataaa 840cagatggtcc agtacattta taagtatacc agctaccctg
atcctatcct cctcatgaag 900tctgccagaa actcttgttg gagcaaggac
gccgagtatg gactgtactc tatctatcag 960ggggggatct ttgaactcaa
agaaaacgat cgcatctttg tcagcgtcac caatgagcat 1020ctcattgata
tggatcatga agctagtttc ttcggggcat tcctcgtggg aggctccggc
1080tctggcaacg gatctagagt cgccgcacac atcacaggga ccagaggcag
aagcaatacc 1140ctgtcctccc caaatagtaa aaacgaaaag gcactcggcc
gcaaaattaa ttcctgggag 1200agcagcagat ccgggcacag ttttctgtct
aatctccatc tgaggaatgg ggagctggtg 1260attcacgaaa aaggatttta
ctacatttac agtcagactt actttcgttt tcaggaagag 1320attaaggaaa
ataccaaaaa cgacaagcag atggtccagt acatctataa atacacctct
1380tatcctgacc caattctgct catgaagagt gcccgcaaca gctgctggtc
taaagacgcc 1440gaatacgggc tgtattccat ttaccagggg ggaatttttg
agctgaagga aaatgatcgg 1500atttttgtct ctgtcacaaa cgaacacctc
atcgatatgg atcacgaagc ctctttcttt 1560ggcgccttcc tggtcggagg
ccctggctcg agttccagct cctcttctgg ctcctgcgac 1620aagacccaca
cctgtccccc ttgtcctgcc cctgaactgc tgggcggacc ttccgtgttc
1680ctgttccccc caaagcccaa ggacaccctg atgatctccc ggacccccga
agtgacctgc 1740gtggtggtgg atgtgtctca cgaggaccct gaagtgaagt
tcaattggta cgtggacggc 1800gtggaagtgc acaacgccaa gaccaagccc
agagaggaac agtactcctc cacctaccgg 1860gtggtgtctg tgctgaccgt
gctgcaccag gactggctga acggcaaaga gtacaagtgc 1920aaggtgtcca
acaaggccct gcctgccccc atcgaaaaga ccatctccaa ggccaagggc
1980cagccccggg aaccccaggt gtacacactg ccccctagcc gggaagagat
gaccaagaac 2040caggtgtccc tgacctgcct ggtcaagggc ttttacccct
ccgacattgc cgtggaatgg 2100gagtccaacg gccagcctga gaacaactac
aagaccaccc cccctgtgct ggactccgac 2160ggctcattct tcctgtactc
caagctgaca gtggacaagt cccggtggca gcagggcaac 2220gtgttctcct
gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg
2280tccctgagcc ccggcaaatg atagaagctt gatatc 231617217PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
human Fc mutein polypeptide" 17Pro Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75 80Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn145 150 155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205Lys Ser Leu
Ser Leu Ser Pro Gly Lys 210 21518759PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 18Met Glu Thr Asp Thr Leu Leu Val Phe Val Leu Leu Val
Trp Val Pro1 5 10 15Ala Gly Asn Gly Gln Arg Val Ala Ala His Ile Thr
Gly Thr Arg Gly 20 25 30Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys
Asn Glu Lys Ala Leu 35 40 45Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser
Arg Ser Gly His Ser Phe 50 55 60Leu Ser Asn Leu His Leu Arg Asn Gly
Glu Leu Val Ile His Glu Lys65 70 75 80Gly Phe Tyr Tyr Ile Tyr Ser
Gln Thr Tyr Phe Arg Phe Gln Glu Glu 85 90 95Ile Lys Glu Asn Thr Lys
Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr 100 105 110Lys Tyr Thr Ser
Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg 115 120 125Asn Ser
Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr 130 135
140Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val
Ser145 150 155 160Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu
Ala Ser Phe Phe 165 170 175Gly Ala Phe Leu Val Gly Gly Ser Gly Ser
Gly Asn Gly Ser Arg Val 180 185 190Ala Ala His Ile Thr Gly Thr Arg
Gly Arg Ser Asn Thr Leu Ser Ser 195 200 205Pro Asn Ser Lys Asn Glu
Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp 210 215 220Glu Ser Ser Arg
Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg225 230 235 240Asn
Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser 245 250
255Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn
260 265 270Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr
Pro Asp 275 280 285Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys
Trp Ser Lys Asp 290 295 300Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln
Gly Gly Ile Phe Glu Leu305 310 315 320Lys Glu Asn Asp Arg Ile Phe
Val Ser Val Thr Asn Glu His Leu Ile 325 330 335Asp Met Asp His Glu
Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly 340 345 350Ser Gly Ser
Gly Asn Gly Ser Arg Val Ala Ala His Ile Thr Gly Thr 355 360 365Arg
Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys 370 375
380Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly
His385 390 395 400Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu
Leu Val Ile His 405 410 415Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln
Thr Tyr Phe Arg Phe Gln 420 425 430Glu Glu Ile Lys Glu Asn Thr Lys
Asn Asp Lys Gln Met Val Gln Tyr 435 440 445Ile Tyr Lys Tyr Thr Ser
Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser 450 455 460Ala Arg Asn Ser
Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser465 470 475 480Ile
Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe 485 490
495Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser
500 505 510Phe Phe Gly Ala Phe Leu Val Gly Gly Pro Gly Ser Ser Ser
Ser Ser 515 520 525Ser Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala 530 535 540Pro Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys545 550 555 560Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val 565 570 575Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 580 585 590Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 595 600 605Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 610 615
620Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu625 630 635 640Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg 645 650 655Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys 660 665 670Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp 675 680 685Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 690 695 700Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser705 710 715 720Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 725 730
735Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
740 745 750Leu Ser Leu Ser Pro Gly Lys 75519740PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 19Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg
Ser Asn Thr1 5 10 15Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu
Gly Arg Lys Ile 20 25 30Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser
Phe Leu Ser Asn Leu 35 40 45His Leu Arg Asn Gly Glu Leu Val Ile His
Glu Lys Gly Phe Tyr Tyr 50 55 60Ile Tyr Ser Gln Thr Tyr Phe Arg Phe
Gln Glu Glu Ile Lys Glu Asn65 70 75 80Thr Lys Asn Asp Lys Gln Met
Val Gln Tyr Ile Tyr Lys Tyr Thr Ser 85 90 95Tyr Pro Asp Pro Ile Leu
Leu Met Lys Ser Ala Arg Asn Ser Cys Trp 100 105 110Ser Lys Asp Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile 115 120 125Phe Glu
Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu 130 135
140His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe
Leu145 150 155 160Val Gly Gly Ser Gly Ser Gly Asn Gly Ser Arg Val
Ala Ala His Ile 165 170 175Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu
Ser Ser Pro Asn Ser Lys 180 185 190Asn Glu Lys Ala Leu Gly Arg Lys
Ile Asn Ser Trp Glu Ser Ser Arg 195 200 205Ser Gly His Ser Phe Leu
Ser Asn Leu His Leu Arg Asn Gly Glu Leu 210 215 220Val Ile His Glu
Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe225 230 235 240Arg
Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met 245 250
255Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu
260 265 270Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu
Tyr Gly 275 280 285Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu
Lys Glu Asn Asp 290 295 300Arg Ile Phe Val Ser Val Thr Asn Glu His
Leu Ile Asp Met Asp His305 310 315 320Glu Ala Ser Phe Phe Gly Ala
Phe Leu Val Gly Gly Ser Gly Ser Gly 325 330 335Asn Gly Ser Arg Val
Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser 340 345 350Asn Thr Leu
Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg 355 360 365Lys
Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser 370 375
380Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly
Phe385 390 395 400Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln
Glu Glu Ile Lys 405 410 415Glu Asn Thr Lys Asn Asp Lys Gln Met Val
Gln Tyr Ile Tyr Lys Tyr 420 425 430Thr Ser Tyr Pro Asp Pro Ile Leu
Leu Met Lys Ser Ala Arg Asn Ser 435 440 445Cys Trp Ser Lys Asp Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly 450 455 460Gly Ile Phe Glu
Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr465 470 475 480Asn
Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala 485 490
495Phe Leu Val Gly Gly Pro Gly Ser Ser Ser Ser Ser Ser Ser Gly Ser
500 505 510Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu 515 520 525Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 530 535 540Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser545 550 555 560His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 565 570 575Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Ser Ser Thr 580 585 590Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 595 600 605Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 610 615
620Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln625 630 635 640Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 645 650 655Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 660 665 670Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro 675 680 685Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 690 695 700Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val705 710 715 720Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 725 730
735Ser Pro Gly Lys 74020754PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 20Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg
Ser Asn Thr1 5 10 15Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu
Gly Arg Lys Ile 20 25 30Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser
Phe Leu Ser Asn Leu 35 40 45His Leu Arg Asn Gly Glu Leu Val Ile His
Glu Lys Gly Phe Tyr Tyr 50 55 60Ile Tyr Ser Gln Thr Tyr Phe Arg Phe
Gln Glu Glu Ile Lys Glu Asn65 70 75 80Thr Lys Asn Asp Lys Gln Met
Val Gln Tyr Ile Tyr Lys Tyr Thr Ser 85 90 95Tyr Pro Asp Pro Ile Leu
Leu Met Lys Ser Ala Arg Asn Ser Cys Trp 100 105 110Ser Lys Asp Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile 115 120 125Phe Glu
Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu 130 135
140His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe
Leu145 150 155 160Val Gly Gly Ser Gly Ser Gly Asn Gly Ser Arg Val
Ala Ala His Ile 165 170 175Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu
Ser Ser Pro Asn Ser Lys 180 185 190Asn Glu Lys Ala Leu Gly Arg Lys
Ile Asn Ser Trp Glu Ser Ser Arg 195 200 205Ser Gly His Ser Phe Leu
Ser Asn Leu His Leu Arg Asn Gly Glu Leu 210 215 220Val Ile His Glu
Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe225 230 235 240Arg
Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met 245 250
255Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu
260 265 270Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu
Tyr Gly 275 280 285Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu
Lys Glu Asn Asp 290 295 300Arg Ile Phe Val Ser Val Thr Asn Glu His
Leu Ile Asp Met Asp His305 310 315 320Glu Ala Ser Phe Phe Gly Ala
Phe Leu Val Gly Gly Ser Gly Ser Gly 325 330 335Asn Gly Ser Arg Val
Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser 340 345 350Asn Thr Leu
Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg 355 360 365Lys
Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser 370 375
380Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly
Phe385 390 395 400Tyr Tyr Ile Tyr
Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys 405 410 415Glu Asn
Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr 420 425
430Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser
435 440 445Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr
Gln Gly 450 455 460Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe
Val Ser Val Thr465 470 475 480Asn Glu His Leu Ile Asp Met Asp His
Glu Ala Ser Phe Phe Gly Ala 485 490 495Phe Leu Val Gly Gly Pro Gly
Ser Ser Ser Ser Ser Ser Ser Gly Ser 500 505 510Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 515 520 525Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 530 535 540Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser545 550
555 560His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu 565 570 575Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Ser Ser Thr 580 585 590Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 595 600 605Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro 610 615 620Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln625 630 635 640Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 645 650 655Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 660 665
670Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
675 680 685Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr 690 695 700Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val705 710 715 720Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 725 730 735Ser Pro Gly Ser Ser Ser Ser
Ser Ser Ala Trp Ser His Pro Gln Phe 740 745 750Glu
Lys21739PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 21Gln Arg Val Ala Ala
His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr1 5 10 15Leu Ser Ser Pro
Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile 20 25 30Asn Ser Trp
Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu 35 40 45His Leu
Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr 50 55 60Ile
Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn65 70 75
80Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser
85 90 95Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys
Trp 100 105 110Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln
Gly Gly Ile 115 120 125Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val
Ser Val Thr Asn Glu 130 135 140His Leu Ile Asp Met Asp His Glu Ala
Ser Phe Phe Gly Ala Phe Leu145 150 155 160Val Gly Gly Ser Gly Ser
Gly Asn Gly Ser Arg Val Ala Ala His Ile 165 170 175Thr Gly Thr Arg
Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys 180 185 190Asn Glu
Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg 195 200
205Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu
210 215 220Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr
Tyr Phe225 230 235 240Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys
Asn Asp Lys Gln Met 245 250 255Val Gln Tyr Ile Tyr Lys Tyr Thr Ser
Tyr Pro Asp Pro Ile Leu Leu 260 265 270Met Lys Ser Ala Arg Asn Ser
Cys Trp Ser Lys Asp Ala Glu Tyr Gly 275 280 285Leu Tyr Ser Ile Tyr
Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp 290 295 300Arg Ile Phe
Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His305 310 315
320Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly Ser Gly Ser Gly
325 330 335Asn Gly Ser Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly
Arg Ser 340 345 350Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys
Ala Leu Gly Arg 355 360 365Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser
Gly His Ser Phe Leu Ser 370 375 380Asn Leu His Leu Arg Asn Gly Glu
Leu Val Ile His Glu Lys Gly Phe385 390 395 400Tyr Tyr Ile Tyr Ser
Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys 405 410 415Glu Asn Thr
Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr 420 425 430Thr
Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser 435 440
445Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly
450 455 460Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser
Val Thr465 470 475 480Asn Glu His Leu Ile Asp Met Asp His Glu Ala
Ser Phe Phe Gly Ala 485 490 495Phe Leu Val Gly Gly Pro Gly Ser Ser
Ser Ser Ser Ser Ser Gly Ser 500 505 510Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala 515 520 525Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 530 535 540Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His545 550 555
560Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
565 570 575His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 580 585 590Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 595 600 605Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile 610 615 620Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val625 630 635 640Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 645 650 655Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 660 665 670Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 675 680
685Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
690 695 700Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met705 710 715 720His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 725 730 735Pro Gly Lys226PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 22Gly Ser Gly Ser Gly Ser1 5234PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 23Gly Ser Gly Ser1243PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 24Gly Ser Gly12521PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 25Gly Pro Gly Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys
Thr His1 5 10 15Thr Cys Pro Pro Cys 2026751PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 26Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg
Ser Asn Thr1 5 10 15Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu
Gly Arg Lys Ile 20 25 30Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser
Phe Leu Ser Asn Leu 35 40 45His Leu Arg Asn Gly Glu Leu Val Ile His
Glu Lys Gly Phe Tyr Tyr 50 55 60Ile Tyr Ser Gln Thr Tyr Phe Arg Phe
Gln Glu Glu Ile Lys Glu Asn65 70 75 80Thr Lys Asn Asp Lys Gln Met
Val Gln Tyr Ile Tyr Lys Tyr Thr Ser 85 90 95Tyr Pro Asp Pro Ile Leu
Leu Met Lys Ser Ala Arg Asn Ser Cys Trp 100 105 110Ser Lys Asp Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile 115 120 125Phe Glu
Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu 130 135
140His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe
Leu145 150 155 160Val Gly Gly Ser Gly Ser Gly Asn Gly Ser Arg Val
Ala Ala His Ile 165 170 175Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu
Ser Ser Pro Asn Ser Lys 180 185 190Asn Glu Lys Ala Leu Gly Arg Lys
Ile Asn Ser Trp Glu Ser Ser Arg 195 200 205Ser Gly His Ser Phe Leu
Ser Asn Leu His Leu Arg Asn Gly Glu Leu 210 215 220Val Ile His Glu
Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe225 230 235 240Arg
Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met 245 250
255Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu
260 265 270Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu
Tyr Gly 275 280 285Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu
Lys Glu Asn Asp 290 295 300Arg Ile Phe Val Ser Val Thr Asn Glu His
Leu Ile Asp Met Asp His305 310 315 320Glu Ala Ser Phe Phe Gly Ala
Phe Leu Val Gly Gly Ser Gly Ser Gly 325 330 335Asn Gly Ser Arg Val
Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser 340 345 350Asn Thr Leu
Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg 355 360 365Lys
Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser 370 375
380Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly
Phe385 390 395 400Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln
Glu Glu Ile Lys 405 410 415Glu Asn Thr Lys Asn Asp Lys Gln Met Val
Gln Tyr Ile Tyr Lys Tyr 420 425 430Thr Ser Tyr Pro Asp Pro Ile Leu
Leu Met Lys Ser Ala Arg Asn Ser 435 440 445Cys Trp Ser Lys Asp Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly 450 455 460Gly Ile Phe Glu
Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr465 470 475 480Asn
Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala 485 490
495Phe Leu Val Gly Gly Pro Gly Ser Ser Ser Ser Ser Ser Gly Ser Asp
500 505 510Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
Gly Pro 515 520 525Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser 530 535 540Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp545 550 555 560Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 565 570 575Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 580 585 590Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 595 600 605Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 610 615
620Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr625 630 635 640Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr 645 650 655Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 660 665 670Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu 675 680 685Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 690 695 700Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu705 710 715 720Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 725 730
735Ser Ser Ser Ser Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys 740
745 75027750PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 27Gln Arg Val Ala Ala
His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr1 5 10 15Leu Ser Ser Pro
Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile 20 25 30Asn Ser Trp
Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu 35 40 45His Leu
Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr 50 55 60Ile
Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn65 70 75
80Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser
85 90 95Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys
Trp 100 105 110Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln
Gly Gly Ile 115 120 125Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val
Ser Val Thr Asn Glu 130 135 140His Leu Ile Asp Met Asp His Glu Ala
Ser Phe Phe Gly Ala Phe Leu145 150 155 160Val Gly Gly Ser Gly Ser
Gly Asn Gly Ser Arg Val Ala Ala His Ile 165 170 175Thr Gly Thr Arg
Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys 180 185 190Asn Glu
Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg 195 200
205Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu
210 215 220Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr
Tyr Phe225 230 235 240Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys
Asn Asp Lys Gln Met 245 250 255Val Gln Tyr Ile Tyr Lys Tyr Thr Ser
Tyr Pro Asp Pro Ile Leu Leu 260 265 270Met Lys Ser Ala Arg Asn Ser
Cys Trp Ser Lys Asp Ala Glu Tyr Gly 275 280 285Leu Tyr Ser Ile Tyr
Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp 290 295 300Arg Ile Phe
Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His305 310 315
320Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly Ser Gly Ser Arg
325 330 335Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr
Leu Ser 340 345 350Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg
Lys Ile Asn Ser 355 360 365Trp Glu Ser Ser Arg Ser Gly His Ser Phe
Leu Ser Asn Leu His Leu 370 375 380Arg Asn Gly Glu Leu Val Ile His
Glu Lys Gly Phe Tyr Tyr Ile Tyr385 390 395 400Ser Gln Thr Tyr Phe
Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys 405 410 415Asn Asp Lys
Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro 420 425 430Asp
Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys 435 440
445Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu
450 455 460Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu
His Leu465 470 475 480Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly
Ala Phe Leu Val Gly 485 490 495Gly Pro Gly Ser Ser Ser Ser
Ser Ser Ser Gly Ser Cys Asp Lys Thr 500 505 510His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 515 520 525Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 530 535 540Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro545 550
555 560Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 565 570 575Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser Ser Thr Tyr
Arg Val Val 580 585 590Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr 595 600 605Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr 610 615 620Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu625 630 635 640Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 645 650 655Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 660 665
670Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
675 680 685Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser 690 695 700Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala705 710 715 720Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Ser 725 730 735Ser Ser Ser Ser Ser Ala Trp
Ser His Pro Gln Phe Glu Lys 740 745 75028750PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 28Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg
Ser Asn Thr1 5 10 15Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu
Gly Arg Lys Ile 20 25 30Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser
Phe Leu Ser Asn Leu 35 40 45His Leu Arg Asn Gly Glu Leu Val Ile His
Glu Lys Gly Phe Tyr Tyr 50 55 60Ile Tyr Ser Gln Thr Tyr Phe Arg Phe
Gln Glu Glu Ile Lys Glu Asn65 70 75 80Thr Lys Asn Asp Lys Gln Met
Val Gln Tyr Ile Tyr Lys Tyr Thr Ser 85 90 95Tyr Pro Asp Pro Ile Leu
Leu Met Lys Ser Ala Arg Asn Ser Cys Trp 100 105 110Ser Lys Asp Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile 115 120 125Phe Glu
Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu 130 135
140His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe
Leu145 150 155 160Val Gly Gly Ser Gly Ser Gly Ser Arg Val Ala Ala
His Ile Thr Gly 165 170 175Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser
Pro Asn Ser Lys Asn Glu 180 185 190Lys Ala Leu Gly Arg Lys Ile Asn
Ser Trp Glu Ser Ser Arg Ser Gly 195 200 205His Ser Phe Leu Ser Asn
Leu His Leu Arg Asn Gly Glu Leu Val Ile 210 215 220His Glu Lys Gly
Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe225 230 235 240Gln
Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 245 250
255Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys
260 265 270Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly
Leu Tyr 275 280 285Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu
Asn Asp Arg Ile 290 295 300Phe Val Ser Val Thr Asn Glu His Leu Ile
Asp Met Asp His Glu Ala305 310 315 320Ser Phe Phe Gly Ala Phe Leu
Val Gly Gly Ser Gly Ser Gly Ser Arg 325 330 335Val Ala Ala His Ile
Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser 340 345 350Ser Pro Asn
Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser 355 360 365Trp
Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu 370 375
380Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile
Tyr385 390 395 400Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys
Glu Asn Thr Lys 405 410 415Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr
Lys Tyr Thr Ser Tyr Pro 420 425 430Asp Pro Ile Leu Leu Met Lys Ser
Ala Arg Asn Ser Cys Trp Ser Lys 435 440 445Asp Ala Glu Tyr Gly Leu
Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu 450 455 460Leu Lys Glu Asn
Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu465 470 475 480Ile
Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 485 490
495Gly Pro Gly Ser Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr
500 505 510His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser 515 520 525Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 530 535 540Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro545 550 555 560Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 565 570 575Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Ser Ser Thr Tyr Arg Val Val 580 585 590Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 595 600 605Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 610 615
620Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu625 630 635 640Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 645 650 655Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 660 665 670Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp 675 680 685Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 690 695 700Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala705 710 715 720Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 725 730
735Ser Ser Ser Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys 740 745
75029736PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 29Gln Arg Val Ala Ala
His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr1 5 10 15Leu Ser Ser Pro
Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile 20 25 30Asn Ser Trp
Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu 35 40 45His Leu
Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr 50 55 60Ile
Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn65 70 75
80Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser
85 90 95Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys
Trp 100 105 110Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln
Gly Gly Ile 115 120 125Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val
Ser Val Thr Asn Glu 130 135 140His Leu Ile Asp Met Asp His Glu Ala
Ser Phe Phe Gly Ala Phe Leu145 150 155 160Val Gly Gly Ser Gly Ser
Gly Asn Gly Ser Arg Val Ala Ala His Ile 165 170 175Thr Gly Thr Arg
Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys 180 185 190Asn Glu
Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg 195 200
205Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu
210 215 220Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr
Tyr Phe225 230 235 240Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys
Asn Asp Lys Gln Met 245 250 255Val Gln Tyr Ile Tyr Lys Tyr Thr Ser
Tyr Pro Asp Pro Ile Leu Leu 260 265 270Met Lys Ser Ala Arg Asn Ser
Cys Trp Ser Lys Asp Ala Glu Tyr Gly 275 280 285Leu Tyr Ser Ile Tyr
Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp 290 295 300Arg Ile Phe
Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His305 310 315
320Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly Gly Ser Gly Ser Arg
325 330 335Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr
Leu Ser 340 345 350Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg
Lys Ile Asn Ser 355 360 365Trp Glu Ser Ser Arg Ser Gly His Ser Phe
Leu Ser Asn Leu His Leu 370 375 380Arg Asn Gly Glu Leu Val Ile His
Glu Lys Gly Phe Tyr Tyr Ile Tyr385 390 395 400Ser Gln Thr Tyr Phe
Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys 405 410 415Asn Asp Lys
Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro 420 425 430Asp
Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys 435 440
445Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu
450 455 460Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu
His Leu465 470 475 480Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly
Ala Phe Leu Val Gly 485 490 495Gly Pro Gly Ser Ser Ser Ser Ser Ser
Ser Gly Ser Cys Asp Lys Thr 500 505 510His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser 515 520 525Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 530 535 540Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro545 550 555
560Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
565 570 575Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser Ser Thr Tyr Arg
Val Val 580 585 590Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr 595 600 605Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr 610 615 620Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu625 630 635 640Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 645 650 655Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 660 665 670Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 675 680
685Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
690 695 700Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala705 710 715 720Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 725 730 73530736PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 30Gln Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg
Ser Asn Thr1 5 10 15Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu
Gly Arg Lys Ile 20 25 30Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser
Phe Leu Ser Asn Leu 35 40 45His Leu Arg Asn Gly Glu Leu Val Ile His
Glu Lys Gly Phe Tyr Tyr 50 55 60Ile Tyr Ser Gln Thr Tyr Phe Arg Phe
Gln Glu Glu Ile Lys Glu Asn65 70 75 80Thr Lys Asn Asp Lys Gln Met
Val Gln Tyr Ile Tyr Lys Tyr Thr Ser 85 90 95Tyr Pro Asp Pro Ile Leu
Leu Met Lys Ser Ala Arg Asn Ser Cys Trp 100 105 110Ser Lys Asp Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile 115 120 125Phe Glu
Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu 130 135
140His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe
Leu145 150 155 160Val Gly Gly Ser Gly Ser Gly Ser Arg Val Ala Ala
His Ile Thr Gly 165 170 175Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser
Pro Asn Ser Lys Asn Glu 180 185 190Lys Ala Leu Gly Arg Lys Ile Asn
Ser Trp Glu Ser Ser Arg Ser Gly 195 200 205His Ser Phe Leu Ser Asn
Leu His Leu Arg Asn Gly Glu Leu Val Ile 210 215 220His Glu Lys Gly
Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe225 230 235 240Gln
Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 245 250
255Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys
260 265 270Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly
Leu Tyr 275 280 285Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu
Asn Asp Arg Ile 290 295 300Phe Val Ser Val Thr Asn Glu His Leu Ile
Asp Met Asp His Glu Ala305 310 315 320Ser Phe Phe Gly Ala Phe Leu
Val Gly Gly Ser Gly Ser Gly Ser Arg 325 330 335Val Ala Ala His Ile
Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser 340 345 350Ser Pro Asn
Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser 355 360 365Trp
Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu 370 375
380Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile
Tyr385 390 395 400Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys
Glu Asn Thr Lys 405 410 415Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr
Lys Tyr Thr Ser Tyr Pro 420 425 430Asp Pro Ile Leu Leu Met Lys Ser
Ala Arg Asn Ser Cys Trp Ser Lys 435 440 445Asp Ala Glu Tyr Gly Leu
Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu 450 455 460Leu Lys Glu Asn
Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu465 470 475 480Ile
Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 485 490
495Gly Pro Gly Ser Ser Ser Ser Ser Ser Ser Gly Ser Cys Asp Lys Thr
500 505 510His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser 515 520 525Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 530 535 540Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro545 550 555 560Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 565 570 575Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Ser Ser Thr Tyr Arg Val Val 580 585 590Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 595 600 605Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 610 615
620Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu625 630 635 640Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 645 650 655Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 660 665 670Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp 675 680
685Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
690 695 700Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala705 710 715 720Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 725 730 735314PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 31Asp Glu Val Asp1
* * * * *