U.S. patent application number 12/482151 was filed with the patent office on 2010-04-15 for thrombopoietic activity of tyrosyl-trna synthetase polypeptides.
This patent application is currently assigned to ATYR PHARMA, INC.. Invention is credited to Rajesh Belani, Alain Philippe Vasserot, Jeffry Dean Watkins, Wei Zhang.
Application Number | 20100092434 12/482151 |
Document ID | / |
Family ID | 41334588 |
Filed Date | 2010-04-15 |
United States Patent
Application |
20100092434 |
Kind Code |
A1 |
Belani; Rajesh ; et
al. |
April 15, 2010 |
THROMBOPOIETIC ACTIVITY OF TYROSYL-TRNA SYNTHETASE POLYPEPTIDES
Abstract
Thrombopoietic compositions are provided comprising tyrosyl tRNA
synthetase polypeptides, including truncations and/or variants
thereof. Also provided are methods of using such compositions in
the treatment of conditions that benefit from increased
thrombopoiesis, such as thrombocytopenia.
Inventors: |
Belani; Rajesh; (San Diego,
CA) ; Watkins; Jeffry Dean; (Encinitas, CA) ;
Zhang; Wei; (San Diego, CA) ; Vasserot; Alain
Philippe; (Carlsbad, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
ATYR PHARMA, INC.
San Diego
CA
|
Family ID: |
41334588 |
Appl. No.: |
12/482151 |
Filed: |
June 10, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61060747 |
Jun 11, 2008 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
424/94.5; 435/320.1; 435/325; 435/375; 435/7.4 |
Current CPC
Class: |
A61P 7/00 20180101; Y02A
50/30 20180101; C12N 9/96 20130101; Y02A 50/385 20180101; A61P
29/00 20180101; A61P 7/06 20180101; A61P 11/00 20180101; A61K 38/53
20130101; A61P 7/04 20180101; C12Y 601/01001 20130101; C12N 9/93
20130101 |
Class at
Publication: |
424/93.7 ;
424/94.5; 435/7.4; 435/320.1; 435/325; 435/375 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61K 38/45 20060101 A61K038/45; A61P 7/00 20060101
A61P007/00; G01N 33/573 20060101 G01N033/573; C12N 15/63 20060101
C12N015/63; C12N 5/00 20060101 C12N005/00; C12N 5/02 20060101
C12N005/02; A61P 29/00 20060101 A61P029/00 |
Claims
1. A method of increasing platelet count in a subject, comprising
administering to the subject a composition comprising a
thrombopoietically-effective concentration of a tyrosyl-tRNA
synthetase polypeptide, thereby increasing platelet count in the
subject.
2. A method of treating, or reducing the risk of developing,
thrombocytopenia in subject, comprising administering to the
subject a composition comprising a thrombopoietically-effective
concentration of a tyrosyl-tRNA synthetase polypeptide, thereby
treating or reducing the risk of developing thrombocytopenia in the
subject.
3. A method of stimulating thrombopoiesis in a subject, comprising
administering to the subject a composition comprising a
thrombopoietically-effective concentration of a tyrosyl-tRNA
synthetase polypeptide, thereby stimulating thrombopoiesis in the
subject.
4. A method of maintaining platelet count in a subject, comprising
administering to the subject a thrombopoietically-effective
concentration of a tyrosyl-tRNA synthetase polypeptide, thereby
maintaining platelet count in the subject.
5. A method of stimulating megakaryocyte proliferation, migration,
and/or differentiation in a subject, comprising administering to
the subject a thrombopoietically-effective concentration of a
tyrosyl-tRNA synthetase polypeptide, thereby stimulating
megakaryocyte proliferation and/or differentiation in the
subject.
6. A method of stimulating neutrophil proliferation in a subject,
comprising administering to the subject a
thrombopoietically-effective concentration of a tyrosyl-tRNA
synthetase polypeptide, thereby stimulating neutrophil
proliferation in the subject.
7. The method of claim 1, wherein the subject has, or is at risk
for having, a disease or condition associated with a decreased or
reduced platelet count.
8. The method of claim 1, wherein the subject has a platelet count
of about 150,000/mm.sup.3 or lower.
9. The method of claim 7, wherein the disease or condition
associated with a decreased or reduced platelet count is selected
from bleeding, epistaxis, hypersplenism, hypothermia, Epstein-Barr
virus infection, infectious mononucleosis, Wiskott-Aldrich
syndrome, maternal ingestion of thiazides, congenital
amegakaryocytic thrombocytopenia, thrombocytopenia absent radius
syndrome, Fanconi anemia, Bernard-Soulier syndrome, May-Hegglin
anomaly, Grey platelet syndrome, Alport syndrome, neonatal rubella,
aplastic anemia, myeolodysplastic syndrome, leukemia, lymphoma,
tumor, cancer of the bone marrow, nutritional deficiency, radiation
exposure, liver failure, bacterial sepsis, measles, dengue fever,
HIV infection or AIDS, prematurity, erythroblastosis fetalis,
idiopathic thrombocytopenic purpura (ITP), maternal ITP,
hemolytic-uremic syndrome, disseminated intravascular coagulation,
thrombotic thrombocytopenic purpura (TTP), post-transfusion
purpura, systemic lupus erythrematosus, rheumatoid arthritis,
neonatal alloimmune thrombocytopenia, and paroxysmal nocturnal
hemoglobinuria, hepatitis C virus infection (HCV), medication
induced thrombocytopenia, and chemotherapy induced thrombocytosis
(CIT).
10. The method of claim 7, wherein the disease or condition
associated with a decreased or reduced platelet count is induced by
a medication or drug.
11. The method of claim 10, wherein the medication or drug is
selected from chemotherapeutic agents, nonsteroidal
anti-inflammatory agents, sulfonamides, vancomycin, clopidogrel,
glycoprotein IIb/IIIa inhibitors, interferons, valproic acid,
abciximab, linezolid, famotidine, mebeverine, histamine blockers,
alkylating agents, heparin, alcohol, and antibiotic
chemotherapeutic agents.
12. The method of claim 11, wherein the chemotherapeutic agent is
selected from cisplatin (CDDP), carboplatine, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding
agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase
inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine
and methotrexate, temazolomide, and derivatives thereof.
13. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises a mammalian tyrosyl-tRNA synthetase truncated
at its C-terminus.
14. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, 10, 12, or 14, wherein at least about 1-50 amino acid
residues are truncated from its C-terminus.
15. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, 10, 12, or 14, wherein at least about 50-100 amino acid
residues are truncated from its C-terminus.
16. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, 10, 12, or 14, wherein at least about 100-150 amino acid
residues are truncated from its C-terminus.
17. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, or 10, wherein at least about 150-200 residues are
truncated from its C-terminus.
18. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, or 10, wherein at least about 200-250 amino acid residues
are truncated from its C-terminus.
19. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises a mammalian tyrosyl-tRNA synthetase truncated
at its N-terminus.
20. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, 10, 12, or 14, wherein at least about 1-50 amino acid
residues are truncated from its N-terminus.
21. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, 10, 12, or 14, wherein at least about 50-100 amino acid
residues are truncated from its N-terminus.
22. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, 10, 12, or 14, wherein at least about 100-150 amino acid
residues are truncated from its N-terminus.
23. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, or 10, wherein at least about 150-200 residues are
truncated from its N-terminus.
24. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, or 10, wherein at least about 200-250 amino acid residues
are truncated from its N-terminus.
25. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide is selected from: (a) a polypeptide comprising an amino
acid sequence at least 80% identical to the amino acid sequence set
forth in SEQ ID NO:2, wherein the alanine at position 341 is not
substituted with a tyrosine; (b) a polypeptide comprising an amino
acid sequence at least 90% identical to the amino acid sequence set
forth in SEQ ID NO:2, wherein the alanine at position 341 is not
substituted with a tyrosine; (c) a polypeptide comprising an amino
acid sequence at least 95% identical to the amino acid sequence set
forth in SEQ ID NO:2, wherein the alanine at position 341 is not
substituted with a tyrosine; (d) a polypeptide comprising an amino
acid sequence at least 98% identical to the amino acid sequence set
forth in SEQ ID NO:2, wherein the alanine at position 341 is not
substituted with a tyrosine; and (e) a polypeptide comprising the
amino acid sequence set forth in SEQ ID NO:2.
26. The method of claim 1, wherein the tyrosyl-tRNA synthetase
polypeptide is selected from: (a) a polypeptide comprising an amino
acid sequence at least 80% identical to the amino acid sequence set
forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14; (b) a polypeptide
comprising an amino acid sequence at least 90% identical to the
amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12,
or 14; (c) a polypeptide comprising an amino acid sequence at least
95% identical to the amino acid sequence set forth in SEQ ID NO: 1,
2, 3, 6, 8, 10, 12, or 14; (d) a polypeptide comprising an amino
acid sequence at least 98% identical to the amino acid sequence set
forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14; and (e) a
polypeptide comprising the amino acid sequence set forth in SEQ ID
NO: 1, 2, 3, 6, 8, 10, 12, or 14.
27. A composition adapted for administration, comprising a
physiologically acceptable excipient and/or carrier and a
thrombopoietically-effective concentration of a tyrosyl-tRNA
synthetase polypeptide, wherein the composition is capable of
stimulating thrombopoiesis and/or increasing the platelet count in
a subject.
28. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises a mammalian tyrosyl-tRNA
synthetase truncated at its C-terminus.
29. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, 10, 12, or 14, wherein at least about 1-50 amino
acid residues are truncated from its C-terminus.
30. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, 10, 12, or 14, wherein at least about 50-100
amino acid residues are truncated from its C-terminus.
31. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, 10, 12, or 14, wherein at least about 100-150
amino acid residues are truncated from its C-terminus.
32. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, or 10, wherein at least about 150-200 residues
are truncated from its C-terminus.
33. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, or 10, wherein at least about 200-250 amino acid
residues are truncated from its C-terminus.
34. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises a mammalian tyrosyl-tRNA
synthetase truncated at its N-terminus.
35. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, 10, 12, or 14, wherein at least about 1-50 amino
acid residues are truncated from its N-terminus.
36. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, 10, 12, or 14, wherein at least about 50-100
amino acid residues are truncated from its N-terminus.
37. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, 10, 12, or 14, wherein at least about 100-150
amino acid residues are truncated from its N-terminus.
38. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, or 10, wherein at least about 150-200 residues
are truncated from its N-terminus.
39. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, or 10, wherein at least about 200-250 amino acid
residues are truncated from its N-terminus.
40. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide is selected from: (a) a polypeptide
comprising an amino acid sequence at least 80% identical to the
amino acid sequence set forth in SEQ ID NO:2, wherein the alanine
at position 341 is not substituted with a tyrosine; (b) a
polypeptide comprising an amino acid sequence at least 90%
identical to the amino acid sequence set forth in SEQ ID NO:2,
wherein the alanine at position 341 is not substituted with a
tyrosine; (c) a polypeptide comprising an amino acid sequence at
least 95% identical to the amino acid sequence set forth in SEQ ID
NO:2, wherein the alanine at position 341 is not substituted with a
tyrosine; (d) a polypeptide comprising an amino acid sequence at
least 98% identical to the amino acid sequence set forth in SEQ ID
NO:2, wherein the alanine at position 341 is not substituted with a
tyrosine; and (e) a polypeptide comprising the amino acid sequence
set forth in SEQ ID NO:2.
41. The composition of claim 27, wherein the tyrosyl-tRNA
synthetase polypeptide is selected from: (a) a polypeptide
comprising an amino acid sequence at least 80% identical to the
amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12,
or 14; (b) a polypeptide comprising an amino acid sequence at least
90% identical to the amino acid sequence set forth in SEQ ID NO: 1,
2, 3, 6, 8, 10, 12, or 14; (c) a polypeptide comprising an amino
acid sequence at least 95% identical to the amino acid sequence set
forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14; (d) a polypeptide
comprising an amino acid sequence at least 98% identical to the
amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12,
or 14; and (e) a polypeptide comprising the amino acid sequence set
forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14.
42. The composition of claim 27, further comprising a second
tyrosyl-tRNA synthetase polypeptide, wherein the two tyrosyl-tRNA
synthetase polypeptides form a dimer.
43. The composition of claim 42, wherein the dimer is a
homodimer.
44. The composition of claim 43, wherein the dimer is a
heterodimer.
45. The composition of claim 44, wherein the heterodimer comprises
a full-length tyrosyl-tRNA synthetase polypeptide and a truncated
tyrosyl-tRNA synthetase polypeptide.
46. The composition of claim 27, further comprising a heterologous
polypeptide, wherein the tyrosyl-tRNA synthetase polypeptide and
the heterologous polypeptide form a heterodimer.
47. A composition comprising a physiologically acceptable excipient
and/or carrier and a thrombopoietically-effective concentration of
a chimeric tyrosyl-tRNA synthetase polypeptide, wherein the
chimeric polypeptide comprises two or more biologically active
fragments of a tyrosyl-tRNA synthetase polypeptide, wherein the two
or more fragments comprise at least 10 contiguous amino acids of a
polypeptide according to any one of claims 27-41, wherein the two
or more fragments are linked to form a chimeric polypeptide, and
wherein the chimeric tyrosyl-tRNA synthetase polypeptide is capable
of stimulating thrombopoiesis and/or increasing the platelet count
in a subject.
48. A composition comprising a physiologically acceptable excipient
and/or carrier and a thrombopoietically-effective concentration of
a chimeric tyrosyl-tRNA synthetase polypeptide, wherein the
chimeric polypeptide comprises (a) one or more biologically active
fragments of a tyrosyl-tRNA synthetase polypeptide, wherein the one
or more fragments comprise at least 10 contiguous amino acids of a
polypeptide according to any one of claims 27-41; and (b) one or
more heterologous polypeptides, wherein the one or more fragments
of (a) and the one or more heterologous polypeptides of (b) are
linked to form a chimeric polypeptide, and wherein the chimeric
polypeptide is capable of stimulating thrombopoiesis and/or
increasing the platelet count in a subject.
49. An antibody, or antigen-binding fragment, that specifically
binds to a tyrosyl tRNA synthetase polypeptide of any one of claims
27-48.
50. A method of identifying or characterizing a YRS polypeptide in
a sample, comprising: (a) obtaining a biological sample; (b)
contacting the biological sample with an antibody, or
antigen-binding fragment, according to claim 49; and (c) detecting
the presence or absence of specific binding by the antibody, or
antigen-binding fragment, to the biological sample, thereby
identifying or characterizing the YRS polypeptide in the
sample.
51. The method of claim 50, wherein the biological sample is
obtained from a subject.
52. A composition comprising an isolated polynucleotide, wherein
the polynucleotide is selected from: (a) a polynucleotide
comprising a nucleotide sequence at least 80% identical to the
nucleotide sequence set forth in SEQ ID NO: 4, 7, 9, 11, 13, or 15;
(b) a polynucleotide comprising a nucleotide sequence at least 90%
identical to the nucleotide sequence set forth in SEQ ID NO: 4, 7,
9, 11, 13, or 15; (c) a polynucleotide comprising a nucleotide
sequence at least 95% identical to the nucleotide sequence set
forth in SEQ ID NO: 4, 7, 9, 11, 13, or 15; (d) a polynucleotide
comprising a nucleotide sequence at least 98% identical to the
nucleotide sequence set forth in SEQ ID NO: 4, 7, 9, 11, 13, or 15;
(e) a polynucleotide comprising the nucleotide sequence set forth
in SEQ ID NO: 4, 7, 9, 11, 13, or 15, wherein the polynucleotide
encodes a tyrosyl-tRNA synthetase polypeptide that is capable of
stimulating thrombopoiesis and/or increasing the platelet count in
a subject.
53. A vector comprising the polynucleotide of claim 52.
54. A host cell comprising the vector of claim 53.
55. A method of stimulating proliferation and/or differentiation of
early megakaryocyte progenitor cells, comprising incubating a
culture of hematopoietic stem cells with a tyrosyl-tRNA synthetase
polypeptide for a time sufficient to allow proliferation of the
early megakaryocyte progenitor cells, thereby stimulating
proliferation and/or differentiation of early megakaryocyte
progenitor cells.
56. The method of claim 55, wherein the method is performed ex vivo
or in vitro.
57. The method of claim 56, wherein the culture is obtained from
bone marrow.
58. The method of claim 56, wherein the culture is obtained from
cord blood.
59. The method of claim 56, further comprising administering the
cells to a subject in need thereof.
60. A method of stimulating migration of a CXCR-2 expressing cell,
comprising contacting the cell with a tyrosyl-tRNA synthetase
polypeptide, thereby stimulating migration of the CXCR-2 expressing
cell.
61. The method of claim 60, wherein the step of contacting the cell
occurs in vitro or ex vivo.
62. The method of claim 60, wherein the step of contacting
comprises administering to a subject in need thereof a composition
comprising an effective concentration of a tyrosyl-tRNA synthetase
polypeptide.
63. A method of reducing pulmonary inflammation, and/or its
symptoms, in a subject, comprising administering to the subject an
effective concentration of a tyrosyl-tRNA synthetase polypeptide,
thereby reducing pulmonary inflammation, and/or its symptoms, in
the subject.
64. The method of claim 63, wherein the subject has a chronic
obstructive pulmonary disease (COPD).
65. The method of claim 63, wherein the administration of the
tyrosyl-tRNA synthetase polypeptide is effective to achieve
desensitization of circulating neutrophils to an allergen.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application No. 61/060,747
filed Jun. 11, 2008, which is incorporated herein by reference in
its entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
120161.sub.--409_SEQ.txt. The text file is 37 KB, was created on
Jun. 10, 2009, and is being submitted electronically via
EFS-Web.
BACKGROUND
[0003] 1. Technical Field
[0004] The present invention relates generally to thrombopoietic
compositions comprising tyrosyl-tRNA synthetase polypeptides,
including truncations and/or variants thereof, and methods of using
such compositions in the treatment of diseases or conditions that
benefit from increased thrombopoiesis, such as diseases or
conditions associated with thrombocytopenia.
[0005] 2. Description of the Related Art
[0006] Thrombocytopenia relates generally to a condition in which
the number of platelets per unit volume of peripheral blood in a
subject is lower than normal. For example, normal platelet counts
generally range from about 150,000 mm.sup.3 to about 450,000
mm.sup.3, and thrombocytopenia is typically characterized by a
decrease in the platelet count to about 100,000/mm.sup.3 or
less.
[0007] Platelets, or thrombocytes, are colorless blood cells that
play an important role in blood clotting by clumping together and
forming plugs in blood vessel holes. Thrombopoiesis refers to the
process by which platelets are formed from precursor hematopoietic
cells, such as megakaryocytes. Thrombopoiesis is primarily
regulated by thrombopoietin, which is in turn regulated by a
variety of mechanisms, such as receptor-mediated uptake and
destruction in response to increased platelet levels, among other
factors.
[0008] Thrombocytopenia is associated with many underlying causes,
such as increased destruction of platelets, decreased production of
platelets, consumption of platelets, trapping of platelets, in
addition to medication-induced thrombocytopenia. Given the central
role of platelets in blood clotting, initial symptoms of
thrombocytopenia normally involve various forms of bleeding and
purpura. Since subjects are at increased risk for bleeding, early
diagnosis and treatment are important, especially for the
prevention of progress to more serious symptoms, such as cerebral
bleeding.
[0009] Treatment for conditions of reduced platelet count is often
guided by etiology and disease severity. Currently available
treatments for thrombocytopenia and related conditions include, for
example, corticosteroids, IVIG, splenectomy, and platelet
transfusion, which methods are either palliative and non-specific,
or drastic and expensive. In addition, previous efforts to utilize
thrombopoietin, the primary biological mediator of thrombopoiesis,
have failed in the clinic due to the serious effects observed in
patients who developed an immune response to the drug and,
consequently, to their own endogenous thrombopoietin.
Thrombopoietin mimetics and small molecule activators of the
thrombopoietin receptor are in development but have not been
approved by the Food and Drug Administration (FDA).
[0010] Aminoacyl-tRNA synthetases, which catalyze the
aminoacylation of tRNA molecules, are essential for decoding
genetic information during the process of translation. In higher
eukaryotes, aminoacyl-tRNA synthetases associate with other
polypeptides to form supramolecular multienzyme complexes. Each of
the eukaryotic tRNA synthetases consists of a core enzyme, which is
closely related to the prokaryotic counterpart of the tRNA
synthetase, and an additional domain that is appended to the
amino-terminal or carboxyl-terminal end of the core enzyme. Human
tyrosyl-tRNA synthetase (YRS), for example, has a carboxyl-terminal
domain that is not part of prokaryotic and lower eukaryotic YRS
molecules.
[0011] Aminoacyl tRNA synthetases, such as tyrosyl-tRNA synthetase,
are currently associated with expanded functions in mammalian
cells, including activities in signal transduction pathways, among
others.
BRIEF SUMMARY
[0012] The present invention stems from the unexpected finding that
compositions comprising tyrosyl-tRNA synthetase (YRS) polypeptides,
including truncated and/or variant polypeptides thereof, stimulate
thrombopoiesis in vivo (i.e., increased platelet formation).
Accordingly, embodiments of the present invention may be utilized
generally to treat and/or reduce the risk of developing diseases or
conditions associated with thrombocytopenia, or reduced platelet
levels.
[0013] Certain embodiments include methods of increasing the
platelet count in a subject, comprising administering to the
subject a composition comprising a thrombopoietically-effective
concentration of a tyrosyl-tRNA synthetase polypeptide, thereby
increasing the platelet count in the subject. Certain embodiments
include methods of treating, or reducing the risk of developing,
thrombocytopenia in subject, comprising administering to the
subject a composition comprising a thrombopoietically-effective
concentration of a tyrosyl-tRNA synthetase polypeptide, thereby
treating or reducing the risk of developing thrombocytopenia in the
subject. Certain embodiments contemplate methods of stimulating
thrombopoiesis in a subject, comprising administering to the
subject a composition comprising a thrombopoietically-effective
concentration of a tyrosyl-tRNA synthetase polypeptide, thereby
stimulating thrombopoiesis in the subject. Certain embodiments
include methods of maintaining platelet count in a subject (e.g., a
subject undergoing a therapy associated with reduced platelet
count), comprising administering to the subject a
thrombopoietically-effective concentration of a tyrosyl-tRNA
synthetase polypeptide, thereby maintaining platelet count in the
subject.
[0014] Certain embodiments encompass methods of stimulating
megakaryocyte migration, proliferation and/or differentiation in a
subject, comprising administering to the subject a
thrombopoietically-effective concentration of a tyrosyl-tRNA
synthetase polypeptide, thereby stimulating megakaryocyte
proliferation and/or differentiation in the subject. Certain
embodiments include methods of stimulating neutrophil migration or
proliferation in a subject, comprising administering to the subject
a thrombopoietically-effective concentration of a tyrosyl-tRNA
synthetase polypeptide, thereby stimulating neutrophil
proliferation in the subject.
[0015] In certain aspects, the subject has, or is at risk for
having, a disease or condition associated with a decreased or
reduced platelet count. In certain aspects, the subject has a
platelet count of about 100,000/mm.sup.3 or lower, about
110,000/mm.sup.3 or lower, about 120,000/mm.sup.3 or lower, about
130,000/mm.sup.3 or lower, about 140,000/mm.sup.3 or lower, or
about 150,000/mm.sup.3 or lower. In certain embodiments, the
disease or condition associated with a decreased or reduced
platelet count includes, but is not limited to, bleeding, bruising,
epistaxis (nose bleeds), hypersplenism, hypothermia, Epstein-Barr
virus infection, infectious mononucleosis, Wiskott-Aldrich
syndrome, maternal ingestion of thiazides, congenital
amegakaryocytic thrombocytopenia, thrombocytopenia absent radius
syndrome, Fanconi anemia, Bernard-Soulier syndrome, May-Hegglin
anomaly, Grey platelet syndrome, Alport syndrome, neonatal rubella,
aplastic anemia, myeolodysplastic syndrome, leukemia, lymphoma,
tumor, cancer of the bone marrow, nutritional deficiency, radiation
exposure, liver failure, bacterial sepsis, measles, dengue fever,
HIV infection or AIDS, prematurity, erythroblastosis fetalis,
idiopathic thrombocytopenic purpura (ITP), maternal ITP,
hemolytic-uremic syndrome, disseminated intravascular coagulation,
thrombotic thrombocytopenic purpura (TTP), post-transfusion
purpura, systemic lupus erythematosus, rheumatoid arthritis,
neonatal alloimmune thrombocytopenia, and paroxysmal nocturnal
hemoglobinuria, hepatitis C virus (HCV) infection, medication
induced thrombocytopenia, and chemotherapy induced thrombocytosis
(CIT), among others known in the art. In certain aspects, the
subject is a platelet donor.
[0016] In certain embodiments, the condition associated with a
decreased or reduced platelet count is induced by a medication or
drug (e.g., medication induced thrombocytopenia, chemotherapy
induced thrombocytosis). Examples of medications or drugs that
reduce platelet count may be selected from chemotherapeutic agents,
nonsteroidal anti-inflammatory agents, sulfonamides, vancomycin,
clopidogrel, glycoprotein IIb/IIIa inhibitors, interferons,
valproic acid, abciximab, linezolid, famotidine, mebeverine,
histamine blockers, alkylating agents, heparin, alcohol, and
antibiotics. In certain embodiments, the chemotherapeutic agents
may be selected from cisplatin (CDDP), carboplatine, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding
agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase
inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine
and methotrexate, Temazolomide (an aqueous form of DTIC), or any
analog or derivative variant of the foregoing.
[0017] In certain embodiments of the claimed methods, the
tyrosyl-tRNA synthetase polypeptide comprises a mammalian
tyrosyl-tRNA synthetase, including a mammalian tyrosyl-tRNA
synthetase truncated at its C-terminus. In certain of the methods
provided herein, the tyrosyl-tRNA synthetase polypeptide comprises
the amino acid sequence of SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14,
wherein about 1-50 amino acid residues are truncated from its
C-terminus. In certain of the methods provided herein, the
tyrosyl-tRNA synthetase polypeptide comprises the amino acid
sequence of SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14, wherein about
50-100 amino acid residues are truncated from its C-terminus. In
certain embodiments, the tyrosyl-tRNA synthetase polypeptide
comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 6, 8, 10,
12, or 14, wherein about 100-150 amino acid residues are truncated
from its C-terminus. In other embodiments, the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, or 10, wherein about 150-200 residues are
truncated from its C-terminus. In other embodiments, the methods
provided herein encompass wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, or 10, wherein about 200-250 amino acid residues are
truncated from its C-terminus. Particular examples of C-terminally
truncated tyrosyl-tRNA synthetase polypeptides include polypeptides
comprising or consisting of amino acids 1-343, amino acids 1-344,
amino acids 1-350, amino acids 1-353, or amino acids 1-364 of the
amino acid sequence set forth in SEQ ID NOS:1, 2, or 3. Additional
examples of C-terminally truncated tyrosyl-tRNA synthetase
polypeptides include the polypeptides of SEQ ID NOS:3 and 8.
[0018] In certain embodiments of the claimed methods, the
tyrosyl-tRNA synthetase polypeptide comprises a mammalian
tyrosyl-tRNA synthetase truncated at its N-terminus. In certain of
the methods provided herein, the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, 10, 12, or 14, wherein about 1-50 amino acid residues are
truncated from its N-terminus. In certain of the methods provided
herein, the tyrosyl-tRNA synthetase polypeptide comprises the amino
acid sequence of SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14, wherein
about 50-100 amino acid residues are truncated from its N-terminus.
In certain embodiments, the tyrosyl-tRNA synthetase polypeptide
comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 6, 8, 10,
12, or 14, wherein about 100-150 amino acid residues are truncated
from its N-terminus. In other embodiments, the tyrosyl-tRNA
synthetase polypeptide comprises the amino acid sequence of SEQ ID
NO: 1, 2, 3, 6, 8, or 10, wherein about 150-200 residues are
truncated from its N-terminus. In other embodiments, the methods
provided herein encompass wherein the tyrosyl-tRNA synthetase
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,
3, 6, 8, or 10, wherein about 200-250 amino acid residues are
truncated from its N-terminus. Particular examples of N-terminally
truncated tyrosyl-tRNA synthetase polypeptides include the
polypeptides of SEQ ID NOS:6, 10, 12, and 14.
[0019] In certain of the methods described herein, the tyrosyl-tRNA
synthetase polypeptide is selected from: (a) a polypeptide
comprising an amino acid sequence at least 80% identical to the
amino acid sequence set forth in SEQ ID NO:2, wherein the alanine
at position 341 is not substituted with a tyrosine; (b) a
polypeptide comprising an amino acid sequence at least 90%
identical to the amino acid sequence set forth in SEQ ID NO:2,
wherein the alanine at position 341 is not substituted with a
tyrosine; (c) a polypeptide comprising an amino acid sequence at
least 95% identical to the amino acid sequence set forth in SEQ ID
NO:2, wherein the alanine at position 341 is not substituted with a
tyrosine; (d) a polypeptide comprising an amino acid sequence at
least 98% identical to the amino acid sequence set forth in SEQ ID
NO:2, wherein the alanine at position 341 is not substituted with a
tyrosine; and (e) a polypeptide comprising the amino acid sequence
set forth in SEQ ID NO:2.
[0020] In certain embodiments of the methods provided herein, the
tyrosyl-tRNA synthetase polypeptide is selected from: (a) a
polypeptide comprising an amino acid sequence at least 80%
identical to the amino acid sequence set forth in SEQ ID NO: 1, 2,
3, 6, 8, 10, 12, or 14; (b) a polypeptide comprising an amino acid
sequence at least 90% identical to the amino acid sequence set
forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14; (c) a polypeptide
comprising an amino acid sequence at least 95% identical to the
amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12,
or 14; (d) a polypeptide comprising an amino acid sequence at least
98% identical to the amino acid sequence set forth in SEQ ID NO: 1,
2, 3, 6, 8, 10, 12, or 14; and (e) a polypeptide comprising the
amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12,
or 14.
[0021] In addition to the methods described herein, certain
embodiments of the present invention encompass compositions adapted
for administration comprising a physiologically acceptable
excipient and/or carrier and a thrombopoietically-effective
concentration of a tyrosyl-tRNA synthetase polypeptide, as
described herein, wherein the composition is capable of stimulating
thrombopoiesis (i.e., increasing or maintaining the platelet count
in a subject), stimulating the proliferation and/or differentiation
of megakaryocytes, and/or stimulating the proliferation of
neutrophils in a subject. In certain compositions, the tyrosyl-tRNA
synthetase polypeptide comprises a mammalian tyrosyl-tRNA
synthetase truncated at its C-terminus, as described above and
elsewhere herein. In certain compositions, the tyrosyl-tRNA
synthetase polypeptide comprises a mammalian tyrosyl-tRNA
synthetase truncated at its N-terminus, as described above and
elsewhere herein.
[0022] In certain embodiments, the thrombopoietic compositions
described herein comprise a tyrosyl-tRNA synthetase polypeptide
selected from: (a) a polypeptide comprising an amino acid sequence
at least 80% identical to the amino acid sequence set forth in SEQ
ID NO:2, wherein the alanine at position 341 is not substituted
with a tyrosine; (b) a polypeptide comprising an amino acid
sequence at least 90% identical to the amino acid sequence set
forth in SEQ ID NO:2, wherein the alanine at position 341 is not
substituted with a tyrosine; (c) a polypeptide comprising an amino
acid sequence at least 95% identical to the amino acid sequence set
forth in SEQ ID NO:2, wherein the alanine at position 341 is not
substituted with a tyrosine; (d) a polypeptide comprising an amino
acid sequence at least 98% identical to the amino acid sequence set
forth in SEQ ID NO:2, wherein the alanine at position 341 is not
substituted with a tyrosine; and (e) a polypeptide comprising the
amino acid sequence set forth in SEQ ID NO:2.
[0023] In certain embodiments, the thrombopoietic compositions
described herein comprise a tyrosyl-tRNA synthetase polypeptide
selected from: (a) a polypeptide comprising an amino acid sequence
at least 80% identical to the amino acid sequence set forth in SEQ
ID NO: 1, 2, 3, 6, 8, 10, 12, or 14; (b) a polypeptide comprising
an amino acid sequence at least 90% identical to the amino acid
sequence set forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14; (c)
a polypeptide comprising an amino acid sequence at least 95%
identical to the amino acid sequence set forth in SEQ ID NO: 1, 2,
3, 6, 8, 10, 12, or 14; (d) a polypeptide comprising an amino acid
sequence at least 98% identical to the amino acid sequence set
forth in SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14; and (e) a
polypeptide comprising the amino acid sequence set forth in SEQ ID
NO: 1, 2, 3, 6, 8, 10, 12, or 14.
[0024] In certain embodiments, the compositions of the present
invention further comprise a second tyrosyl-tRNA synthetase
polypeptide, including wherein the two tyrosyl-tRNA synthetase
polypeptides form a dimer. In certain aspects, the dimer is a
homodimer. In other aspects, the dimer is a heterodimer, such as a
heterodimer between a full-length tyrosyl-tRNA synthetase
polypeptide and a truncated tyrosyl-tRNA synthetase polypeptide. In
certain embodiments, the compositions of the present invention
further comprise a heterologous polypeptide, wherein the
tyrosyl-tRNA synthetase polypeptide and the heterologous
polypeptide form a heterodimer, such as a bi-functional
heterodimer.
[0025] In certain embodiments, the thrombopoietic compositions
provided herein comprise a physiologically acceptable excipient
and/or carrier and a thrombopoietically-effective concentration of
a chimeric tyrosyl-tRNA synthetase polypeptide, wherein the
chimeric polypeptide comprises two or more biologically active
fragments of a tyrosyl-tRNA synthetase polypeptide, wherein the two
or more fragments comprise at least 10 contiguous amino acids of a
YRS polypeptide, wherein the two or more fragments are linked to
form a chimeric polypeptide, and wherein the chimeric tyrosyl-tRNA
synthetase polypeptide is capable of stimulating thrombopoiesis
and/or increasing the platelet count in a subject.
[0026] In certain embodiments, the thrombopoietic compositions
provided herein comprise a physiologically acceptable excipient
and/or carrier and a thrombopoietically-effective concentration of
a chimeric tyrosyl-tRNA synthetase polypeptide, wherein the
chimeric polypeptide comprises (a) one or more biologically active
fragments of a tyrosyl-tRNA synthetase polypeptide, wherein the one
or more fragments comprise at least 10 contiguous amino acids of a
YRS polypeptide; and (b) one or more heterologous polypeptides,
wherein the one or more fragments of (a) and the one or more
heterologous polypeptides of (b) are linked to form a chimeric
polypeptide, and wherein the chimeric polypeptide is capable of
stimulating thrombopoiesis (i.e., increasing or maintaining the
platelet count in a subject), stimulating the proliferation and/or
differentiation of megakaryocytes, and/or stimulating the
proliferation of neutrophils in a subject.
[0027] Certain embodiments relate to methods of stimulating
proliferation and/or differentiation of early megakaryocyte
progenitor cells, comprising incubating a culture of hematopoietic
stem cells with a tyrosyl-tRNA synthetase polypeptide for a time
sufficient to allow proliferation of the early megakaryocyte
progenitor cells, thereby stimulating proliferation and/or
differentiation of early megakaryocyte progenitor cells. In certain
embodiments, the method is performed ex vivo or in vitro. In
certain embodiments, the culture is obtained from bone marrow. In
certain embodiments, the culture is obtained from cord blood. In
certain embodiments, such methods further comprise administering
the cells to a subject in need thereof.
[0028] Certain embodiments relate to methods of stimulating
migration of a CXCR-2 expressing cell, comprising contacting the
cell with a tyrosyl-tRNA synthetase polypeptide, thereby
stimulating migration of the CXCR-2 expressing cell. In certain
embodiments, the step of contacting the cell occurs in vitro or ex
vivo. In certain embodiments, the step of contacting comprises
administering to a subject in need thereof a composition comprising
an effective concentration of a tyrosyl-tRNA synthetase
polypeptide.
[0029] Certain embodiments relate to methods of reducing pulmonary
inflammation, and/or its symptoms, in a subject, comprising
administering to the subject an effective concentration of a
tyrosyl-tRNA synthetase polypeptide, thereby reducing pulmonary
inflammation, and/or its symptoms, in the subject. In certain
embodiments, the subject has a chronic obstructive pulmonary
disease (COPD). In certain embodiments, the administration of the
tyrosyl-tRNA synthetase polypeptide is effective to achieve
desensitization of circulating neutrophils to an allergen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows the full-length amino acid sequence of human
tyrosyl-tRNA synthetase (SEQ ID NO:1).
[0031] FIG. 2 shows the amino acid sequence of a Y341A variant of
full-length human tyrosyl-tRNA synthetase (SEQ ID NO:2).
[0032] FIG. 3 shows the amino acid sequence of a C-terminally
truncated (amino acids 1-364) human tyrosyl-tRNA synthetase having
thrombopoietic activity (SEQ ID NO:3).
[0033] FIG. 4 shows a polynucleotide sequence that encodes the
full-length amino acid sequence of human tyrosyl-tRNA synthetase
(SEQ ID NO:4).
[0034] FIGS. 5(a) and 5(b) show the in vivo effects on platelet
number following administration of a truncated human tyrosyl-tRNA
synthetase. For FIG. 5(a), mice were injected subcutaneously twice
daily for seven days with 1, 3 and 10 .mu.g/kg a C-terminally
truncated tyrosyl-tRNA synthetase polypeptide (SEQ ID NO:3) having
an eight amino acid C-terminal tag, L-E-H-H-H-H-H-H (SEQ ID NO:5),
and platelet count was determined at the end of the study. For FIG.
5(b), mice were injected subcutaneously twice daily for 7 days with
3 .mu.g/kg of the same C-terminally truncated polypeptide as in
FIG. 5(a), and platelet count was determined at the end of the
study.
[0035] FIG. 6 shows the in vivo effects on megakaryocyte number
following administration of a C-terminally truncated
human-tyrosyl-tRNA synthetase polypeptide (SEQ ID NO:3) having an
eight amino acid C-terminal tag, L-E-H-H-H-H-H-H (SEQ ID NO:5).
Animals were injected subcutaneously twice daily with 3 and 300
.mu.g/kg a tyrosyl-tRNA synthetase polypeptide of SEQ ID NO:3
having an eight amino acid C-terminal tag (SEQ ID NO:5) for 6 days
and bone marrow and spleen histology were examined at the end of
the study.
[0036] FIG. 7 shows the amino acid sequence of the SP1 human
tyrosyl-tRNA synthetase splice variant (SEQ ID NO:6), which
represents an N-terminally truncated variant of the full-length
wild-type YRS polypeptide sequence. The SP1 splice variant has 8 or
9 N-terminal amino acids that show no sequence similarity to the
wild-type sequence. "X" represents any amino acid.
[0037] FIG. 8 shows the nucleic acid sequence (SEQ ID NO:7) that
encodes the SP1 human tyrosyl-tRNA synthetase polypeptide of SEQ ID
NO:6.
[0038] FIG. 9 shows the amino acid sequence of the SP2 human
tyrosyl-tRNA synthetase splice variant (SEQ ID NO:8), which
represents a C-terminally truncated variant of the full-length
wild-type YRS polypeptide sequence. The SP2 variant has about 35
C-terminal amino acids that show no sequence similarity to the
wild-type sequence. "X" represents any amino acid.
[0039] FIG. 10 shows the nucleic acid sequence (SEQ ID NO:9) that
encodes the SP2 human tyrosyl-tRNA synthetase polypeptide of SEQ ID
NO:8.
[0040] FIG. 11 shows the amino acid sequence of the SP3 human
tyrosyl-tRNA synthetase splice variant (SEQ ID NO:10), which
represents an N-terminally truncated variant of the full-length
wild-type YRS polypeptide sequence.
[0041] FIG. 12 shows the nucleic acid sequence (SEQ ID NO:11) that
encodes the SP3 human tyrosyl-tRNA synthetase polypeptide of SEQ ID
NO:10.
[0042] FIG. 13 shows the amino acid sequence of the SP4 human
tyrosyl-tRNA synthetase splice variant (SEQ ID NO:12), which
represents an N-terminally truncated variant of the full-length
wild-type YRS polypeptide sequence.
[0043] FIG. 14 shows the nucleic acid sequence (SEQ ID NO:13) that
encodes the SP4 human tyrosyl-tRNA synthetase polypeptide of SEQ ID
NO:12.
[0044] FIG. 15 shows the amino acid sequence of the SP5 human
tyrosyl-tRNA synthetase splice variant (SEQ ID NO:14), which
represents an N-terminally truncated variant of the full-length
wild-type YRS polypeptide sequence. The SP5 variant has about 8
N-terminal amino acids that show no similarity to the wild-type
sequence. "X" represents any amino acid.
[0045] FIG. 16 shows the nucleic acid sequence (SEQ ID NO:15) that
encodes the SP5 human tyrosyl-tRNA synthetase polypeptide of SEQ ID
NO:14.
[0046] FIG. 17 illustrates the alternate gene splicing of wild-type
(WT) human tyrosyl-tRNA synthetase, as represented by the cDNA
sequence of alternate splice variants SP1 to SP5.
[0047] FIG. 18 provides the NCBI annotation of the cDNA sequences
for human tyrosyl-tRNA synthetase splice variants SP1 to SP5.
[0048] FIG. 19 depicts the protein sequence alignment of the
predicted and reported open reading frames for the SP1 to SP5 YRS
polypeptides as compared to the full-length human YRS
polypeptide.
[0049] FIG. 20 shows the thrombopoietic activity of YRS
polypeptides in rats (see Example 4).
[0050] FIG. 21 shows the migration of MO7e megakaryoblasts in
response to stimulation by YRS polypeptides (see Example 5).
[0051] FIG. 22 shows that tyrosyl-tRNA synthetase polypeptides
promote cell adhesion of THP-1 cells to endothelial monolayers of
HUVEC-2 cells (see Example 6).
[0052] FIG. 23 shows that tyrosyl-tRNA synthetase polypeptides
increase expression of adhesion molecule VCAM-1 in endothelial
monolayers of HUVEC-2 cells (see Example 6).
[0053] FIG. 24 shows that tyrosyl-tRNA synthetase polypeptides
stimulate migration of 293 and CHO cell lines transfected with the
CXCR-2 receptor (see Example 7). The left graph in FIG. 24 shows
the results for 293/CXCR-2 cells, and the right graph in FIG. 24
shows the results for CHO/CXCR-2 cells.
[0054] FIG. 25 shows the stimulatory effects of YRS polypeptides on
polymorphonuclear (PMN) cell migration (see Example 8).
[0055] FIG. 26 shows the effects of tyrosyl-tRNA synthetase
polypeptides on megakaryocyte progenitor cells in bone marrow cell
cultures, as measured by the number of colonies (see Example 10).
FIG. 26(A) shows the stimulatory effects of YRS polypeptides on
colony formation of primitive lineage-restricted progenitors, or
early progenitors, and FIGS. 26(B) and (C) show the inhibitory
effects of YRS polypeptides on relatively mature intermediate
progenitors (B) and late progenitors (C), respectively.
DETAILED DESCRIPTION
[0056] The present invention relates to the unexpected discovery
that tyrosyl-tRNA synthetase (YRS) polypeptides, including
truncations and variants thereof, are capable of mimicking and
stimulating the normal thrombopoiesis process, and, thus, possess
therapeutically beneficial thrombopoietic activity. Certain
embodiments of the present invention, therefore, relate to the use
of YRS polypeptides to stimulate the natural thrombopoiesis
process, and thereby increase platelet production in subjects in
need thereof, such as subjects suffering from a condition
associated with thrombocytopenia (i.e., reduced platelet count).
Advantages of the use of YRS polypeptides over other treatments
include, for example, a different mechanism of action than
traditional treatments, synergism with thrombopoietin signaling,
higher potency, and the benefits associated with using a
de-immunized molecule (e.g., no impact of potential adverse immune
response against thrombopoietin). Other advantages will be apparent
to a person skilled in the art.
[0057] The practice of the present invention will employ, unless
indicated specifically to the contrary, conventional methods of
molecular biology and recombinant DNA techniques within the skill
of the art, many of which are described below for the purpose of
illustration. Such techniques are explained fully in the
literature. See, e.g., Sambrook, et al., Molecular Cloning: A
Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular
Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical
Approach, vol. I & II (D. Glover, ed.); Oligonucleotide
Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B.
Hames & S. Higgins, eds., 1985); Transcription and Translation
(B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.
Freshney, ed., 1986); A Practical Guide to Molecular Cloning (B.
Perbal, ed., 1984).
[0058] All publications, patents and patent applications cited
herein are hereby incorporated by reference in their entirety.
DEFINITIONS
[0059] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which the invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, preferred methods and materials are described.
For the purposes of the present invention, the following terms are
defined below.
[0060] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0061] By "about" is meant a quantity, level, value, number,
frequency, percentage, dimension, size, amount, weight or length
that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3,
2 or 1% to a reference quantity, level, value, number, frequency,
percentage, dimension, size, amount, weight or length.
[0062] The term "biologically active fragment", as applied to
fragments of a reference polynucleotide or polypeptide sequence,
refers to a fragment that has at least about 0.1, 0.5, 1, 2, 5, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 110, 120, 150, 200,
300, 400, 500, 600, 700, 800, 900, 1000% or more of the activity of
a reference sequence. Included within the scope of the present
invention are biologically active fragments of at least about 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80,
90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320,
340, 360, 380, 400, or more contiguous nucleotides or amino acid
residues in length, including all integers in between, which
comprise or encode a thrombopoietic activity of a reference
polynucleotide or polypeptide, such as the reference polypeptide
sequences of SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12 and 14, or the
reference nucleotide sequences of SEQ ID NOS: 4, 7, 9, 11, 13, and
15. Particular examples of biologically active fragments include,
but are not limited to, C-terminally truncated tyrosyl-tRNA
synthetase polypeptides comprising or consisting of amino acids
1-343, amino acids 1-344, amino acids 1-350, amino acids 1-353, or
amino acids 1-364 of the amino acid sequence set forth in SEQ ID
NO:1, in addition to the polypeptides of SEQ ID NOS:3 and 6.
Additional examples of biologically active fragments include, but
are not limited to, N-terminally truncated tyrosyl-tRNA synthetase
polypeptides comprising or consisting of the amino acid sequences
set forth in SEQ ID NOS: 6, 10, 12, and 14. Representative
biologically active fragments generally participate in an
interaction, e.g., an intramolecular or an inter-molecular
interaction. An inter-molecular interaction can be a specific
binding interaction or an enzymatic interaction. An inter-molecular
interaction can be between a YRS polypeptide and target molecule,
such as a target molecule involved in regulating the process of
thrombopoiesis. Biologically active fragments of a YRS polypeptide
include polypeptide fragments comprising amino acid sequences with
sufficient similarity or identity to, or which are derived from,
the amino acid sequences of any of SEQ ID NOS: 1, 2, 3, 6, 8, 10,
12 or 14, including thrombopoietically effective portions thereof,
or are encoded by a nucleotide sequences of SEQ ID NOS: 4, 7, 9,
11, 13, and 15.
[0063] By "coding sequence" is meant any nucleic acid sequence that
contributes to the code for the polypeptide product of a gene. By
contrast, the term "non-coding sequence" refers to any nucleic acid
sequence that does not contribute to the code for the polypeptide
product of a gene.
[0064] Throughout this specification, unless the context requires
otherwise, the words "comprise", "comprises" and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements.
[0065] By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of." Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements may be present. By
"consisting essentially of" is meant including any elements listed
after the phrase, and limited to other elements that do not
interfere with or contribute to the activity or action specified in
the disclosure for the listed elements. Thus, the phrase
"consisting essentially of" indicates that the listed elements are
required or mandatory, but that other elements are optional and may
or may not be present depending upon whether or not they affect the
activity or action of the listed elements.
[0066] The terms "complementary" and "complementarity" refer to
polynucleotides (i.e., a sequence of nucleotides) related by the
base-pairing rules. For example, the sequence "A-G-T," is
complementary to the sequence "T-C-A." Complementarity may be
"partial," in which only some of the nucleic acids' bases are
matched according to the base pairing rules. Or, there may be
"complete" or "total" complementarity between the nucleic acids.
The degree of complementarity between nucleic acid strands has
significant effects on the efficiency and strength of hybridization
between nucleic acid strands.
[0067] By "corresponds to" or "corresponding to" is meant (a) a
polynucleotide having a nucleotide sequence that is substantially
identical or complementary to all or a portion of a reference
polynucleotide sequence or encoding an amino acid sequence
identical to an amino acid sequence in a peptide or protein; or (b)
a peptide or polypeptide having an amino acid sequence that is
substantially identical to a sequence of amino acids in a reference
peptide or protein.
[0068] By "derivative" is meant a polypeptide that has been derived
from the basic sequence by modification, for example by conjugation
or complexing with other chemical moieties (e.g., pegylation) or by
post-translational modification techniques as would be understood
in the art. The term "derivative" also includes within its scope
alterations that have been made to a parent sequence including
additions or deletions that provide for functionally equivalent
molecules.
[0069] As used herein, the terms "function" and "functional" and
the like refer to a biological, enzymatic, or therapeutic
function.
[0070] By "gene" is meant a unit of inheritance that occupies a
specific locus on a chromosome and consists of transcriptional
and/or translational regulatory sequences and/or a coding region
and/or non-translated sequences (i.e., introns, 5' and 3'
untranslated sequences).
[0071] "Homology" refers to the percentage number of amino acids
that are identical or constitute conservative substitutions.
Homology may be determined using sequence comparison programs such
as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395),
which is incorporated herein by reference. In this way sequences of
a similar or substantially different length to those cited herein
could be compared by insertion of gaps into the alignment, such
gaps being determined, for example, by the comparison algorithm
used by GAP.
[0072] The term "host cell" includes an individual cell or cell
culture which can be or has been a recipient of any recombinant
vector(s) or isolated polynucleotide of the invention. Host cells
include progeny of a single host cell, and the progeny may not
necessarily be completely identical (in morphology or in total DNA
complement) to the original parent cell due to natural, accidental,
or deliberate mutation and/or change. A host cell includes cells
transfected or infected in vivo or in vitro with a recombinant
vector or a polynucleotide of the invention. A host cell which
comprises a recombinant vector of the invention is a recombinant
host cell.
[0073] By "isolated" is meant material that is substantially or
essentially free from components that normally accompany it in its
native state. For example, an "isolated polynucleotide", as used
herein, refers to a polynucleotide, which has been purified from
the sequences which flank it in a naturally-occurring state, e.g.,
a DNA fragment which has been removed from the sequences that are
normally adjacent to the fragment. Alternatively, an "isolated
peptide" or an "isolated polypeptide" and the like, as used herein,
refer to in vitro isolation and/or purification of a peptide or
polypeptide molecule from its natural cellular environment, and
from association with other components of the cell; i.e., it is not
associated with in vivo substances.
[0074] By "obtained from" is meant that a sample such as, for
example, a polynucleotide extract or polypeptide extract is
isolated from, or derived from, a particular source of the subject.
For example, the extract can be obtained from a tissue or a
biological fluid isolated directly from the subject.
[0075] The term "oligonucleotide" as used herein refers to a
polymer composed of a multiplicity of nucleotide residues
(deoxyribonucleotides or ribonucleotides, or related structural
variants or synthetic analogues thereof) linked via phosphodiester
bonds (or related structural variants or synthetic analogues
thereof). Thus, while the term "oligonucleotide" typically refers
to a nucleotide polymer in which the nucleotide residues and
linkages between them are naturally occurring, it will be
understood that the term also includes within its scope various
analogues including, but not restricted to, peptide nucleic acids
(PNAs), phosphoramidates, phosphorothioates, methyl phosphonates,
2-O-methyl ribonucleic acids, and the like. The exact size of the
molecule can vary depending on the particular application. An
oligonucleotide is typically rather short in length, generally from
about 10 to 30 nucleotide residues, but the term can refer to
molecules of any length, although the term "polynucleotide" or
"nucleic acid" is typically used for large oligonucleotides.
[0076] The term "operably linked" as used herein means placing a
structural gene under the regulatory control of a promoter, which
then controls the transcription and optionally translation of the
gene. In the construction of heterologous promoter/structural gene
combinations, it is generally preferred to position the genetic
sequence or promoter at a distance from the gene transcription
start site that is approximately the same as the distance between
that genetic sequence or promoter and the gene it controls in its
natural setting; i.e., the gene from which the genetic sequence or
promoter is derived. As is known in the art, some variation in this
distance can be accommodated without loss of function. Similarly,
the preferred positioning of a regulatory sequence element with
respect to a heterologous gene to be placed under its control is
defined by the positioning of the element in its natural setting;
i.e., the genes from which it is derived.
[0077] The recitation "polynucleotide" or "nucleic acid" as used
herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically
refers to polymeric form of nucleotides of at least 10 bases in
length, either ribonucleotides or deoxynucleotides or a modified
form of either type of nucleotide. The term includes single and
double stranded forms of DNA.
[0078] The terms "polynucleotide variant" and "variant" and the
like refer to polynucleotides displaying substantial sequence
identity with a reference polynucleotide sequence or
polynucleotides that hybridize with a reference sequence under
stringent conditions that are defined hereinafter. These terms also
encompass polynucleotides that are distinguished from a reference
polynucleotide by the addition, deletion or substitution of at
least one nucleotide. Accordingly, the terms "polynucleotide
variant" and "variant" include polynucleotides in which one or more
nucleotides have been added or deleted, or replaced with different
nucleotides. In this regard, it is well understood in the art that
certain alterations inclusive of mutations, additions, deletions
and substitutions can be made to a reference polynucleotide whereby
the altered polynucleotide retains the biological function or
activity of the reference polynucleotide. Polynucleotide variants
include, for example, polynucleotides having at least 50% (and at
least 51% to at least 99% and all integer percentages in between)
sequence identity with the sequence set forth in SEQ ID NO:4, or
portions thereof that encode a biologically active fragment of a
thrombopoietic tyrosyl-tRNA synthetase polypeptide. The terms
"polynucleotide variant" and "variant" also include naturally
occurring allelic variants.
[0079] "Polypeptide," "polypeptide fragment," "peptide" and
"protein" are used interchangeably herein to refer to a polymer of
amino acid residues and to variants and synthetic analogues of the
same. Thus, these terms apply to amino acid polymers in which one
or more amino acid residues are synthetic non-naturally occurring
amino acids, such as a chemical analogue of a corresponding
naturally occurring amino acid, as well as to naturally-occurring
amino acid polymers.
[0080] The terms "tyrosine RNA synthetase" and "tyrosyl-tRNA
synthetase" are used interchangeably herein, and refer to a "YRS"
polypeptide of the invention.
[0081] The recitations "YRS polypeptides" "YRS polypeptide
fragments," "truncated YRS polypeptides" or "variants thereof"
encompass, without limitation, polypeptides having the amino acid
sequence that shares at least 50% (and at least 51% to at least 99%
and all integer percentages in between) sequence identity with a
reference sequence set forth in any one of SEQ ID NOS: 1, 2, 3, 6,
8, 10, 12, or 14, including biologically active fragments thereof,
such as fragments having at least 10, 20, 30, 40, 50, 60, 70, 80,
90, 100, 120, 140, 160, 180, 200, or more contiguous amino acids of
the reference sequences, including all integers in between. These
recitations further encompass natural allelic variation of YRS
polypeptides that may exist and occur from one genus or species to
another.
[0082] YRS polypeptides, including truncations and/or variants
thereof, encompass polypeptides that exhibit at least about 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%,
140%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%
or more of the specific biological activity of a reference YRS
polypeptide (i.e., such as having a thrombopoietic activity in a
subject or in vitro). For purposes of the present application,
YRS-related biological activity may be quantified, for example, by
measuring the ability of a YRS polypeptide to either increase the
platelet count in a subject, or to increase the megakaryocyte
number in a subject (see, e.g., Example 1). In addition, suitable
animal models for measuring human platelet production are described
in Suzuki et al., European Journal of Haemotology 78:123-130, 2007,
herein incorporated by reference. Suitable in vitro models for
measuring thrombopoietic activity are described in Example 2, and
further include assaying megakaryocyte colony formation, as
exemplified in Dessypris et al., Exp Hematol. 18:754-7, 1990. YRS
polypeptides, including truncations and/or variants thereof, having
substantially reduced biological activity relative to a wild-type
reference YRS polypeptide are those that exhibit less than about
25%, 10%, 5% or 1% of the specific activity of wild-type YRS.
[0083] The recitation polypeptide "variant" refers to polypeptides
that are distinguished from a reference polypeptide by the
addition, deletion or substitution of at least one amino acid
residue. In certain embodiments, a polypeptide variant is
distinguished from a reference polypeptide by one or more
substitutions, which may be conservative or non-conservative. In
certain embodiments, the polypeptide variant comprises conservative
substitutions and, in this regard, it is well understood in the art
that some amino acids may be changed to others with broadly similar
properties without changing the nature of the activity of the
polypeptide. Polypeptide variants also encompass polypeptides in
which one or more amino acids have been added or deleted, or
replaced with different amino acid residues.
[0084] The present invention contemplates the use in the methods
described herein of variants of full-length YRS polypeptides (e.g.,
a full-length polypeptide having a Y341A substitution), truncated
fragments of full-length YRS polypeptides, variants of truncated
fragments, as well as their related biologically active fragments.
Typically, biologically active fragments of a YRS polypeptide may
participate in an interaction, for example, an intra-molecular or
an inter-molecular interaction. An inter-molecular interaction can
be a specific binding interaction or an enzymatic interaction
(e.g., the interaction can be transient and a covalent bond is
formed or broken). Biologically active fragments of a YRS
polypeptide include peptides comprising amino acid sequences
sufficiently similar to, or derived from, the amino acid sequences
of a (putative) full-length YRS polypeptide sequence, such as SEQ
ID NO:1, or portions thereof, such as the polypeptides of SEQ ID
NOS: 3, 6, 8, 10, 12, and 14. Typically, biologically active
fragments comprise a domain or motif with at least one activity of
a YRS polypeptide and may include one or more (and in some cases
all) of the various active domains, and include fragments having a
thrombopoietic activity. In some cases, biologically active
fragments of a YRS polypeptide have a biological activity (e.g.,
thrombopoietic activity) that is unique to the particular,
truncated fragment, such that the full-length YRS polypeptide may
not have that activity. In certain cases, the biological activity
may be revealed by separating the biologically active YRS
polypeptide fragment from the other full-length YRS polypeptide
sequences, or by altering certain residues (e.g., Y341A) of the
full-length YRS wild-type polypeptide sequence to unmask the
thrombopoietically active domains. A biologically active fragment
of a truncated YRS polypeptide can be a polypeptide fragment which
is, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260,
280, 300, 320, 340, 360, 380, 400 or more contiguous amino acids,
including all integers in between, of the amino acid sequences set
forth in any one of SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, or 14. In
certain embodiments, a biologically active fragment comprises a
thrombopoiesis stimulating sequence, domain, or motif. Suitably,
the biologically-active fragment has no less than about 1%, 10%,
25%, 50% of an activity of the wild-type polypeptide from which it
is derived.
[0085] The recitations "sequence identity" or, for example,
comprising a "sequence 50% identical to," as used herein, refer to
the extent that sequences are identical on a
nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis
over a window of comparison. Thus, a "percentage of sequence
identity" may be calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical nucleic acid base (e.g., A, T, C,
G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,
Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu,
Asn, Gln, Cys and Met) occurs in both sequences to yield the number
of matched positions, dividing the number of matched positions by
the total number of positions in the window of comparison (i.e.,
the window size), and multiplying the result by 100 to yield the
percentage of sequence identity.
[0086] Terms used to describe sequence relationships between two or
more polynucleotides or polypeptides include "reference sequence",
"comparison window", "sequence identity", "percentage of sequence
identity" and "substantial identity". A "reference sequence" is at
least 12 but frequently 15 to 18 and often at least 25 monomer
units, inclusive of nucleotides and amino acid residues, in length.
Because two polynucleotides may each comprise (1) a sequence (i.e.,
only a portion of the complete polynucleotide sequence) that is
similar between the two polynucleotides, and (2) a sequence that is
divergent between the two polynucleotides, sequence comparisons
between two (or more) polynucleotides are typically performed by
comparing sequences of the two polynucleotides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window" refers to a conceptual segment of
at least 6 contiguous positions, usually about 50 to about 100,
more usually about 100 to about 150 in which a sequence is compared
to a reference sequence of the same number of contiguous positions
after the two sequences are optimally aligned. The comparison
window may comprise additions or deletions (i.e., gaps) of about
20% or less as compared to the reference sequence (which does not
comprise additions or deletions) for optimal alignment of the two
sequences. Optimal alignment of sequences for aligning a comparison
window may be conducted by computerized implementations of
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Drive Madison, Wis., USA) or by inspection and the best
alignment (i.e., resulting in the highest percentage homology over
the comparison window) generated by any of the various methods
selected. Reference also may be made to the BLAST family of
programs as for example disclosed by Altschul et al., 1997, Nucl.
Acids Res. 25:3389. A detailed discussion of sequence analysis can
be found in Unit 19.3 of Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter
15.
[0087] A "subject," as used herein, includes any animal that
exhibits a symptom, or is at risk for exhibiting a symptom, that
can be treated with a thrombopoietic YRS polypeptide of the
invention. Suitable subjects (patients) include laboratory animals
(such as mouse, rat, rabbit, or guinea pig), farm animals, and
domestic animals or pets (such as a cat or dog). Non-human primates
and, preferably, human patients, are included. Typical subjects
include animals that exhibit, or are at risk for exhibiting,
aberrant amounts of one or more physiological activities that can
be modulated by a thrombopoietic polypeptide, such as decreased or
reduced platelet counts (i.e., thrombocytopenia). Typically, a
subject having thrombocytopenia, or a "reduced" platelet count, as
used herein, refers to a subject having a decrease in the platelet
count to about 100,000/mm.sup.3 or lower, about 110,000/mm.sup.3 or
lower, about 120,000/mm.sup.3 or lower, about 130,000/mm.sup.3 or
lower, about 140,000/mm.sup.3 or lower, or about 150,000/mm.sup.3
or lower, as compared to a normal platelet count. As used herein, a
"normal" platelet count generally ranges from about
150,000/mm.sup.3 to about 450,000/mm.sup.3 in a subject. As one
example, a "subject" may also be about to undergo, is undergoing,
or has undergone, a transplant procedure, such as a stem cell or
bone marrow transplant. A subject may also have a pulmonary
disorder or disease, such as chronic obstructive pulmonary disease
(COPD), and/or be suffering from pulmonary inflammation.
[0088] "Thrombopoiesis," as used herein, refers to the formation of
blood platelets, or thrombocytes.
[0089] A "thrombopoietically-effective concentration" of a
tyrosyl-tRNA synthetase polypeptide, as described herein, refers to
an amount that is capable of "treating" a subject, such as by being
"effective" to stimulate or enhance thrombopoiesis, as typically
measured by increased platelet levels, maintained platelet levels,
increased megakaryocyte numbers, and/or increased neutrophil
production.
[0090] A "megakaryocyte" refers generally to a bone marrow cell
that is responsible for the production of blood thrombocytes (i.e.,
platelets), which are necessary for normal blood clotting.
Megakaryocytes typically account for 1 out of 10,000 bone marrow
cells. Megakaryocytes are derived from pluripotent hematopoietic
stem cell precursor cells in the bone marrow. Thrombopoietin (TPO)
is the primary signal for megakaryocyte production, i.e., TPO is
sufficient but not absolutely necessary for inducing
differentiation of progenitor cells in the bone marrow towards a
final megakaryocyte phenotype. Other molecular signals for
megakaryocyte differentiation include GM-CSF, IL-3, IL-6, IL-11,
chemokines (SDF-1; FGF-4), and erythropoietin.
[0091] Megakaryocytes are believed to develop through the following
lineage: CFU-Me (pluripotential hemopoietic stem cell or
hemocytoblast)->megakaryoblast->promegakaryocyte->megakaryocyte.
At the megakaryoblast stage, the cell loses its ability to divide,
but is still able to replicate its DNA and continue development,
becoming polyploid. Upon maturation, megakaryocytes begin the
process of producing platelets. Thrombopoietin plays a role in
inducing the megakaryocyte to form small proto-platelet processes,
or cytoplasmic internal membranes for storing platelets prior to
release. Upon release, each of these proto-platelet processes can
give rise to 2000-5000 new platelets. Overall, about 2/3 of the
newly-released platelets will remain in circulation and about 1/3
will be sequestered by the spleen. After releasing the platelets,
the remaining cell nucleus typically crosses the bone marrow
barrier to the blood and is consumed in the lung by alveolar
macrophages.
[0092] A "neutrophil," or neutrophil granulocyte, refers generally
to an abundant type of white blood cells in humans, which, together
with basophils and eosinophils, form part of the polymorphonuclear
cell family (PMNs). Neutrophils can be readily identified according
to their unique staining characteristics on hematoxylin and eosin
(H&E) histological or cytological preparations. Neutrophils are
normally found in the blood stream, but are one of the first group
of inflammatory cells to migrate toward inflammation sites during
the beginning (i.e., acute) phase of inflammation, mainly as a
result of infection or cancer. Typically, neutrophils first migrate
through the blood vessels, and then through interstitial tissues,
following chemical signals (e.g., interleukin-8 (IL-8),
interferon-gamma (IFN-gamma), and C5a) that originate at the site
of inflammation. "Neutropenia" refers to the presence of low
neutrophil counts, which can result from a congenital (genetic)
disorder, it can develop due to other conditions, as in the case of
aplastic anemia or some kinds of leukemia. Certain medications,
such as chemotherapeutics, may also cause neutropenia. Neutropenia
predisposes heavily for infection. Neutropenia can also result from
the colonization of intracellular neutrophilic parasites.
[0093] By "enhance" or "enhancing," or "increase" or "increasing,"
or "stimulate" or "stimulating," refers generally to the ability of
one or agents or compositions to produce or cause a greater
physiological response (i.e., downstream effects) in a cell, as
compared to the response caused by either no YRS polypeptide or a
control molecule/composition. A measurable physiological response
may include greater cell growth, expansion, or migration, among
others apparent from the understanding in the art and the
description herein. Among other methods known in the art, in vitro
colony formation assays represent one way to measure cellular
responses to agents provided herein. An "increased" or "enhanced"
amount is typically a "statistically significant" amount, and may
include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 30 or more times (e.g., 500, 1000 times) (including all
integers and decimal points in between and above 1), e.g., 1.5,
1.6, 1.7. 1.8, etc.) the amount produced by no YRS polypeptide (the
absence of an agent) or a control composition.
[0094] The term "reduce" may relate generally to the ability of one
or more YRS polypeptides of the invention to "decrease" a relevant
physiological or cellular response, such as a symptom of a disease
or condition (e.g., pulmonary inflammation, etc.), as measured
according to routine techniques in the diagnostic art. One specific
example of a relevant response includes the migration of immune
cells (e.g., neutrophils) to certain tissues, such as the lung.
Other relevant physiological or cellular responses (in vivo or in
vitro) will be apparent to persons skilled in the art. A "decrease"
in a response may be statistically significant as compared to the
response produced by no YRS polypeptide or a control
composition.
[0095] "Migration" refers to cellular migration, a process that can
be measured according to routine in vitro assays, as described
herein and known in the art (see, e.g., Example 8). Migration also
refers to in vivo migration, such as the migration of cells from
one tissue to another tissue (e.g., from bone marrow to peripheral
blood, or from peripheral blood to lung tissue), or from a site
within one tissue to another site within the same tissue. Migration
in vivo (e.g., chemotaxis) often occurs in a response to infection
or damaged/irritated tissue.
[0096] "Differentiation" refers to the process by which a less
specialized (e.g., pluripotent, totipotent, multipotent, etc.) cell
becomes a more specialized cell type.
[0097] "Desensitization" refers generally to the reduction or
elimination of an organism's negative (pathological) immune
reaction to a substance or stimulus, such as an allergen or
irritant, including foreign antigens as well as "self-antigens."
For instance, certain pulmonary diseases or conditions are
associated with a negative reaction to foreign irritants such as
smoke, such that desensitizing neutrophils to these irritants may
prevent (i.e., reduce the risk of developing) or reduce such
diseases or conditions, and/or their symptoms.
[0098] "Treatment" or "treating," as used herein, includes any
desirable effect on the symptoms or pathology of a disease or
condition associated with thrombocytopenia (i.e., reduced platelet
levels), or a risk of developing thrombocytopenia, and may include
even minimal changes or improvements in one or more measurable
markers of the disease or condition being treated. "Treatment" or
"treating" does not necessarily indicate complete eradication or
cure of the disease or condition, or associated symptoms thereof.
The subject receiving this treatment is any animal in need,
including primates, in particular humans, and other mammals such as
equines, cattle, swine and sheep; and poultry and pets in general.
Exemplary markers of clinical improvement include either increased
platelet counts, maintenance of normal platelet counts, and/or
increased megakaryocyte numbers, following administration of a
thrombopoietic YRS polypeptide, as described herein.
[0099] By "vector" is meant a polynucleotide molecule, preferably a
DNA molecule derived, for example, from a plasmid, bacteriophage,
yeast or virus, into which a polynucleotide can be inserted or
cloned. A vector preferably contains one or more unique restriction
sites and can be capable of autonomous replication in a defined
host cell including a target cell or tissue or a progenitor cell or
tissue thereof, or be integrable with the genome of the defined
host such that the cloned sequence is reproducible. Accordingly,
the vector can be an autonomously replicating vector, i.e., a
vector that exists as an extra-chromosomal entity, the replication
of which is independent of chromosomal replication, e.g., a linear
or closed circular plasmid, an extra-chromosomal element, a
mini-chromosome, or an artificial chromosome. The vector can
contain any means for assuring self-replication. Alternatively, the
vector can be one which, when introduced into the host cell, is
integrated into the genome and replicated together with the
chromosome(s) into which it has been integrated. A vector system
can comprise a single vector or plasmid, two or more vectors or
plasmids, which together contain the total DNA to be introduced
into the genome of the host cell, or a transposon. The choice of
the vector will typically depend on the compatibility of the vector
with the host cell into which the vector is to be introduced. In
the present case, the vector is preferably one which is operably
functional in a bacterial cell. The vector can also include a
selection marker such as an antibiotic resistance gene that can be
used for selection of suitable transformants.
[0100] The terms "wild-type" and "naturally occurring" are used
interchangeably to refer to a gene or gene product that has the
characteristics of that gene or gene product when isolated from a
naturally occurring source. A wild-type gene or gene product (e.g.,
a polypeptide) is that which is most frequently observed in a
population and is thus arbitrarily designed the "normal" or
"wild-type" form of the gene.
Thrombopoietic Tyrosyl-tRNA Polypeptides and Variants Thereof
[0101] The present invention relates in part to the unexpected
observation that certain tyrosyl-tRNA synthetase polypeptides,
including truncations and/or variants thereof, mimic and stimulate
the natural thrombopoietic process in vivo. Accordingly,
thrombopoietic polypeptides of the present invention include a
full-length tyrosyl-tRNA synthetase polypeptide, in addition to any
biologically active fragment, or variant or modification thereof,
of a tyrosyl-tRNA synthetase polypeptide, wherein the polypeptide
is capable of stimulating thrombopoiesis (i.e., platelet
formation), megakaryocyte proliferation and/or differentiation,
and/or neutrophil proliferation in a subject or in vitro.
[0102] Aminoacyl-tRNA synthetases, such as tyrosyl-tRNA synthetase,
typically catalyze the aminoacylation of tRNA by their cognate
amino acid. Because of their central role in linking amino acids
with nucleotide triplets contained in tRNAs, aminoacyl-tRNA
synthetases are thought to be among the first proteins that
appeared in evolution. Tyrosyl-tRNA synthetases in particular
belong to the class I tRNA synthetase family, which has two highly
conserved sequence motifs at the active site, HIGH and KMSKS. Class
I tRNA synthetases aminoacylate at the 2'-OH of an adenosine
nucleotide, and are usually monomeric or dimeric (one or two
subunits, respectively).
[0103] The human tyrosyl-tRNA synthetase is composed of three
domains: 1) an amino-terminal Rossmann fold domain that is
responsible for formation of the activated E.cndot.Tyr-AMP
intermediate and is conserved among bacteria, archeae, and
eukaryotes; 2) a tRNA anticodon recognition domain that has not
been conserved between bacteria and eukaryotes; and 3) a
carboxyl-terminal domain that is unique to the human tyrosyl-tRNA
synthetase, and whose primary structure is 49% identical to the
putative human cytokine endothelial monocyte-activating protein II,
50% identical to the carboxyl-terminal domain of methionyl-tRNA
synthetase from Caenorhabditis elegans, and 43% identical to the
carboxyl-terminal domain of Arc1p from Saccharomyces
cerevisiae.
[0104] The first two domains of the human tyrosyl-tRNA synthetase
are 52, 36, and 16% identical to tyrosyl-tRNA synthetases from S.
cerevisiae, Methanococcus jannaschii, and Bacillus
stearothermophilus, respectively. Nine of fifteen amino acids known
to be involved in the formation of the tyrosyl-adenylate complex in
B. stearothermophilus are conserved across all of the organisms,
whereas amino acids involved in the recognition of tRNA.sup.Tyr are
not conserved. Kinetic analyses of recombinant human and B.
stearothermophilus tyrosyl-tRNA synthetases expressed in
Escherichia coli indicate that human tyrosyl-tRNA synthetase
aminoacylates human but not B. stearothermophilus tRNA.sup.Tyr, and
vice versa. It is believed that the carboxyl-terminal domain of
human tyrosyl-tRNA synthetase evolved from gene duplication of the
carboxyl-terminal domain of methionyl-tRNA synthetase and may
direct tRNA to the active site of the enzyme.
[0105] Biological fragments of eukaryotic tyrosyl-tRNA synthetases
connect protein synthesis to cell-signaling pathways, such as
thrombopoiesis. These fragments may be produced naturally by either
alternative splicing or proteolysis. For example, as provided in
the present invention, the pro-thrombopoietic N-terminal fragment
mini-YRS is capable of stimulating thrombopoiesis in vivo. In
addition, certain mutations in the full-length YRS polypeptide
sequence confer increased thrombopoietic activity on the wild-type
reference sequence (e.g., Y341A). Examples of truncated splice
variants of the full-length YRS polypeptide sequence include the
SP1-SP5 polypeptides described in FIGS. 17-19.
[0106] The structure of human mini-YRS (i.e., SEQ ID NO:3; or
mini-Tyr), which contains both the catalytic and the anticodon
recognition domain, has been reported to a resolution of 1.18
.ANG.. Whereas the catalytic domains of the human and bacterial
enzymes superimpose, the spatial disposition of the anticodon
recognition domain relative to the catalytic domain is unique in
mini-YRS relative to the bacterial orthologs. Without wishing to be
bound by any one theory, the unique orientation of the
anticodon-recognition domain may explain why the fragment mini-YRS
is more active in various cell-signaling pathways.
[0107] Accordingly, embodiments of the present invention
contemplate the use of compositions comprising thrombopoietic YRS
polypeptides, including truncated, variant and/or modified
polypeptides thereof, for stimulating thrombopoiesis in a subject.
Variant proteins encompassed by the present application are
biologically active, that is, they continue to possess the
thrombopoietic activity of a reference YRS polypeptide sequence
(e.g., SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, and 14). Such variants
may result from, for example, genetic polymorphism or from human
manipulation. Biologically active variants of a reference YRS
polypeptide fragment will have at least 40%, 50%, 60%, 70%,
generally at least 75%, 80%, 85%, usually about 90% to 95% or more,
and typically about 98% or more sequence similarity or identity
with the amino acid sequence for a reference protein as determined
by sequence alignment programs described elsewhere herein using
default parameters. A biologically active variant of a reference
YRS polypeptide may differ from that protein generally by as much
200, 100, 50 or 20 amino acid residues or suitably by as few as
1-15 amino acid residues, as few as 1-10, such as 6-10, as few as
5, as few as 4, 3, 2, or even 1 amino acid residue. In some
embodiments, a YRS polypeptide differs from the reference sequences
in SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, and 14 by at least one but by
less than 15, 10 or 5 amino acid residues. In other embodiments, it
differs from the reference sequences in SEQ ID NOS: 1, 2, 3, 6, 8,
10, 12, and 14 by at least one residue but less than 20%, 15%, 10%
or 5% of the residues.
[0108] A YRS polypeptide may be altered in various ways including
amino acid substitutions, deletions, truncations, and insertions.
Methods for such manipulations are generally known in the art. For
example, amino acid sequence variants of a truncated and/or variant
YRS polypeptide can be prepared by mutations in the DNA. Methods
for mutagenesis and nucleotide sequence alterations are well known
in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci.
USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154:
367-382), U.S. Pat. No. 4,873,192, Watson, J. D. et al.,
("Molecular Biology of the Gene", Fourth Edition,
Benjamin/Cummings, Menlo Park, Calif., 1987) and the references
cited therein. Guidance as to appropriate amino acid substitutions
that do not affect biological activity of the protein of interest
may be found in the model of Dayhoff et al., (1978) Atlas of
Protein Sequence and Structure (Natl. Biomed. Res. Found.,
Washington, D.C.). Methods for screening gene products of
combinatorial libraries made by point mutations or truncation, and
for screening cDNA libraries for gene products having a selected
property are known in the art. Such methods are adaptable for rapid
screening of the gene libraries generated by combinatorial
mutagenesis of YRS polypeptides. Recursive ensemble mutagenesis
(REM), a technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify YRS polypeptide variants (Arkin and
Yourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave
et al., (1993) Protein Engineering, 6: 327-331). Conservative
substitutions, such as exchanging one amino acid with another
having similar properties, may be desirable as discussed in more
detail below.
[0109] Thrombopoietically active truncated and/or variant YRS
polypeptides may contain conservative amino acid substitutions at
various locations along their sequence, as compared to a reference
YRS amino acid sequence (e.g., SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12,
or 14). A "conservative amino acid substitution" is one in which
the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art, which can be
generally sub-classified as follows:
[0110] Acidic: The residue has a negative charge due to loss of H
ion at physiological pH and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH. Amino acids having an acidic side chain
include glutamic acid and aspartic acid.
[0111] Basic: The residue has a positive charge due to association
with H ion at physiological pH or within one or two pH units
thereof (e.g., histidine) and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH. Amino acids having a basic side chain
include arginine, lysine and histidine.
[0112] Charged: The residues are charged at physiological pH and,
therefore, include amino acids having acidic or basic side chains
(i.e., glutamic acid, aspartic acid, arginine, lysine and
histidine).
[0113] Hydrophobic: The residues are not charged at physiological
pH and the residue is repelled by aqueous solution so as to seek
the inner positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium. Amino acids having
a hydrophobic side chain include tyrosine, valine, isoleucine,
leucine, methionine, phenylalanine and tryptophan.
[0114] Neutral/polar: The residues are not charged at physiological
pH, but the residue is not sufficiently repelled by aqueous
solutions so that it would seek inner positions in the conformation
of a peptide in which it is contained when the peptide is in
aqueous medium. Amino acids having a neutral/polar side chain
include asparagine, glutamine, cysteine, histidine, serine and
threonine.
[0115] This description also characterizes certain amino acids as
"small" since their side chains are not sufficiently large, even if
polar groups are lacking, to confer hydrophobicity. With the
exception of proline, "small" amino acids are those with four
carbons or less when at least one polar group is on the side chain
and three carbons or less when not. Amino acids having a small side
chain include glycine, serine, alanine and threonine. The
gene-encoded secondary amino acid proline is a special case due to
its known effects on the secondary conformation of peptide chains.
The structure of proline differs from all the other
naturally-occurring amino acids in that its side chain is bonded to
the nitrogen of the .alpha.-amino group, as well as the
.alpha.-carbon. Several amino acid similarity matrices (e.g.,
PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff
et al., (1978), A model of evolutionary change in proteins.
Matrices for determining distance relationships In M. O. Dayhoff,
(ed.), Atlas of protein sequence and structure, Vol. 5, pp.
345-358, National Biomedical Research Foundation, Washington D.C.;
and by Gonnet et al., (Science, 256: 14430-1445, 1992), however,
include proline in the same group as glycine, serine, alanine and
threonine. Accordingly, for the purposes of the present invention,
proline is classified as a "small" amino acid.
[0116] The degree of attraction or repulsion required for
classification as polar or nonpolar is arbitrary and, therefore,
amino acids specifically contemplated by the invention have been
classified as one or the other. Most amino acids not specifically
named can be classified on the basis of known behaviour.
[0117] Amino acid residues can be further sub-classified as cyclic
or non-cyclic, and aromatic or non-aromatic, self-explanatory
classifications with respect to the side-chain substituent groups
of the residues, and as small or large. The residue is considered
small if it contains a total of four carbon atoms or less,
inclusive of the carboxyl carbon, provided an additional polar
substituent is present; three or less if not. Small residues are,
of course, always non-aromatic. Dependent on their structural
properties, amino acid residues may fall in two or more classes.
For the naturally-occurring protein amino acids, sub-classification
according to this scheme is presented in Table A.
TABLE-US-00001 TABLE A Amino acid sub-classification Sub-classes
Amino acids Acidic Aspartic acid, Glutamic acid Basic Noncyclic:
Arginine, Lysine; Cyclic: Histidine Charged Aspartic acid, Glutamic
acid, Arginine, Lysine, Histidine Small Glycine, Serine, Alanine,
Threonine, Proline Polar/neutral Asparagine, Histidine, Glutamine,
Cysteine, Serine, Threonine Polar/large Asparagine, Glutamine
Hydrophobic Tyrosine, Valine, Isoleucine, Leucine, Methionine,
Phenylalanine, Tryptophan Aromatic Tryptophan, Tyrosine,
Phenylalanine Residues that Glycine and Proline influence chain
orientation
[0118] Conservative amino acid substitution also includes groupings
based on side chains. For example, a group of amino acids having
aliphatic side chains is glycine, alanine, valine, leucine, and
isoleucine; a group of amino acids having aliphatic-hydroxyl side
chains is serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulphur-containing side chains is cysteine and
methionine. For example, it is reasonable to expect that
replacement of a leucine with an isoleucine or valine, an aspartate
with a glutamate, a threonine with a serine, or a similar
replacement of an amino acid with a structurally related amino acid
will not have a major effect on the properties of the resulting
variant polypeptide. Whether an amino acid change results in a
functional truncated and/or variant YRS polypeptide can readily be
determined by assaying its activity, as described herein (see,
e.g., Examples 1 and 2). Conservative substitutions are shown in
Table B under the heading of exemplary substitutions. Amino acid
substitutions falling within the scope of the invention, are, in
general, accomplished by selecting substitutions that do not differ
significantly in their effect on maintaining (a) the structure of
the peptide backbone in the area of the substitution, (b) the
charge or hydrophobicity of the molecule at the target site, or (c)
the bulk of the side chain. After the substitutions are introduced,
the variants are screened for biological activity.
TABLE-US-00002 TABLE B Exemplary Amino Acid Substitutions Original
Preferred Residue Exemplary Substitutions Substitutions Ala Val,
Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp
Glu Glu Cys Ser Ser Gln Asn, His, Lys, Asn Glu Asp, Lys Asp Gly Pro
Pro His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Leu
Norleu Leu Norleu, Ile, Val, Met, Ala, Phe Ile Lys Arg, Gln, Asn
Arg Met Leu, Ile, Phe Leu Phe Leu, Val, Ile, Ala Leu Pro Gly Gly
Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe, Thr, Ser Phe Val
Ile, Leu, Met, Phe, Ala, Leu Norleu
[0119] Alternatively, similar amino acids for making conservative
substitutions can be grouped into three categories based on the
identity of the side chains. The first group includes glutamic
acid, aspartic acid, arginine, lysine, histidine, which all have
charged side chains; the second group includes glycine, serine,
threonine, cysteine, tyrosine, glutamine, asparagine; and the third
group includes leucine, isoleucine, valine, alanine, proline,
phenylalanine, tryptophan, methionine, as described in Zubay, G.,
Biochemistry, third edition, Wm. C. Brown Publishers (1993).
[0120] Thus, a predicted non-essential amino acid residue in a
truncated and/or variant YRS polypeptide is typically replaced with
another amino acid residue from the same side chain family.
Alternatively, mutations can be introduced randomly along all or
part of a YRS coding sequence, such as by saturation mutagenesis,
and the resultant mutants can be screened for an activity of the
parent polypeptide to identify mutants which retain that activity.
Following mutagenesis of the coding sequences, the encoded peptide
can be expressed recombinantly and the activity of the peptide can
be determined. A "non-essential" amino acid residue is a residue
that can be altered from the wild-type sequence of an embodiment
polypeptide without abolishing or substantially altering one or
more of its activities. Suitably, the alteration does not
substantially abolish one of these activities, for example, the
activity is at least 20%, 40%, 60%, 70% or 80% 100%, 500%, 1000% or
more of wild-type. An "essential" amino acid residue is a residue
that, when altered from the wild-type sequence of a reference
truncated YRS polypeptide, results in abolition of an activity of
the parent molecule such that less than 20% of the wild-type
activity is present. For example, such essential amino acid
residues include those that are conserved in YRS polypeptides
across different species, including those sequences that are
conserved in the thrombopoiesis stimulating-binding site(s) or
motif(s) of YRS polypeptides from various sources.
[0121] Accordingly, the present invention also contemplates
variants of the naturally-occurring YRS polypeptide sequences or
their biologically-active fragments, wherein the variants are
distinguished from the naturally-occurring sequence by the
addition, deletion, or substitution of one or more amino acid
residues. In general, variants will display at least about 30, 40,
50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99% similarity or sequence identity to a reference YRS polypeptide
sequences, for example, as set forth in SEQ ID NOS: 1, 2, 3, 6, 8,
10, 12, and 14. Moreover, sequences differing from the native or
parent sequences by the addition, deletion, or substitution of 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
30, 40, 50, 60, 70, 80, 90, 100 or more amino acids but which
retain the properties of a parent or reference YRS polypeptide
sequence are contemplated. In certain embodiments, the C-terminal
or N-terminal region of any of SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12,
or 14 may be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more
amino acids, including all integers in between (e.g., 101, 102,
103, 104, 105), so long as the truncated YRS polypeptide is capable
of stimulating thrombopoiesis (i.e., platelet formation),
megakaryocyte proliferation and/or differentiation, and/or
neutrophil proliferation in a subject or in vitro.
[0122] In some embodiments, variant polypeptides differ from a
reference YRS sequence by at least one but by less than 50, 40, 30,
20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In other
embodiments, variant polypeptides differ from the corresponding
sequences of SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, or 14 by at least
1% but less than 20%, 15%, 10% or 5% of the residues. (If this
comparison requires alignment, the sequences should be aligned for
maximum similarity. "Looped" out sequences from deletions or
insertions, or mismatches, are considered differences.) The
differences are, suitably, differences or changes at a
non-essential residue or a conservative substitution.
[0123] In certain embodiments, a variant polypeptide includes an
amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more
sequence identity or similarity to a corresponding sequence of a
YRS polypeptide as, for example, set forth in SEQ ID NOS: 1, 2, 3,
6, 8, 10, 12, or 14, and has the ability to stimulate
thrombopoiesis in a subject, stimulate the proliferation and/or
differentiation of megakaryocytes in a subject, and/or stimulate
the proliferation of neutrophils in a subject. Examples of YRS
polypeptide variants include, but are not limited to, a full-length
YRS polypeptide, or a truncation or splice variant thereof, having
one or more amino acid substitutions selected from an R93Q
substitution, an I14L substitution, an N17G substitution, an L271
substitution, an A85S substitution, and a V156L substitution, in
addition to combinations thereof. Particular examples of YRS
polypeptide variants include, but are not limited to, a YRS
polypeptide having amino acids 1-364 of SEQ ID NO:1 with an R93Q
substitution, a YRS polypeptide having amino acids 1-353 of SEQ ID
NO:1 with an I14L substitution, a YRS polypeptide having amino
acids 1-353 of SEQ ID NO:1 with an N17G substitution, a YRS
polypeptide having amino acids 1-353 of SEQ ID NO:1 with an L27I
substitution, a YRS polypeptide having amino acids 1-353 of SEQ ID
NO:1 with an A85S substitution, and a YRS polypeptide having amino
acids 1-353 of SEQ ID NO:1 with a V156L substitution.
[0124] Calculations of sequence similarity or sequence identity
between sequences (the terms are used interchangeably herein) are
performed as follows. To determine the percent identity of two
amino acid sequences, or of two nucleic acid sequences, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in one or both of a first and a second amino acid
or nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In certain
embodiments, the length of a reference sequence aligned for
comparison purposes is at least 30%, preferably at least 40%, more
preferably at least 50%, 60%, and even more preferably at least
70%, 80%, 90%, 100% of the length of the reference sequence. The
amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position.
[0125] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences,
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences.
[0126] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm
which has been incorporated into the GAP program in the GCG
software package (available at http://www.gcg.com), using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of parameters (and the one that should be used unless otherwise
specified) are a Blossum 62 scoring matrix with a gap penalty of
12, a gap extend penalty of 4, and a frameshift gap penalty of
5.
[0127] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers and W.
Miller (1989, Cabios, 4: 11-17) which has been incorporated into
the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[0128] The nucleic acid and protein sequences described herein can
be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al., (1990, J. Mol.
Biol, 215: 403-10). BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to nucleic acid molecules of the invention.
BLAST protein searches can be performed with the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences homologous to
protein molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al., (1997, Nucleic Acids Res, 25: 3389-3402). When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs (e.g., XBLAST and NBLAST) can be
used.
[0129] Variants of a YRS polypeptide can be identified by screening
combinatorial libraries of mutants of a YRS polypeptide. Libraries
or fragments e.g., N terminal, C terminal, or internal fragments,
of YRS protein coding sequence can be used to generate a variegated
population of fragments for screening and subsequent selection of
variants of a YRS polypeptide.
[0130] Methods for screening gene products of combinatorial
libraries made by point mutation or truncation, and for screening
cDNA libraries for gene products having a selected property are
known in the art. Such methods are adaptable for rapid screening of
the gene libraries generated by combinatorial mutagenesis of YRS
polypeptides.
[0131] The present invention also contemplates the use of YRS
chimeric or fusion proteins for stimulating thrombopoiesis. As used
herein, a YRS "chimeric protein" or "fusion protein" includes a YRS
polypeptide or polypeptide fragment linked to either another
YRS-polypeptide (e.g., to create multiple fragments), to a non-YRS
polypeptide, or to both. A "non-YRS polypeptide" refers to a
"heterologous polypeptide" having an amino acid sequence
corresponding to a protein which is different from the YRS protein,
and which is derived from the same or a different organism. The YRS
polypeptide of the fusion protein can correspond to all or a
portion of a biologically active YRS amino acid sequence. In
certain embodiments, a YRS fusion protein includes at least one (or
two) biologically active portion of an YRS protein. The
polypeptides forming the fusion protein are typically linked
C-terminus to N-terminus, although they can also be linked
C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus
to C-terminus. The polypeptides of the fusion protein can be in any
order.
[0132] The fusion partner may be designed and included for
essentially any desired purpose provided they do not adversely
affect the thrombopoietic activity of the polypeptide. For example,
in one embodiment, a fusion partner may comprise a sequence that
assists in expressing the protein (an expression enhancer) at
higher yields than the native recombinant protein. Other fusion
partners may be selected so as to increase the solubility of the
protein or to enable the protein to be targeted to desired
intracellular compartments.
[0133] The fusion protein can include a moiety which has a high
affinity for a ligand. For example, the fusion protein can be a
GST-YRS fusion protein in which the YRS sequences are fused to the
C-terminus of the GST sequences. As another example, a YRS
polypeptide may be fused to an eight amino acid tag at the
C-terminus, such as an L-E-H-H-H-H-H-H (SEQ ID NO:5) tag. In
certain embodiments, amino acids 1-364 of a YRS polypeptide are
fused to a 365-L-E-H-H-H-H-H-H-372 (SEQ ID NO:5) tag at the
C-terminus. Such fusion proteins can facilitate the purification
and/or identification of a YRS polypeptide. Alternatively, the
fusion protein can be a YRS protein containing a heterologous
signal sequence at its N-terminus. In certain host cells,
expression and/or secretion of YRS proteins can be increased
through use of a heterologous signal sequence.
[0134] More generally, fusion to heterologous sequences, such as an
Fc fragment, may be utilized to remove unwanted characteristics or
to improve the desired characteristics (e.g., pharmacokinetic
properties) of a thrombopoietic YRS polypeptide. For example,
fusion to a heterologous sequence may increase chemical stability,
decrease immunogenicity, improve in vivo targeting, and/or increase
half-life in circulation of a thrombopoietic YRS polypeptide.
[0135] Fusion to heterologous sequences may also be used to create
bi-functional fusion proteins, such as bi-functional proteins that
are not only capable of stimulating thrombopoiesis, megakaryocyte
proliferation and/or differentiation, and/or neutrophil
proliferation through the YRS polypeptide, but are also capable of
modifying (i.e., stimulating or inhibiting) other pathways through
the heterologous polypeptide. Examples of such pathways include,
but are not limited to, various immune system-related pathways,
such as innate or adaptive immune activation pathways, or
cell-growth regulatory pathways, such as hematopoiesis and
angiogenesis. In certain aspects, the heterologous polypeptide may
act synergistically with the YRS polypeptide to stimulate
thrombopoietic-related and/or hematopoietic-related pathways in a
subject. Examples of heterologous polypeptides that may be utilized
to create a bi-functional fusion protein include, but are not
limited to, thrombopoietin, cytokines (e.g., IL-11), chemokines,
and various hematopoietic growth factors, in addition to
biologically active fragments and/or variants thereof.
[0136] Fusion proteins may generally be prepared using standard
techniques. For example, DNA sequences encoding the polypeptide
components of a desired fusion may be assembled separately, and
ligated into an appropriate expression vector. The 3' end of the
DNA sequence encoding one polypeptide component is ligated, with or
without a peptide linker, to the 5' end of a DNA sequence encoding
the second polypeptide component so that the reading frames of the
sequences are in phase. This permits translation into a single
fusion protein that retains the biological activity of both
component polypeptides.
[0137] A peptide linker sequence may be employed to separate the
first and second polypeptide components by a distance sufficient to
ensure that each polypeptide folds into its secondary and tertiary
structures, if desired. Such a peptide linker sequence is
incorporated into the fusion protein using standard techniques well
known in the art. Certain peptide linker sequences may be chosen
based on the following factors: (1) their ability to adopt a
flexible extended conformation; (2) their inability to adopt a
secondary structure that could interact with functional epitopes on
the first and second polypeptides; and (3) the lack of hydrophobic
or charged residues that might react with the polypeptide
functional epitopes. Preferred peptide linker sequences contain
Gly, Asn and Ser residues. Other near neutral amino acids, such as
Thr and Ala may also be used in the linker sequence. Amino acid
sequences which may be usefully employed as linkers include those
disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al.,
Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No.
4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may
generally be from 1 to about 50 amino acids in length. Linker
sequences are not required when the first and second polypeptides
have non-essential N-terminal amino acid regions that can be used
to separate the functional domains and prevent steric
interference.
[0138] The ligated DNA sequences may be operably linked to suitable
transcriptional or translational regulatory elements. The
regulatory elements responsible for expression of DNA are located
5' to the DNA sequence encoding the first polypeptides. Similarly,
stop codons required to end translation and transcription
termination signals are present 3' to the DNA sequence encoding the
second polypeptide.
[0139] In general, polypeptides and fusion polypeptides (as well as
their encoding polynucleotides) are isolated. An "isolated"
polypeptide or polynucleotide is one that is removed from its
original environment. For example, a naturally-occurring protein is
isolated if it is separated from some or all of the coexisting
materials in the natural system. Preferably, such polypeptides are
at least about 90% pure, more preferably at least about 95% pure
and most preferably at least about 99% pure. A polynucleotide is
considered to be isolated if, for example, it is cloned into a
vector that is not a part of the natural environment.
[0140] Certain embodiments also encompass dimers of YRS
polypeptides. Dimers may include, for example, homodimers between
two identical YRS polypeptides, heterodimers between two different
YRS polypeptides (e.g., a full-length YRS polypeptide and a
truncated YRS polypeptide), and/or heterodimers between a YRS
polypeptide and a heterologous polypeptide. Certain heterodimers,
such as those between a YRS polypeptide and a heterologous
polypeptide, may be bi-functional, as described herein.
[0141] Certain embodiments of the present invention also
contemplate the use of modified YRS polypeptides, including
modifications that improved desired characteristics of a YRS
polypeptide, as described herein. Modifications of YRS polypeptides
of the invention include chemical and/or enzymatic derivatizations
at one or more constituent amino acid, including side chain
modifications, backbone modifications, and N- and C-terminal
modifications including acetylation, hydroxylation, methylation,
amidation, and the attachment of carbohydrate or lipid moieties,
cofactors, and the like. Exemplary modifications also include
pegylation of a YRS-polypeptide (see, e.g., Veronese and Harris,
Advanced Drug Delivery Reviews 54: 453-456, 2002, herein
incorporated by reference).
[0142] In certain aspects, chemoselective ligation technology may
be utilized to modify truncated YRS polypeptides of the invention,
such as by attaching polymers in a site-specific and controlled
manner. Such technology typically relies on the incorporation of
chemoselective anchors into the protein backbone by either chemical
or recombinant means, and subsequent modification with a polymer
carrying a complementary linker. As a result, the assembly process
and the covalent structure of the resulting protein-polymer
conjugate may be controlled, enabling the rational optimization of
drug properties, such as efficacy and pharmacokinetic properties
(see, e.g., Kochendoerfer, Current Opinion in Chemical Biology
9:555-560, 2005).
[0143] The truncated and/or variant YRS polypeptides of the
invention may be prepared by any suitable procedure known to those
of skill in the art, such as by recombinant techniques. For
example, YRS polypeptides may be prepared by a procedure including
the steps of: (a) preparing a construct comprising a polynucleotide
sequence that encodes a truncated YRS polypeptide and that is
operably linked to a regulatory element; (b) introducing the
construct into a host cell; (c) culturing the host cell to express
the truncated YRS polypeptide; and (d) isolating the truncated
and/or variant YRS polypeptide from the host cell. In illustrative
examples, the nucleotide sequence encodes at least a biologically
active portion of a polypeptide sequence set forth in, or derived
from, SEQ ID NOS:1, 2, 3, 6, 8, 10, 12, or 14, or a biologically
active variant or fragment thereof. Recombinant YRS polypeptides
can be conveniently prepared using standard protocols as described
for example in Sambrook, et al., (1989, supra), in particular
Sections 16 and 17; Ausubel et al., (1994, supra), in particular
Chapters 10 and 16; and Coligan et al., Current Protocols in
Protein Science (John Wiley & Sons, Inc. 1995-1997), in
particular Chapters 1, 5 and 6.
[0144] In addition to recombinant production methods, polypeptides
of the invention, and fragments thereof, may be produced by direct
peptide synthesis using solid-phase techniques (Merrifield, J. Am.
Chem. Soc. 85:2149-2154 (1963)). Protein synthesis may be performed
using manual techniques or by automation. Automated synthesis may
be achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (Perkin Elmer). Alternatively, various fragments may be
chemically synthesized separately and combined using chemical
methods to produce the desired molecule.
Polynucleotide Compositions
[0145] The present invention also provides isolated polynucleotides
that encode the tyrosyl-tRNA synthetase polypeptides of the
invention, including truncations and/or variants thereof, as well
as compositions comprising such polynucleotides.
[0146] As used herein, the terms "DNA" and "polynucleotide" and
"nucleic acid" refer to a DNA molecule that has been isolated free
of total genomic DNA of a particular species. Therefore, a DNA
segment encoding a polypeptide refers to a DNA segment that
contains one or more coding sequences yet is substantially isolated
away from, or purified free from, total genomic DNA of the species
from which the DNA segment is obtained. Included within the terms
"DNA segment" and "polynucleotide" are DNA segments and smaller
fragments of such segments, and also recombinant vectors,
including, for example, plasmids, cosmids, phagemids, phage,
viruses, and the like.
[0147] As will be understood by those skilled in the art, the
polynucleotide sequences of this invention can include genomic
sequences, extra-genomic and plasmid-encoded sequences and smaller
engineered gene segments that express, or may be adapted to
express, proteins, polypeptides, peptides and the like. Such
segments may be naturally isolated, or modified synthetically by
the hand of man.
[0148] As will be recognized by the skilled artisan,
polynucleotides may be single-stranded (coding or antisense) or
double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA
molecules. Additional coding or non-coding sequences may, but need
not, be present within a polynucleotide of the present invention,
and a polynucleotide may, but need not, be linked to other
molecules and/or support materials.
[0149] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes a tyrosyl-tRNA synthetase or a
portion thereof) or may comprise a variant, or a biological
functional equivalent of such a sequence. Polynucleotide variants
may contain one or more substitutions, additions, deletions and/or
insertions, as further described below, preferably such that the
thrombopoietic activity of the encoded polypeptide is not
substantially diminished relative to the unmodified polypeptide.
The effect on the thrombopoietic activity of the encoded
polypeptide may generally be assessed as described herein.
[0150] In additional embodiments, the present invention provides
isolated polynucleotides comprising various lengths of contiguous
stretches of sequence identical to or complementary to a
tyrosyl-tRNA synthetase, wherein the isolated polynucleotides
encode a truncated tyrosyl tRNA synthetase as described herein.
[0151] Exemplary nucleotide sequences that encode the YRS
polypeptides of the application encompass full-length YRS genes,
such as the polynucleotide sequences of SEQ ID NOS:4, 7, 9, 11, 13,
and 15, as well as portions of the full-length or substantially
full-length nucleotide sequences of the YRS genes or their
transcripts or DNA copies of these transcripts. Portions of a YRS
nucleotide sequence may encode polypeptide portions or segments
that retain the biological activity of the reference polypeptide. A
portion of a YRS nucleotide sequence that encodes a biologically
active fragment of a YRS polypeptide may encode at least about 20,
21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300
or 400 contiguous amino acid residues, or almost up to the total
number of amino acids present in a full-length YRS polypeptide. It
will be readily understood that "intermediate lengths," in this
context and in all other contexts used herein, means any length
between the quoted values, such as 101, 102, 103, etc.; 151, 152,
153, etc.; 201, 202, 203, etc.
[0152] The polynucleotides of the present invention, regardless of
the length of the coding sequence itself, may be combined with
other DNA sequences, such as promoters, polyadenylation signals,
additional restriction enzyme sites, multiple cloning sites, other
coding segments, and the like, such that their overall length may
vary considerably. It is therefore contemplated that a
polynucleotide fragment of almost any length may be employed, with
the total length preferably being limited by the ease of
preparation and use in the intended recombinant DNA protocol.
[0153] The invention also contemplates variants of the YRS
nucleotide sequences. Nucleic acid variants can be
naturally-occurring, such as allelic variants (same locus),
homologs (different locus), and orthologs (different organism) or
can be non naturally-occurring. Naturally occurring variants such
as these can be identified with the use of well-known molecular
biology techniques, as, for example, with polymerase chain reaction
(PCR) and hybridization techniques as known in the art.
Non-naturally occurring variants can be made by mutagenesis
techniques, including those applied to polynucleotides, cells, or
organisms. The variants can contain nucleotide substitutions,
deletions, inversions and insertions. Variation can occur in either
or both the coding and non-coding regions. The variations can
produce both conservative and non-conservative amino acid
substitutions (as compared in the encoded product). For nucleotide
sequences, conservative variants include those sequences that,
because of the degeneracy of the genetic code, encode the amino
acid sequence of a reference YRS polypeptide, such as the sequences
set forth in SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, and 14. Variant
nucleotide sequences also include synthetically derived nucleotide
sequences, such as those generated, for example, by using
site-directed mutagenesis but which still encode a YRS polypeptide.
Generally, variants of a particular YRS nucleotide sequence will
have at least about 30%, 40% 50%, 55%, 60%, 65%, 70%, generally at
least about 75%, 80%, 85%, desirably about 90% to 95% or more, and
more suitably about 98% or more sequence identity to that
particular nucleotide sequence as determined by sequence alignment
programs described elsewhere herein using default parameters.
[0154] YRS nucleotide sequences can be used to isolate
corresponding sequences and alleles from other organisms,
particularly other organisms or microorganisms. Methods are readily
available in the art for the hybridization of nucleic acid
sequences. Coding sequences from other organisms may be isolated
according to well known techniques based on their sequence identity
with the coding sequences set forth herein. In these techniques all
or part of the known coding sequence is used as a probe which
selectively hybridizes to other YRS-coding sequences present in a
population of cloned genomic DNA fragments or cDNA fragments (i.e.,
genomic or cDNA libraries) from a chosen organism.
[0155] Accordingly, the present invention also contemplates
polynucleotides that hybridize to reference YRS nucleotide
sequences, or to their complements, under stringency conditions
described below. As used herein, the term "hybridizes under low
stringency, medium stringency, high stringency, or very high
stringency conditions" describes conditions for hybridization and
washing. Guidance for performing hybridization reactions can be
found in Ausubel et al., (1998, supra), Sections 6.3.1-6.3.6.
Aqueous and non-aqueous methods are described in that reference and
either can be used. Reference herein to low stringency conditions
include and encompass from at least about 1% v/v to at least about
15% v/v formamide and from at least about 1 M to at least about 2 M
salt for hybridization at 42.degree. C., and at least about 1 M to
at least about 2 M salt for washing at 42.degree. C. Low stringency
conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM
EDTA, 0.5 M NaHPO.sub.4 (pH 7.2), 7% SDS for hybridization at
65.degree. C., and (i) 2.times.SSC, 0.1% SDS; or (ii) 0.5% BSA, 1
mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 5% SDS for washing at room
temperature. One embodiment of low stringency conditions includes
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by two washes in 0.2.times.SSC, 0.1%
SDS at least at 50.degree. C. (the temperature of the washes can be
increased to 55.degree. C. for low stringency conditions). Medium
stringency conditions include and encompass from at least about 16%
v/v to at least about 30% v/v formamide and from at least about 0.5
M to at least about 0.9 M salt for hybridization at 42.degree. C.,
and at least about 0.1 M to at least about 0.2 M salt for washing
at 55.degree. C. Medium stringency conditions also may include 1%
Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO.sub.4 (pH 7.2),
7% SDS for hybridization at 65.degree. C., and (i) 2.times.SSC,
0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2),
5% SDS for washing at 60-65.degree. C. One embodiment of medium
stringency conditions includes hybridizing in 6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 60.degree. C. High stringency conditions include and
encompass from at least about 31% v/v to at least about 50% v/v
formamide and from about 0.01 M to about 0.15 M salt for
hybridization at 42.degree. C., and about 0.01 M to about 0.02 M
salt for washing at 55.degree. C. High stringency conditions also
may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO.sub.4 (pH 7.2), 7% SDS
for hybridization at 65.degree. C., and (i) 0.2.times.SSC, 0.1%
SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 1%
SDS for washing at a temperature in excess of 65.degree. C. One
embodiment of high stringency conditions includes hybridizing in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0156] In certain embodiments, a YRS polypeptide is encoded by a
polynucleotide that hybridizes to a disclosed nucleotide sequence
under very high stringency conditions. One embodiment of very high
stringency conditions includes hybridizing in 0.5 M sodium
phosphate, 7% SDS at 65.degree. C., followed by one or more washes
in 0.2.times.SSC, 1% SDS at 65.degree. C.
[0157] Other stringency conditions are well known in the art and a
skilled addressee will recognize that various factors can be
manipulated to optimize the specificity of the hybridization.
Optimization of the stringency of the final washes can serve to
ensure a high degree of hybridization. For detailed examples, see
Ausubel et al., supra at pages 2.10.1 to 2.10.16 and Sambrook et
al. (1989, supra) at sections 1.101 to 1.104.
[0158] While stringent washes are typically carried out at
temperatures from about 42.degree. C. to 68.degree. C., one skilled
in the art will appreciate that other temperatures may be suitable
for stringent conditions. Maximum hybridization rate typically
occurs at about 20.degree. C. to 25.degree. C. below the T.sub.m
for formation of a DNA-DNA hybrid. It is well known in the art that
the T.sub.m is the melting temperature, or temperature at which two
complementary polynucleotide sequences dissociate. Methods for
estimating T.sub.m are well known in the art (see Ausubel et al.,
supra at page 2.10.8).
[0159] In general, the T.sub.m of a perfectly matched duplex of DNA
may be predicted as an approximation by the formula:
T.sub.m=81.5+16.6 (log.sub.10 M)+0.41 (% G+C)-0.63 (%
formamide)-(600/length) wherein: M is the concentration of
Na.sup.+, preferably in the range of 0.01 molar to 0.4 molar; % G+C
is the sum of guanosine and cytosine bases as a percentage of the
total number of bases, within the range between 30% and 75% G+C; %
formamide is the percent formamide concentration by volume; length
is the number of base pairs in the DNA duplex. The T.sub.m of a
duplex DNA decreases by approximately 1.degree. C. with every
increase of 1% in the number of randomly mismatched base pairs.
Washing is generally carried out at T.sub.m-15.degree. C. for high
stringency, or T.sub.m-30.degree. C. for moderate stringency.
[0160] In one example of a hybridization procedure, a membrane
(e.g., a nitrocellulose membrane or a nylon membrane) containing
immobilized DNA is hybridized overnight at 42.degree. C. in a
hybridization buffer (50% deionized formamide, 5.times.SSC,
5.times.Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrollidone
and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured
salmon sperm DNA) containing a labeled probe. The membrane is then
subjected to two sequential medium stringency washes (i.e.,
2.times.SSC, 0.1% SDS for 15 min at 45.degree. C., followed by
2.times.SSC, 0.1% SDS for 15 min at 50.degree. C.), followed by two
sequential higher stringency washes (i.e., 0.2.times.SSC, 0.1% SDS
for 12 min at 55.degree. C. followed by 0.2.times.SSC and 0.1% SDS
solution for 12 min at 65-68.degree. C.
[0161] Polynucleotides and fusions thereof may be prepared,
manipulated and/or expressed using any of a variety of well
established techniques known and available in the art. For example,
polynucleotide sequences which encode polypeptides of the
invention, or fusion proteins or functional equivalents thereof,
may be used in recombinant DNA molecules to direct expression of a
truncated and/or variant tyrosyl-tRNA synthetase polypeptide in
appropriate host cells. Due to the inherent degeneracy of the
genetic code, other DNA sequences that encode substantially the
same or a functionally equivalent amino acid sequence may be
produced and these sequences may be used to clone and express a
given polypeptide.
[0162] As will be understood by those of skill in the art, it may
be advantageous in some instances to produce polypeptide-encoding
nucleotide sequences possessing non-naturally occurring codons. For
example, codons preferred by a particular prokaryotic or eukaryotic
host can be selected to increase the rate of protein expression or
to produce a recombinant RNA transcript having desirable
properties, such as a half-life which is longer than that of a
transcript generated from the naturally occurring sequence.
[0163] Moreover, the polynucleotide sequences of the present
invention can be engineered using methods generally known in the
art in order to alter polypeptide encoding sequences for a variety
of reasons, including but not limited to, alterations which modify
the cloning, processing, expression and/or activity of the gene
product.
[0164] In order to express a desired polypeptide, a nucleotide
sequence encoding the polypeptide, or a functional equivalent, may
be inserted into appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence. Methods which are well
known to those skilled in the art may be used to construct
expression vectors containing sequences encoding a polypeptide of
interest and appropriate transcriptional and translational control
elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook et al.,
Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,
Current Protocols in Molecular Biology (1989).
[0165] A variety of expression vector/host systems are known and
may be utilized to contain and express polynucleotide sequences.
These include, but are not limited to, microorganisms such as
bacteria transformed with recombinant bacteriophage, plasmid, or
cosmid DNA expression vectors; yeast transformed with yeast
expression vectors; insect cell systems infected with virus
expression vectors (e.g., baculovirus); plant cell systems
transformed with virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or with bacterial
expression vectors (e.g., Ti or pBR322 plasmids); or animal cell
systems.
[0166] The "control elements" or "regulatory sequences" present in
an expression vector are those non-translated regions of the
vector--enhancers, promoters, 5' and 3' untranslated regions--which
interact with host cellular proteins to carry out transcription and
translation. Such elements may vary in their strength and
specificity. Depending on the vector system and host utilized, any
number of suitable transcription and translation elements,
including constitutive and inducible promoters, may be used. For
example, when cloning in bacterial systems, inducible promoters
such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid
(Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL,
Gaithersburg, Md.) and the like may be used. In mammalian cell
systems, promoters from mammalian genes or from mammalian viruses
are generally preferred. If it is necessary to generate a cell line
that contains multiple copies of the sequence encoding a
polypeptide, vectors based on SV40 or EBV may be advantageously
used with an appropriate selectable marker.
[0167] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for the expressed
polypeptide. For example, when large quantities are needed, vectors
which direct high level expression of fusion proteins that are
readily purified may be used. Such vectors include, but are not
limited to, the multifunctional E. coli cloning and expression
vectors such as BLUESCRIPT (Stratagene), in which the sequence
encoding the polypeptide of interest may be ligated into the vector
in frame with sequences for the amino-terminal Met and the
subsequent 7 residues of .beta.-galactosidase so that a hybrid
protein is produced; pIN vectors (Van Heeke & Schuster, J.
Biol. Chem. 264:5503 5509 (1989)); and the like. pGEX Vectors
(Promega, Madison, Wis.) may also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily
be purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione.
Proteins made in such systems may be designed to include heparin,
thrombin, or factor XA protease cleavage sites so that the cloned
polypeptide of interest can be released from the GST moiety at
will.
[0168] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH may be used. For reviews, see
Ausubel et al. (supra) and Grant et al., Methods Enzymol.
153:516-544 (1987).
[0169] In cases where plant expression vectors are used, the
expression of sequences encoding polypeptides may be driven by any
of a number of promoters. For example, viral promoters such as the
35S and 19S promoters of CaMV may be used alone or in combination
with the omega leader sequence from TMV (Takamatsu, EMBO J.
6:307-311 (1987)). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi et
al., EMBO J. 3:1671-1680 (1984); Broglie et al., Science
224:838-843 (1984); and Winter et al., Results Probl. Cell Differ.
17:85-105 (1991)). These constructs can be introduced into plant
cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, e.g., Hobbs in McGraw Hill,
Yearbook of Science and Technology, pp. 191-196 (1992)).
[0170] An insect system may also be used to express a polypeptide
of interest. For example, in one such system, Autographa
californica nuclear polyhedrosis virus (AcNPV) is used as a vector
to express foreign genes in Spodoptera frugiperda cells or in
Trichoplusia larvae. The sequences encoding the polypeptide may be
cloned into a non-essential region of the virus, such as the
polyhedrin gene, and placed under control of the polyhedrin
promoter. Successful insertion of the polypeptide-encoding sequence
will render the polyhedrin gene inactive and produce recombinant
virus lacking coat protein. The recombinant viruses may then be
used to infect, for example, S. frugiperda cells or Trichoplusia
larvae in which the polypeptide of interest may be expressed
(Engelhard et al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227
(1994)).
[0171] In mammalian host cells, a number of viral-based expression
systems are generally available. For example, in cases where an
adenovirus is used as an expression vector, sequences encoding a
polypeptide of interest may be ligated into an adenovirus
transcription/translation complex consisting of the late promoter
and tripartite leader sequence. Insertion in a non-essential E1 or
E3 region of the viral genome may be used to obtain a viable virus
which is capable of expressing the polypeptide in infected host
cells (Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A.
81:3655-3659 (1984)). In addition, transcription enhancers, such as
the Rous sarcoma virus (RSV) enhancer or immediate/early
cytomegalovirus (CMV) enhancer/promoter region, may be used to
increase expression in mammalian host cells.
[0172] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding a polypeptide of
interest. Such signals include the ATG initiation codon and
adjacent sequences. In cases where sequences encoding the
polypeptide, its initiation codon, and upstream sequences are
inserted into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a portion thereof,
is inserted, exogenous translational control signals including the
ATG initiation codon should be provided. Furthermore, the
initiation codon should be in the correct reading frame to ensure
translation of the entire insert. Exogenous translational elements
and initiation codons may be of various origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers which are appropriate for the particular
cell system which is used, such as those described in the
literature (Scharf. et al., Results Probl. Cell Differ. 20:125-162
(1994)).
[0173] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different
host cells such as CHO, HeLa, MDCK, HEK293, and W138, which have
specific cellular machinery and characteristic mechanisms for such
post-translational activities, may be chosen to ensure the correct
modification and processing of the foreign protein.
[0174] For long-term, high-yield production of recombinant
proteins, stable expression is generally preferred. For example,
cell lines which stably express a polynucleotide of interest may be
transformed using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells which successfully express the introduced
sequences. Resistant clones of stably transformed cells may be
proliferated using tissue culture techniques appropriate to the
cell type.
[0175] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler et al., Cell
11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et
al., Cell 22:817-823 (1990)) genes which can be employed in tk- or
aprt- cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler et
al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70 (1980)); npt, which
confers resistance to the aminoglycosides, neomycin and G-418
(Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or
pat, which confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase, respectively (Murry, supra). Additional
selectable genes have been described, for example, trpB, which
allows cells to utilize indole in place of tryptophan, or hisD,
which allows cells to utilize histinol in place of histidine
(Hartman & Mulligan, Proc. Natl. Acad. Sci. U.S.A. 85:8047-51
(1988)). The use of visible markers has gained popularity with such
markers as anthocyanins, .beta.-glucuronidase and its substrate
GUS, and luciferase and its substrate luciferin, being widely used
not only to identify transformants, but also to quantify the amount
of transient or stable protein expression attributable to a
specific vector system (Rhodes et al., Methods Mol. Biol.
55:121-131 (1995)).
[0176] A variety of protocols for detecting and measuring the
expression of polynucleotide-encoded products, using either
polyclonal or monoclonal antibodies specific for the product are
known in the art. Examples include enzyme-linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), and fluorescence activated
cell sorting (FACS). These and other assays are described, among
other places, in Hampton et al., Serological Methods, a Laboratory
Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216
(1983).
[0177] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides include oligolabeling, nick translation,
end-labeling or PCR amplification using a labeled nucleotide.
Alternatively, the sequences, or any portions thereof may be cloned
into a vector for the production of an mRNA probe. Such vectors are
known in the art, are commercially available, and may be used to
synthesize RNA probes in vitro by addition of an appropriate RNA
polymerase such as T7, T3, or SP6 and labeled nucleotides. These
procedures may be conducted using a variety of commercially
available kits. Suitable reporter molecules or labels, which may be
used include radionuclides, enzymes, fluorescent, chemiluminescent,
or chromogenic agents as well as substrates, cofactors, inhibitors,
magnetic particles, and the like.
[0178] Host cells transformed with a polynucleotide sequence of
interest may be cultured under conditions suitable for the
expression and recovery of the protein from cell culture. The
protein produced by a recombinant cell may be secreted or contained
intracellularly depending on the sequence and/or the vector used.
As will be understood by those of skill in the art, expression
vectors containing polynucleotides of the invention may be designed
to contain signal sequences which direct secretion of the encoded
polypeptide through a prokaryotic or eukaryotic cell membrane.
Other recombinant constructions may be used to join sequences
encoding a polypeptide of interest to nucleotide sequence encoding
a polypeptide domain which will facilitate purification of soluble
proteins.
Antibody Compositions, Fragments Thereof and Other Binding
Agents
[0179] According to another aspect, the present invention further
provides binding agents, such as antibodies and antigen-binding
fragments thereof, that exhibit immunological binding to a
polypeptide disclosed herein, or to a portion, variant or
derivative thereof, and methods of using same. Preferably, such
binding agents are effective for modulating one or more of the
non-canonical activities mediated by a YRS polypeptide of the
invention, or for detecting the presence or absence of selected YRS
polypeptides (e.g., truncations, alternate splice variants,
mutants) in a sample, such as a biological sample obtained from a
subject.
[0180] For example, certain embodiments contemplate a method of
identifying or characterizing a YRS polypeptide in a subject,
comprising obtaining a biological sample from the subject,
contacting the biological sample with an antibody, or
antigen-binding fragment thereof, wherein the antibody or
antigen-fragment specifically binds to a YRS polypeptide of the
invention, and detecting the presence or absence of the bound
antibody, or antigen-binding fragment thereof, thereby identifying
or characterizing the YRS polypeptide in the subject. In certain
aspects, the antibody, or antigen-binding fragment thereof,
specifically binds to a certain variant or truncated YRS
polypeptide, such as a selected YRS mutant or alternate splice
variant, but does not specifically bind to other YRS polypeptides,
such as a full-length, wild type YRS polypeptide.
[0181] An antibody, or antigen-binding fragment thereof, is said to
"specifically bind," "immunologically bind," and/or is
"immunologically reactive" to a polypeptide of the invention if it
reacts at a detectable level (within, for example, an ELISA assay)
with the polypeptide, and does not react detectably with unrelated
polypeptides under similar conditions.
[0182] Immunological binding, as used in this context, generally
refers to the non-covalent interactions of the type which occur
between an immunoglobulin molecule and an antigen for which the
immunoglobulin is specific. The strength, or affinity of
immunological binding interactions can be expressed in terms of the
dissociation constant (K.sub.d) of the interaction, wherein a
smaller K.sub.d represents a greater affinity. Immunological
binding properties of selected polypeptides can be quantified using
methods well known in the art. One such method entails measuring
the rates of antigen-binding site/antigen complex formation and
dissociation, wherein those rates depend on the concentrations of
the complex partners, the affinity of the interaction, and on
geometric parameters that equally influence the rate in both
directions. Thus, both the "on rate constant" (K.sub.on) and the
"off rate constant" (K.sub.off) can be determined by calculation of
the concentrations and the actual rates of association and
dissociation. The ratio of K.sub.off/K.sub.on enables cancellation
of all parameters not related to affinity, and is thus equal to the
dissociation constant K.sub.d. See, generally, Davies et al. (1990)
Annual Rev. Biochem. 59:439-473.
[0183] An "antigen-binding site," or "binding portion" of an
antibody refers to the part of the immunoglobulin molecule that
participates in antigen binding. The antigen binding site is formed
by amino acid residues of the N-terminal variable ("V") regions of
the heavy ("H") and light ("L") chains. Three highly divergent
stretches within the V regions of the heavy and light chains are
referred to as "hypervariable regions" which are interposed between
more conserved flanking stretches known as "framework regions," or
"FRs." Thus, the term "FR" refers to amino acid sequences which are
naturally found between and adjacent to hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs."
[0184] A binding agent may be, for example, a ribosome, with or
without a peptide component, an RNA molecule or a polypeptide. In a
preferred embodiment, a binding agent is an antibody or an
antigen-binding fragment thereof. Antibodies may be prepared by any
of a variety of techniques known to those of ordinary skill in the
art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, 1988. In general, antibodies can be
produced by cell culture techniques, including the generation of
monoclonal antibodies as described herein, or via transfection of
antibody genes into suitable bacterial or mammalian cell hosts, in
order to allow for the production of recombinant antibodies. In one
technique, an immunogen comprising the polypeptide is initially
injected into any of a wide variety of mammals (e.g., mice, rats,
rabbits, sheep or goats). In this step, the polypeptides of this
invention may serve as the immunogen without modification.
Alternatively, particularly for relatively short polypeptides, a
superior immune response may be elicited if the polypeptide is
joined to a carrier protein, such as bovine serum albumin or
keyhole limpet hemocyanin. The immunogen is injected into the
animal host, preferably according to a predetermined schedule
incorporating one or more booster immunizations, and the animals
are bled periodically. Polyclonal antibodies specific for the
polypeptide may then be purified from such antisera by, for
example, affinity chromatography using the polypeptide coupled to a
suitable solid support.
[0185] Monoclonal antibodies specific for an polypeptide of
interest may be prepared, for example, using the technique of
Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and
improvements thereto. Briefly, these methods involve the
preparation of immortal cell lines capable of producing antibodies
having the desired specificity (i.e., reactivity with the
polypeptide of interest). Such cell lines may be produced, for
example, from spleen cells obtained from an animal immunized as
described above. The spleen cells are then immortalized by, for
example, fusion with a myeloma cell fusion partner, preferably one
that is syngeneic with the immunized animal. A variety of fusion
techniques may be employed. For example, the spleen cells and
myeloma cells may be combined with a nonionic detergent for a few
minutes and then plated at low density on a selective medium that
supports the growth of hybrid cells, but not myeloma cells. A
preferred selection technique uses HAT (hypoxanthine, aminopterin,
thymidine) selection. After a sufficient time, usually about 1 to 2
weeks, colonies of hybrids are observed. Single colonies are
selected and their culture supernatants tested for binding activity
against the polypeptide. Hybridomas having high reactivity and
specificity are preferred.
[0186] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the
hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host, such as a mouse. Monoclonal antibodies may then be
harvested from the ascites fluid or the blood. Contaminants may be
removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation, and extraction. The
polypeptides of this invention may be used in the purification
process in, for example, an affinity chromatography step.
[0187] A number of therapeutically useful molecules are known in
the art which comprise antigen-binding sites that are capable of
exhibiting immunological binding properties of an antibody
molecule. The proteolytic enzyme papain preferentially cleaves IgG
molecules to yield several fragments, two of which (the "F(ab)"
fragments) each comprise a covalent heterodimer that includes an
intact antigen-binding site. The enzyme pepsin is able to cleave
IgG molecules to provide several fragments, including the
"F(ab').sub.2" fragment which comprises both antigen-binding sites.
An "Fv" fragment can be produced by preferential proteolytic
cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin
molecule. Fv fragments are, however, more commonly derived using
recombinant techniques known in the art. The Fv fragment includes a
non-covalent V.sub.H::V.sub.L heterodimer including an
antigen-binding site which retains much of the antigen recognition
and binding capabilities of the native antibody molecule. Inbar et
al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al.
(1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem
19:4091-4096.
[0188] A single chain Fv ("sFv") polypeptide is a covalently linked
V.sub.H::V.sub.L heterodimer which is expressed from a gene fusion
including V.sub.H- and V.sub.L-encoding genes linked by a
peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci.
USA 85 (16):5879-5883. A number of methods have been described to
discern chemical structures for converting the naturally
aggregated--but chemically separated--light and heavy polypeptide
chains from an antibody V region into an sFv molecule which will
fold into a three dimensional structure substantially similar to
the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.
5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No.
4,946,778, to Ladner et al.
[0189] Each of the above-described molecules includes a heavy chain
and a light chain CDR set, respectively interposed between a heavy
chain and a light chain FR set which provide support to the CDRS
and define the spatial relationship of the CDRs relative to each
other. As used herein, the term "CDR set" refers to the three
hypervariable regions of a heavy or light chain V region.
Proceeding from the N-terminus of a heavy or light chain, these
regions are denoted as "CDR1," "CDR2," and "CDR3" respectively. An
antigen-binding site, therefore, includes six CDRs, comprising the
CDR set from each of a heavy and a light chain V region. A
polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3)
is referred to herein as a "molecular recognition unit."
Crystallographic analysis of a number of antigen-antibody complexes
has demonstrated that the amino acid residues of CDRs form
extensive contact with bound antigen, wherein the most extensive
antigen contact is with the heavy chain CDR3. Thus, the molecular
recognition units are primarily responsible for the specificity of
an antigen-binding site.
[0190] As used herein, the term "FR set" refers to the four
flanking amino acid sequences which frame the CDRs of a CDR set of
a heavy or light chain V region. Some FR residues may contact bound
antigen; however, FRs are primarily responsible for folding the V
region into the antigen-binding site, particularly the FR residues
directly adjacent to the CDRS. Within FRs, certain amino residues
and certain structural features are very highly conserved. In this
regard, all V region sequences contain an internal disulfide loop
of around 90 amino acid residues. When the V regions fold into a
binding-site, the CDRs are displayed as projecting loop motifs
which form an antigen-binding surface. It is generally recognized
that there are conserved structural regions of FRs which influence
the folded shape of the CDR loops into certain "canonical"
structures--regardless of the precise CDR amino acid sequence.
Further, certain FR residues are known to participate in
non-covalent interdomain contacts which stabilize the interaction
of the antibody heavy and light chains.
[0191] A number of "humanized" antibody molecules comprising an
antigen-binding site derived from a non-human immunoglobulin have
been described, including chimeric antibodies having rodent V
regions and their associated CDRs fused to human constant domains
(Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989)
Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J
Immunol. 138:4534-4538; and Brown et al. (1987) Cancer Res.
47:3577-3583), rodent CDRs grafted into a human supporting FR prior
to fusion with an appropriate human antibody constant domain
(Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al.
(1988) Science 239:1534-1536; and Jones et al. (1986) Nature
321:522-525), and rodent CDRs supported by recombinantly veneered
rodent FRs (European Patent Publication No. 519,596, published Dec.
23, 1992). These "humanized" molecules are designed to minimize
unwanted immunological response toward rodent antihuman antibody
molecules which limits the duration and effectiveness of
therapeutic applications of those moieties in human recipients.
[0192] As used herein, the terms "veneered FRs" and "recombinantly
veneered FRs" refer to the selective replacement of FR residues
from, e.g., a rodent heavy or light chain V region, with human FR
residues in order to provide a xenogeneic molecule comprising an
antigen-binding site which retains substantially all of the native
FR polypeptide folding structure. Veneering techniques are based on
the understanding that the ligand binding characteristics of an
antigen-binding site are determined primarily by the structure and
relative disposition of the heavy and light chain CDR sets within
the antigen-binding surface. Davies et al. (1990) Ann. Rev.
Biochem. 59:439-473. Thus, antigen binding specificity can be
preserved in a humanized antibody only wherein the CDR structures,
their interaction with each other, and their interaction with the
rest of the V region domains are carefully maintained. By using
veneering techniques, exterior (e.g., solvent-accessible) FR
residues which are readily encountered by the immune system are
selectively replaced with human residues to provide a hybrid
molecule that comprises either a weakly immunogenic, or
substantially non-immunogenic veneered surface.
[0193] In another embodiment of the invention, monoclonal
antibodies of the present invention may be coupled to one or more
agents of interest. For example, a therapeutic agent may be coupled
(e.g., covalently bonded) to a suitable monoclonal antibody either
directly or indirectly (e.g., via a linker group). A direct
reaction between an agent and an antibody is possible when each
possesses a substituent capable of reacting with the other. For
example, a nucleophilic group, such as an amino or sulfhydryl
group, on one may be capable of reacting with a carbonyl-containing
group, such as an anhydride or an acid halide, or with an alkyl
group containing a good leaving group (e.g., a halide) on the
other.
[0194] Alternatively, it may be desirable to couple a therapeutic
agent and an antibody via a linker group. A linker group can
function as a spacer to distance an antibody from an agent in order
to avoid interference with binding capabilities. A linker group can
also serve to increase the chemical reactivity of a substituent on
an agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
be possible.
[0195] It will be evident to those skilled in the art that a
variety of bifunctional or polyfunctional reagents, both homo- and
hetero-functional (such as those described in the catalog of the
Pierce Chemical Co., Rockford, Ill.), may be employed as the linker
group. Coupling may be effected, for example, through amino groups,
carboxyl groups, sulfhydryl groups or oxidized carbohydrate
residues. There are numerous references describing such
methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.
[0196] Where a therapeutic agent is more potent when free from the
antibody portion of the immunoconjugates of the present invention,
it may be desirable to use a linker group which is cleavable during
or upon internalization into a cell. A number of different
cleavable linker groups have been described. The mechanisms for the
intracellular release of an agent from these linker groups include
cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.
4,489,710, to Spitler), by irradiation of a photolabile bond (e.g.,
U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045,
to Kohn et al.), by serum complement-mediated hydrolysis (e.g.,
U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed
hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.).
[0197] It may be desirable to couple more than one agent to an
antibody. In one embodiment, multiple molecules of an agent are
coupled to one antibody molecule. In another embodiment, more than
one type of agent may be coupled to one antibody. Regardless of the
particular embodiment, immunoconjugates with more than one agent
may be prepared in a variety of ways. For example, more than one
agent may be coupled directly to an antibody molecule, or linkers
that provide multiple sites for attachment can be used.
Thrombocytopenia and Methods of Use
[0198] As noted above, the present invention generally relates to
methods of treating, and/or reducing the risks of developing,
thrombocytopenia or other conditions associated with decreased
platelet count. Thrombocytopenia is generally characterized by
reduced platelet counts, as compared to a normal range of platelet
counts for a typical subject. For example, thrombocytopenia refers
generally to a decrease in the platelet count to about
100,000/mm.sup.3 or lower compared to a normal platelet count. A
normal platelet count generally ranges from about 150,000 mm.sup.3
to about 450,000 mm.sup.3 in a subject.
[0199] Thrombocytopenia often causes no signs or symptoms, but may
be identified by routine blood tests. If present, possible signs
and symptoms of thrombocytopenia include easy bruising and/or
excessive bleeding. For example, bleeding in the skin may be the
first sign of a low platelet count. Many tiny red dots (petechiae)
often appear in the skin on the lower legs, and minor injuries may
cause small scattered bruises. In addition, the gums may bleed, and
blood may appear in the stool or urine. Menstrual periods may be
unusually heavy. Bleeding may be hard to stop.
[0200] Bleeding typically worsens as the number of platelets
decreases. People who have very few platelets may lose large
amounts of blood into the digestive tract or may develop
life-threatening bleeding in the brain even though they have not
been injured. The rate at which symptoms develop can vary depending
on the cause of thrombocytopenia.
[0201] Thrombocytopenia may be congenital, acquired, and/or
iatrogenic, and may stem from a variety of underlying physiological
causes or conditions. For example, thrombocytopenia may result
generally from decreased production of platelets, increased
destruction of platelets, consumption of platelets,
entrapment/sequestration of platelets due to hypersplenism (i.e.,
enlarged spleen) or hypothermia, and/or from the side-effects of
certain medications (i.e., medication induced thrombocytopenia). In
addition, idiopathic forms of thrombocytopenia occur, especially in
children, transient forms may follow viral infections (e.g.,
Epstein-Barr or infectious mononucleosis), and pregnant women may
develop mild thrombocytopenia, often when close to delivery.
[0202] Examples of congenital conditions associated with the
decreased production (i.e., diminished or defective production) of
platelets include Wiskott-Aldrich syndrome, maternal ingestion of
thiazides, congenital amegakaryocytic thrombocytopenia,
thrombocytopenia absent radius syndrome, Fanconi anemia,
Bernard-Soulier syndrome, May-Hegglin anomaly, Grey platelet
syndrome, Alport syndrome, and neonatal rubella. Examples of
acquired conditions associated with the decreased production of
platelets include aplastic anemia, myeolodysplastic syndrome,
marrow infiltration (e.g., acute and chronic leukemias, tumors,
cancer of the bone marrow), lymphomas, nutritional deficiencies
(e.g., B.sub.12, folic acid), the use of myelosuppressive agents,
the use of drugs that directly influence platelet production (e.g.,
thiazides, alcohol, hormones), radiation exposure (e.g., radiation
therapy), exposure to toxic chemicals (e.g., pesticides, arsenic,
benzene), decreased production of thrombopoietin by the liver in
liver failure, bacterial sepsis, and certain viral infections
(e.g., chickenpox, mumps, parvovirus, measles, dengue, HIV,
HCV).
[0203] Examples of congenital conditions associated with increased
peripheral platelet destruction include nonimmune conditions, such
as prematurity, erythroblastosis fetalis, infection; and immune
conditions, such as drug sensitivity, idiopathic thrombocytopenic
purpura (ITP), and maternal ITP. Examples of acquired conditions
associated with increased peripheral platelet destruction include
nonimmune conditions, such as hemolytic-uremic syndrome,
disseminated intravascular coagulation, thrombotic thrombocytopenic
purpura (TTP); immune conditions, such as drug-induced
thrompocytopenia (e.g., especially with quinine and quinidine),
post-transfusion purpura, systemic lupus erythematosus, rheumatoid
arthritis, neonatal alloimmune thrombocytopenia, paroxysmal
nocturnal hemoglobinuria, acute and chronic ITP, sepsis, and
alcohol; in addition to the use of invasive lines and devices
(e.g., arterial or central venous catheters), intra-aortic ballon
pumps, prosthetic heart valves, as well as the use of
heparin-related therapies.
[0204] Medication-induced thrombocytopenia may result in particular
from certain drugs, such as chemotherapeutic agents, nonsteroidal
anti-inflammatory agents, sulfonamides, vancomycin, clopidogrel,
glycoprotein IIb/IIIa inhibitors, interferons, valproic acid,
abciximab, linezolid, famotidine, mebeverine, histamine blockers,
alkylating agents, heparin, alcohol, antibiotic chemotherapeutic
agents, carbapenems, ureido-penicillins, cefazolin, among others
known in the art. Particular examples of chemotherapeutic agents
include, but are not limited to, cisplatin (CDDP), carboplatine,
procarbazine, mechlorethamine, cyclophosphamide, camptothecin,
ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,
dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,
mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen
receptor binding agents, taxol, gemcitabien, navelbine,
farnesyl-protein transferase inhibitors, transplatinum,
5-fluorouracil, vincristine, vinblastine and methotrexate,
Temazolomide (an aqueous form of DTIC), or any analog or derivative
variant of the foregoing.
[0205] The present invention relates generally to methods of
treating, or reducing the risks of developing, thrombocytopenia
(i.e., decreased platelet count) in a subject, such as in a subject
having one or more of the exemplary diseases or conditions provided
herein, among others known in the art, by administering to the
subject a composition comprising a thrombopoietically-effective
concentration of a truncated and/or variant tyrosyl-tRNA synthetase
polypeptide, or a modified polypeptide thereof. Embodiments of the
present invention encompass methods of treatment intended not only
to increase or improving the platelet count in a subject having a
reduced, decreased, abnormal, or low platelet count, but to
maintain a normal platelet count in a subject at risk for
developing a low platelet count. Certain embodiments also
contemplate the use of YRS polypeptides to increase the platelet
count in a platelet donor, including an otherwise healthy donor
(i.e., a donor with a normal platelet count), such as administering
a YRS polypeptide to the donor prior to, during, and/or after the
platelet donation or apheresis process.
[0206] Accordingly, certain embodiments include methods for
increasing the platelet count in a subject, comprising
administering to the subject a composition comprising a
thrombopoietically-effective concentration of a truncated and/or
tyrosyl-tRNA synthetase polypeptide, or a modified polypeptide
thereof, thereby increasing the platelet count in the subject.
Other embodiments include methods of maintaining a normal platelet
count in subject, comprising administering to the subject a
composition comprising a thrombopoietically-effective concentration
of a truncated and/or variant tyrosyl-tRNA synthetase polypeptide,
such as wherein the subject is at risk for developing a low
platelet count. Certain embodiments may include methods of
stimulating thrombopoiesis in a subject, such as by administering
to the subject a composition comprising a
thrombopoietically-effective concentration of a truncated and/or
variant tyrosyl-tRNA synthetase polypeptide. In certain aspects,
the subject has a reduced, lowered, or abnormal platelet count,
such as a platelet count of about 100,000/mm.sup.3 or less. In
certain aspects, the YRS polypeptides provided herein may be
utilized to stimulate the proliferation and/or differentiation of
megakaryocytes and/or neutrophils in a subject.
[0207] A subject having a reduced platelet count may also be at
risk for developing other problems associated with
thrombocytopenia, such as bleeding or bruising, hemorrhage,
gastrointestinal bleeding, eptistaxis (i.e., nose bleeds), or
intracranial hemorrhage (i.e., bleeding in the brain). As one
particular example, septic patients with thrombocytopenia have
increased bleeding. Accordingly, certain aspects of the invention
may utilize the thrombopoietic compositions provided herein to
reduce the risk of developing these types of thrombocytopenia
associated problems, among others. In other aspects, the subject
may be at risk for developing a reduced, lowered, or otherwise
abnormal platelet count, such as from an acquired condition
associated with lowered platelet levels (e.g., certain medical
therapies, leukemias, among others).
[0208] In certain aspects, the methods of treatments described
herein may be employed independently of other therapeutic
modalities, and may be the only or primary therapeutic modality
relied upon to manage a thrombocytopenic condition and/or otherwise
reduce the risk not only of developing thrombocytopenia, but of
developing other medical problems associated therewith, such as
bleeding. For example, a subject having thrombocytopenia for which
there is no known, underlying cause (e.g., idiopathic
thrombocytopenic purpura), may benefit from the methods of
treatment provided herein to increase and/or manage platelet
levels.
[0209] In certain aspects, the methods and compositions of the
present invention may be employed as part of a combination therapy,
such as by administration with other agents that may stimulate
thrombopoetic and/or hematopoietic pathways in a subject. Examples
of other agents that may be used as part of a combination therapy
include thrombopoetin, cytokines (e.g., IL-11), chemokines, and/or
growth factors involved in thrombopoiesis or hematopoiesis,
including biologically active fragments or variants thereof.
[0210] In certain aspects, the methods of the present invention may
be employed in conjunction with other therapeutic modalities, such
as those involved in treating the underlying condition that causes
the condition associated with thrombocytopenia. For example, a
subject having congenital amegakaryocytic thrombocytopenia (CAMT)
may ultimately undergo a bone marrow transplantation procedure, but
may also benefit from a separate treatment, as provided herein, to
either enhance platelet levels and/or to maintain platelet levels
within a normal range. The thrombopoietic polypeptides of the
present invention may be employed in this and similar regards.
[0211] In certain aspects, the methods provided herein may be
employed in combination with a subject undergoing other medical
treatments, such as treatments that either cause thrombocytopenia
or increase the risk of developing thrombocytopenia. For example,
the methods provided herein may be employed with a subject
undergoing, a subject about to undergo, and/or a subject who as
undergone, radiation therapy, chemotherapy, or other type of
treatment, including various types of pharmaceutical treatments, as
described herein and known in the art, since such treatments are
known to reduce the platelet count in a subject. Accordingly, the
methods provided herein may be utilized before, during, and/or
after other medical treatments to reduce the risk of developing
thrombocytopenia resulting from such treatments, and/or to manage
or improve thrombocytopenia resulting from such treatments.
[0212] In certain embodiments, the methods provided herein may be
utilized to prophylactically treat or manage thrompocytopenic
symptoms associated with such particular conditions as described
herein and known in the art.
Stimulation of Megakaryocyte Progenitor Cells and Methods of
Use
[0213] The YRS polypeptides of the present invention may also be
used to stimulate the growth of megakaryocyte progenitor cells,
including early progenitor cells, i.e., the most primitive
lineage-restricted progenitors of the megakaryocyte lineage.
Included are methods of stimulating proliferation of early
megakaryocyte progenitor cells, comprising incubating a culture of
hematopoietic stem cells with a tyrosyl-tRNA synthetase polypeptide
for a time sufficient to allow proliferation of the early
megakaryocyte progenitor cells, thereby stimulating proliferation
of early megakaryocyte progenitor cells. In these and related
embodiments, the YRS polypeptides of the invention may be incubated
with purified HSCs, partially purified HSCs, whole bone marrow
cultures (e.g., for bone marrow transplants), cord blood, or other
types of cultures used in hematopoietic graft therapies. Such
methods may result in a culture that is enriched for early
megakaryocyte progenitor cells. YRS polypeptides of the invention
may also be administered directly to a subject (in vivo) to
stimulate proliferation of early megakaryocyte progenitors in that
subject.
[0214] "Hematopoietic stem cells (HSCs)" relate generally to either
pluripotent or multipotent "stem cells" that give rise to the blood
cell types, including myeloid (e.g., monocytes and macrophages,
neutrophils, basophils, eosinophils, erythrocytes,
megakaryocytes/platelets, dendritic cells), and lymphoid lineages
(e.g., T-cells, B-cells, NK-cells), and others known in the art.
"Stem cells" are typically defined by their ability to form
multiple cell types (i.e., multipotency) and their ability to
self-renew. In certain embodiments, however, oligopotent and
unipotent progenitors may be included. "Hematopoiesis" refers
generally to the process of cellular differentiation or formation
of particular, specialized blood cells from an HSC.
[0215] HSCs may be obtained according to known techniques in the
art. For instance, HSCs may be found in the bone marrow of adults,
which includes femurs, hip, ribs, sternum, and other bones. HSCs
may be obtained directly by removal from the hip using a needle and
syringe, or from the blood, often following pre-treatment with
cytokines, such as G-CSF (granulocyte colony-stimulating factors),
that induce cells to be released from the bone marrow compartment.
Other sources for clinical and scientific use include umbilical
cord blood, placenta, and mobilized peripheral blood. For
experimental purposes, fetal liver, fetal spleen, and AGM
(Aorta-gonad-mesonephros) of animals are also useful sources of
HSCs.
[0216] HSCs may be identified according to certain phenotypic or
genotypic markers. For example, HSCs may be identified by their
small size, lack of lineage (lin) markers, low staining (side
population) with vital dyes such as rhodamine 123
(rhodamine.sup.DULL, also called rho.sup.lo) or Hoechst 33342, and
presence of various antigenic markers on their surface, many of
which belong to the cluster of differentiation series (e.g., CD34,
CD38, CD90, CD133, CD105, CD45, and c-kit, the receptor for stem
cell factor). HSCs are mainly negative for the markers that are
typically used to detect lineage commitment, and, thus, are often
referred to as lin(-) cells. Most human HSCs may be characterized
as CD34.sup.+, CD59.sup.+, Thy1/CD90.sup.+, CD38.sup.lo/-,
C-kit/CD117.sup.+, and lin(-). However, not all stem cells are
covered by these combinations, as certain HSCs are
CD34.sup.-/CD38.sup.-. Also some studies suggest that earliest stem
cells may lack c-kit on the cell surface. For human HSCs, CD133 may
represent an early marker, as both CD34.sup.+ and CD34.sup.- HSCs
have been shown to be CD133.sup.+.
[0217] For purification of lin(-) HSCs by flow cytometry, or FACS,
an array of mature blood-lineage marker antibodies may be used to
deplete the lin(+) cells or late multipotent progenitors (MPP),
including, for example, antibodies to CD13 and CD33 for human
myeloid cells, CD71 for human erythroid cells, CD19 for human B
cells, CD61 for human megakaryocytic cells, Mac-1 (CD11b/CD18) for
monocytes, Gr-1 for Granulocytes, Il7Ra, CD3, CD4, CD5, and CD8 for
T cells, among others known in the art. Other purification methods
are known in the art, such as those methods that use the particular
signature of the `signaling lymphocyte activation molecules` (SLAM)
family of cell surface molecules.
[0218] HSCs, whether obtained from, or present in, cord blood, bone
marrow, peripheral blood, or other source, may be grown or expanded
in any suitable, commercially available or custom defined medium,
with or without serum, as desired (see, e.g., Hartshorn et al.,
Cell Technology for Cell Products, pages 221-224, R. Smith, Editor;
Springer Netherlands, 2007, herein incorporated by reference in its
entirety). For instance, in certain embodiments, serum free medium
may utilize albumin and/or transferrin, which have been shown to be
useful for the growth and expansion of CD34+ cells in serum free
medium. Also, cytokines may be included, such as Flt-3 ligand, stem
cell factor (SCF), and thrombopoietin (TPO), among others. HSCs may
also be grown in vessels such as bioreactors (see, e.g., Liu et
al., Journal of Biotechnology 124:592-601, 2006, herein
incorporated by reference in its entirety). A suitable medium for
ex vivo expansion of HSCs may also comprise HSC supporting cells,
such as stromal cells (e.g., lymphoreticular stromal cells), which
can be derived, for instance, from the disaggregation of lymphoid
tissue, and which have been show to support the in vitro, ex vivo,
and in vivo maintenance, growth, and differentiation of HSCs, as
well as their progeny.
[0219] HSC growth or expansion can be measured in vitro or in vivo
according to routine techniques known in the art. For example, WO
2008/073748, herein incorporated by references for these methods,
describes methods for measuring in vivo and in vitro expansion of
HSCs, and for distinguishing between the growth/expansion of HSCs
and the growth/expansion of other cells in a potentially
heterogeneous population (e.g., bone marrow), such as intermediate
progenitor cells. The administering or incubation step that results
in the growth or expansion can occur in vivo, ex vivo, or in vitro,
though in certain embodiments, the administration or incubation
occurs during ex vivo treatment of HSCs.
[0220] Growth or proliferation of megakaryocyte progenitor cells
(e.g., early, intermediate, late, etc.) can also be measured
according to routine techniques known in the art and described
herein (see, e.g., Example 10). For instance, among other
characteristics, early megakaryocyte progenitors may be identified
by immuno-staining as Lin.sup.-c-Kit.sup.+CD41.sup.+, and later
stage megakaryocyte progenitors may be identified as
Lin.sup.-c-Kit.sup.-CD41.sup.+ (see, e.g., Perez et al., PLoS ONE.
3:e3565, 2008; and Lefebvre et al., Journal of Hematotherapy &
Stem Cell Research. 9:913-921, 2000, each of which is incorporated
by reference in its entirety).
[0221] "Cord blood" or "umbilical cord blood" relates generally to
the relatively small amount of blood (up to about 180 mL) from a
newborn baby that returns to the neonatal circulation if the
umbilical cord is not prematurely clamped. Cord blood is rich in
HSCs, and may be harvested and stored for later use according to
techniques known in the art (see, e.g., U.S. Pat. Nos. 7,147,626
and 7,131,958, herein incorporated by reference for such
methodologies). Also, if the umbilical cord is ultimately not
clamped, a physiological clamping occurs upon interaction with cold
air, wherein the internal gelatinous substance, called Wharton's
jelly, swells around the umbilical artery and veins. Nonetheless,
Wharton's jelly can still serve as a source of HSCs.
[0222] As noted above, "ex vivo" refers to generally to activities
that take place outside an organism, such as experimentation or
measurements done in or on living tissue in an artificial
environment outside the organism, preferably with minimum
alteration of the natural conditions. Most commonly, "ex vivo"
procedures involve living cells or tissues taken from an organism
and cultured in a laboratory apparatus, usually under sterile
conditions, and typically for a few hours or up to about 24 hours,
but including up to 48 or 72 hours, depending on the circumstances.
In certain embodiments, such tissues or cells can be collected and
frozen, and later thawed for ex vivo treatment. Tissue culture
experiments or procedures lasting longer than a few days using
living cells or tissue are typically considered to be "in vitro,"
though in certain embodiments, this term can be used
interchangeably with ex vivo.
[0223] The terms "ex vivo administration," "ex vivo treatment," or
"ex vivo therapeutic use," relate generally to medical procedures
in which one or more organs, cells, or tissues are obtained from a
living or recently deceased subject, optionally purified/enriched,
exposed to a treatment or procedure to expand the stem cells (e.g.,
an ex vivo administration step that involves incubating the cells
with a composition of the present invention to enhance expansion of
desirable cells, such as HSCs or megakaryocyte progenitors), and
then administered to the same or different living subject after
that optional treatment or procedure. As one example,
thrombocytopenia may be alleviated by infusion of megakaryocyte
progenitor cells (see, e.g., De Bruyn et al., Stem Cells Dev.
14:415-24, 2005, herein incorporated by reference).
[0224] Such ex vivo therapeutic applications may also include an
optional in vivo treatment or procedural step, such as by
administering a YRS polypeptide of the invention one or more times
to the living subject prior to, during, or after administration of
the organ, cells, or tissue. Both local and systemic administration
are contemplated for these embodiments, according to well-known
techniques in the art. The amount of YRS polypeptide administered
to a subject will depend on the characteristics of that subject,
such as general health, age, sex, body weight, and tolerance to
drugs, as well as the degree, severity, and type of reaction to the
polypeptide and/or cell transplant.
Stimulation of CXCR-2 Expressing Cells
[0225] Certain embodiments relate to the discovery that YRS
polypeptides are capable of stimulating the migration of CXCR-2
expressing cells. CXCR-2 is a member of the CXC chemokine receptor
family, expressed on a wide variety of cell types, including
neutrophils and other immune cells. CXC chemokine receptors are
integral membrane proteins that specifically bind and respond to
cytokines of the CXC chemokine family. These CXC-based receptors
represent one subfamily of chemokine receptors, a large family of G
protein-linked receptors, also referred to as seven transmembrane
receptors. There are currently seven known CXC chemokine receptors
in mammals, named CXCR1 through CXCR7. CXCR-2 (and highly related
CXCR-1) is a well-known receptor that recognizes C--X--C chemokines
which possess an E-L-R amino acid motif immediately adjacent to
their C--X--C motif. CXCL8 (i.e., interleukin-8) and CXCL6 can both
bind CXCR1 in humans, while all other ELR-motif-positive
chemokines, such as CXCL1 to CXCL7, bind only CXCR2 (see, e.g.,
Tsai et al., Cell 110:373-383, 2002; and Pelus et al., Exp Hematol.
34:1010-20, 2006, each of which is incorporated by reference in its
entirety). As noted above, CXCR-2 is expressed on the surface of
neutrophils, and can play a role in neutrophil migration (see,
e.g., Rios-Santos et al., American Journal of Respiratory and
Critical Care Medicine 175:490-497, 2007, incorporated by reference
in its entirety).
[0226] Accordingly, given the role of CXCR-2 in cell signaling and
cell migration (e.g., neutrophil signaling/migration), among other
biologically relevant pathways, certain embodiments include methods
of stimulating migration of a CXCR-2 expressing cell, comprising
contacting the cell with a tyrosyl-tRNA synthetase polypeptide,
thereby stimulating migration of the CXCR-2 expressing cell.
Pulmonary Diseases and Methods of Use
[0227] Embodiments of the present invention also relate to the
unexpected discovery that YRS polypeptides may provide benefits in
the treatment of pulmonary diseases, such as chronic obstructive
pulmonary disease (COPD). In this regard, neutrophil migration from
the circulatory system to the lungs is implicated in chronic
pulmonary obstructive disease (COPD) (see, e.g., R. A. Stockley,
Chest 121:151 S-155S, 2002, incorporated by reference in its
entirety). As noted above, CXCR-2 is expressed on the surface of
neutrophils, and can play a role in neutrophil migration (see,
e.g., Rios-Santos et al., American Journal of Respiratory and
Critical Care Medicine 175:490-497, 2007, incorporated by reference
in its entirety). Since CXCR-2 signaling in neutrophils is
implicated in their migration to certain tissues, such as the
lungs, especially in response to foreign matter, such as irritants,
bacteria, lipopolysaccharide (LPS) etc., it may thus be implicated
in various pathological conditions, such as COPD.
[0228] Given the observations that YRS polypeptides of the
invention affect CXCR-2 signaling and polymorphonuclear (PMN) cell
migration (see, e.g., Examples 7 and 8), it is believed that these
polypeptides may be useful in the treatment or management of
pulmonary diseases, such as COPD. For instance, without wishing to
be bound by any one theory, YRS polypeptides may be used to
desensitize circulatory neutrophils to various irritants or
allergens, thereby reducing the migration of these immune cells
into the lungs (see, e.g., Example 9). Hence, certain embodiments
relate to methods of treating or managing (e.g., reducing the
complications of) pulmonary inflammation and/or pulmonary diseases,
such as COPD, comprising administering to a subject with pulmonary
inflammation or COPD an effective concentration of a tyrosyl-tRNA
synthetase polypeptide, thereby reducing COPD, and/or its symptoms,
in the subject. Often, in desensitizing immune cells, multiple
administrations are required (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc),
typically at a defined frequency (number of administrations per
day, per week, per month, etc).
[0229] COPD refers generally to a group of lung diseases that block
airflow and make it increasingly difficult for affected individuals
to breathe normally. Emphysema and chronic bronchitis are the two
main conditions within the group of COPD diseases, but COPD can
also refer to damage caused by chronic asthmatic bronchitis, among
other conditions known in the art. In all cases, damage to the
airways eventually interferes with the exchange of oxygen and
carbon dioxide in the lungs. Treatment focuses mainly on
controlling symptoms and minimizing further damage.
[0230] Emphysema represents one aspect of COPD. Emphysema leads to
inflammation within the fragile walls of the alveoli, which may
destroy some of the walls and elastic fibers, allowing small
airways to collapse upon exhaling, and impairing airflow out of the
lungs. Signs and symptoms of emphysema include, for instance,
shortness of breath, especially during physical activities,
wheezing, and chest tightness.
[0231] Chronic bronchitis represents another aspect of COPD.
Chronic bronchitis is characterized by an ongoing cough, and leads
to inflammation and narrowing of the bronchial tubes. This
condition also causes increased mucus production, which can further
block the narrowed tubes. Chronic bronchitis occurs mainly in
smokers, and is typically defined as a cough that lasts for at
least three months a year for two consecutive years. Signs and
symptoms of chronic bronchitis include, for example, having to
clear the throat first thing in the morning, especially for
smokers, a chronic cough that produces yellowish sputum, shortness
of breath in the later stages, and frequent respiratory
infections.
[0232] As noted above, COPD refers primarily to obstruction in the
lungs resulting from the two above-noted chronic lung conditions.
However, many individuals with COPD have both of these
conditions.
[0233] Chronic asthmatic bronchitis represents another aspect of
COPD. Chronic asthmatic bronchitis is usually characterized as
chronic bronchitis combined with asthma (bronchospasm). Asthma may
occur when inflamed and infected secretions irritate the smooth
muscles in the airways. Symptoms are similar to those of chronic
bronchitis, but also include intermittent, or even daily, episodes
of wheezing.
[0234] Mainly, COPD is ultimately caused by cigarette smoke and
other irritants. In the vast majority of cases, the lung damage
that leads to COPD is caused by long-term cigarette smoking.
However, other irritants may cause COPD, including cigar smoke,
secondhand smoke, pipe smoke, air pollution and certain
occupational fumes. Gastroesophageal reflux disease (GERD), which
occurs when stomach acids wash back up into your esophagus, can not
only aggravate COPD, but may even cause it in some individuals. In
rare cases, COPD results from a genetic disorder that causes low
levels of a protein called alpha-1-antitrypsin. Hence, risk factors
for COPD include exposure to tobacco smoke, occupational exposure
to dusts and chemicals (long-term exposure to chemical fumes,
vapors and dusts irritates and inflames the lungs),
gastroesophageal reflux disease (a severe form of acid reflux--the
backflow of acid and other stomach contents into the esophagus),
age (COPD develops slowly over years, so most people are at least
40 years old when symptoms begin), and genetics (a rare genetic
disorder known as alpha-1-antitrypsin deficiency is the source of a
few cases of COPD).
[0235] Complications of COPD may include respiratory infections,
high blood pressure, heart problems (e.g., heart attacks), lung
cancer (smokers with chronic bronchitis are at a higher risk of
developing lung cancer than are smokers who don't have chronic
bronchitis), and depression, among others known in the art.
[0236] Subjects with COPD may be identified according to routine
diagnostic techniques known in the art. For instance, pulmonary
function tests, such as spirometry, measure how much air the lungs
can hold and how fast an individual can blow the air out of their
lungs. Spirometry can detect COPD before the appearance of
symptoms, and can also be used to track disease progression and
monitor treatment. In addition, chest X-rays show emphysema, one of
the main causes of COPD, and may also rule out other lung problems
or heart failure. In addition, arterial blood gas analysis measures
how effectively the lungs bring oxygen into the blood and remove
carbon dioxide, providing an indication of COPD. Sputum
examination, i.e., the analysis of the cells in the sputum, can
identify the cause of certain lung problems and help rule out
certain lung cancers. Also, computerized tomography (CT) scan
produces highly-detailed images of the internal organs, which can
help detect emphysema, and, thus, COPD.
[0237] As elsewhere herein, the amount of YRS polypeptide
administered to a subject with COPD (or at risk for COPD) will
depend on the characteristics of that subject, such as general
health, age, sex, body weight, and tolerance to drugs, as well as
the degree, severity, and type of reaction to the polypeptide.
Formulations and Pharmaceutical Compositions
[0238] The compositions of the invention comprise tyrosyl-tRNA
synthetase polypeptides, including truncations and/or variants
thereof, formulated in pharmaceutically-acceptable or
physiologically-acceptable solutions for administration to a cell
or an animal, either alone, or in combination with one or more
other modalities of therapy. It will also be understood that, if
desired, the compositions of the invention may be administered in
combination with other agents as well, such as, e.g., other
proteins or polypeptides or various pharmaceutically-active agents.
There is virtually no limit to other components that may also be
included in the compositions, provided that the additional agents
do not adversely affect the thrombopoietic or other effects desired
to be achieved.
[0239] In the pharmaceutical compositions of the invention,
formulation of pharmaceutically-acceptable excipients and carrier
solutions is well-known to those of skill in the art, as is the
development of suitable dosing and treatment regimens for using the
particular compositions described herein in a variety of treatment
regimens, including e.g., oral, parenteral, intravenous,
intranasal, and intramuscular administration and formulation.
[0240] In certain applications, the pharmaceutical compositions
disclosed herein may be delivered via oral administration to a
subject. As such, these compositions may be formulated with an
inert diluent or with an assimilable edible carrier, or they may be
enclosed in hard- or soft-shell gelatin capsule, or they may be
compressed into tablets, or they may be incorporated directly with
the food of the diet.
[0241] In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein parenterally,
intravenously, intramuscularly, or even intraperitoneally as
described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No.
5,641,515 and U.S. Pat. No. 5,399,363 (each specifically
incorporated herein by reference in its entirety). Solutions of the
active compounds as free base or pharmacologically acceptable salts
may be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions may also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0242] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions (U.S. Pat. No. 5,466,468, specifically incorporated
herein by reference in its entirety). In all cases the form should
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and should be preserved against the
contaminating action of microorganisms, such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (e.g., glycerol, propylene glycol,
and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and/or vegetable oils. Proper fluidity may be maintained,
for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be facilitated by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0243] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, a sterile
aqueous medium that can be employed will be known to those of skill
in the art in light of the present disclosure. For example, one
dosage may be dissolved in 1 ml of isotonic NaCl solution and
either added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion (see, e.g., Remington's Pharmaceutical
Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some
variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, and the general
safety and purity standards as required by FDA Office of Biologics
standards.
[0244] Sterile injectable solutions can be prepared by
incorporating the active compounds in the required amount in the
appropriate solvent with the various other ingredients enumerated
above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0245] The compositions disclosed herein may be formulated in a
neutral or salt form. Pharmaceutically-acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective. The formulations are easily administered in a variety of
dosage forms such as injectable solutions, drug-release capsules,
and the like.
[0246] As used herein, "carrier" includes any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0247] The phrase "pharmaceutically-acceptable" refers to molecular
entities and compositions that do not produce an allergic or
similar untoward reaction when administered to a human. The
preparation of an aqueous composition that contains a protein as an
active ingredient is well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to injection can also be prepared. The
preparation can also be emulsified.
[0248] In certain embodiments, the pharmaceutical compositions may
be delivered by intranasal sprays, inhalation, and/or other aerosol
delivery vehicles. Methods for delivering genes, polynucleotides,
and peptide compositions directly to the lungs via nasal aerosol
sprays have been described e.g., in U.S. Pat. No. 5,756,353 and
U.S. Pat. No. 5,804,212 (each specifically incorporated herein by
reference in its entirety). Likewise, the delivery of drugs using
intranasal microparticle resins (Takenaga et al., 1998) and
lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,
specifically incorporated herein by reference in its entirety) are
also well-known in the pharmaceutical arts. Likewise, transmucosal
drug delivery in the form of a polytetrafluoroetheylene support
matrix is described in U.S. Pat. No. 5,780,045 (specifically
incorporated herein by reference in its entirety).
[0249] In certain embodiments, the delivery may occur by use of
liposomes, nanocapsules, microparticles, microspheres, lipid
particles, vesicles, and the like, for the introduction of the
compositions of the present invention into suitable host cells. In
particular, the compositions of the present invention may be
formulated for delivery either encapsulated in a lipid particle, a
liposome, a vesicle, a nanosphere, a nanoparticle or the like. The
formulation and use of such delivery vehicles can be carried out
using known and conventional techniques.
[0250] All publications, patent applications, and issued patents
cited in this specification are herein incorporated by reference as
if each individual publication, patent application, or issued
patent were specifically and individually indicated to be
incorporated by reference.
[0251] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims. The
following examples are provided by way of illustration only and not
by way of limitation. Those of skill in the art will readily
recognize a variety of noncritical parameters that could be changed
or modified to yield essentially similar results.
EXAMPLES
Example 1
Stimulation of Thrombopoiesis In Vivo
[0252] The effects of a tyrosyl-tRNA synthetase polypeptide on
thrombopoiesis were measured in vivo. The tyrosyl-tRNA synthetase
polypeptide utilized in the experiments described below is a
C-terminal truncation that comprises amino acids 1-364 of the
full-length human tyrosyl-tRNA. This C-terminally truncated
polypeptide was fused to an eight amino acid C-terminal tag
(365-L-E-H-H-H-H-H-H-372) (SEQ ID NO:5). The amino acid sequence of
the full-length human tyrosyl-tRNA synthetase is set forth in SEQ
ID NO:1.
[0253] To measure the effects of tyrosyl-tRNA synthetase
polypeptides on thrombopoiesis, in a first set of experiments, mice
were injected subcutaneously twice daily for seven days with 3
.mu.g/kg of the C-terminally truncated tyrosyl-tRNA synthetase
polypeptide. In a second set of experiments, mice were injected
twice daily for seven days with 1, 3, and 10 .mu.g/kg of the
C-terminally truncated tyrosyl-tRNA synthetase polypeptide. In a
third set of experiments, mice were injected subcutaneously twice
daily for six days with (i) 3 and 300 .mu.g/kg of the C-terminally
truncated tyrosyl-tRNA synthetase polypeptide, and one single daily
injection of (ii) 90 .mu.g/kg thrombopoietin (TPO), and (iii) 250
.mu.g/kg G-CSF.
[0254] For the first and second set of experiments described above,
the platelet count for each animal was determined upon completion
of the administration protocol. For the third set of experiments,
bone marrow and spleen histology were examined at the end of the
administration protocol.
[0255] Administration of a truncated tyrosyl-tRNA synthetase for
about one week showed a reproducible, in vivo increase in
thrombopoietic activity, as measured by either increased platelet
count or increased megakaryocyte numbers. FIG. 5(a) shows the
platelet count for the experiment in which mice were injected with
1, 3, and 10 .mu.g/kg of the truncated tyrosyl-tRNA synthetase
polypeptide, as compared to a phosphate-buffer saline (PBS)
control. FIG. 5(b) shows the platelet count for the experiment in
which mice were injected with 3 .mu.g/kg of the truncated
tyrosyl-tRNA synthetase polypeptide, as compared to a PBS control.
In both experiments, mice showed an increase in platelet counts
over control in response to treatment with a tyrosyl-tRNA
polypeptide of the invention. FIG. 6 shows an increase in
megakaryocyte numbers in response to administration of the
truncated tyrosyl-tRNA synthetase polypeptide, as compared to
untreated animals, which is comparable to the increased numbers
observed after administration with TPO. These results show that
tyrosyl-tRNA synthetase polypeptide fragments, and in particular
C-terminally truncated fragments, are capable of stimulating
thrombopoiesis in vivo.
Example 2
In Vitro Measurements of Thrombopoiesis
[0256] Effects on thrombopoiesis may also be measured in vitro.
Stem cells are treated in vitro with a tyrosyl-tRNA synthetase
polypeptide of the invention to determine its effect on
hematopoietic progenitors of the erythroid, myeloid and
megakaryocte lineages using colony-forming cell (CFC) assays (e.g.,
inhibition, stimulation, toxicity, synergism with other cytokines,
hematopoietic defects). In addition, CD34+ megakaryocyte progenitor
cells are treated in vitro with a tyrosyl-tRNA synthetase
polypeptide of the invention to monitor megakaryocyte expansion and
differentiation (e.g., increase in number of progenitor cells,
stimulation of differentiation, increase in polyploidy). Similar
experiments are performed using bone marrow and spleen cells
derived from mice treated with a tyrosyl-tRNA synthetase
polypeptide.
Example 3
Combination Therapy Stimulates Thrombopoiesis
[0257] To assess whether a tyrosyl-tRNA synthetase polypeptide of
the present invention has a synergestic and/or additive effect on
the proliferation and differentiation of megakaryocytes in vitro,
CD34+ cord blood cells are grown in liquid culture medium in the
presence of optimal or sub-optimal formulations of cytokines
(StemCell Technologies, Vancouver), such as IL-11, and treated with
increasing concentrations of a tyrosyl-tRNA synthetase polypeptide.
Additivity or synergism can be determined by monitoring the growth
and differentiation of the progenitor cells in the two formulation
conditions.
[0258] Similarly, in a protocol comparable to that described in
Example 1, mice are injected with a limiting amount of
thrombopoietin and with increasing amounts of a tyrosyl-tRNA
synthetase polypeptide and the effects of the combination therapy
on thrombopoiesis in vivo can be determined by platelet and
megakaryocyte counts. In addition, combination therapy with limited
amounts of other cytokines, chemokines and/or growth factors
involved in hematopoiesis can be evaluated using the same type of
regimen.
Example 4
Thrombopoietic Activity of Tyrosyl-tRNA Synthetase Polypeptides in
Rats
[0259] The effects of two tyrosyl-tRNA synthetase polypeptides on
thrombopoiesis were measured in rats. The tyrosyl-tRNA synthetase
polypeptides utilized in the experiments described below are: i) a
C-terminal truncation that comprises amino acids 1-364 of the
full-length human tyrosyl-tRNA (SEQ ID NO:3) fused to an eight
amino acid C-terminal histidine tag (SEQ ID NO:5) and; ii) a mutant
of the full length human tyrosyl-tRNA synthetase with a single,
tyrosine to alanine, amino acid substitution at position 341,
referred to as "Y341A" (SEQ ID NO: 2).
[0260] To measure the effects of tyrosyl-tRNA synthetase
polypeptides on thrombopoiesis, platelet count for each rat was
determined one day before the first scheduled injection and animals
were grouped in seven cohorts according to their initial platelet
counts. Three groups of rats were injected intravenously once daily
for seven days with 0.1, 10, and 1000 .mu.g/kg of the C-terminally
truncated tyrosyl-tRNA synthetase polypeptide, respectively. Three
additional groups were administered with the same dosages of Y341A.
One control group received a daily injection of buffer only
(0.5.times.PBS, 2 mM DTT) and an additional control group was
injected daily with 90 .mu.g/kg of thrombopoietin (R&D Systems,
Minneapolis, Minn.).
[0261] Administration of the two tyrosyl-tRNA synthetase
polypeptides resulted in a marked elevation in platelet counts,
comparable or superior to that observed in the thrombopoietin group
(See FIG. 20). These results show that tyrosyl-tRNA synthetase
polypeptides are capable of stimulating thrombopoiesis in vivo.
Example 5
Tyrosyl-tRNA Synthetase Polypeptides are Chemoattractants for
Megakaryocytes
[0262] MO7e cells (DSMZ, Braunschweig, Germany) were cultured in
RPMI-1640 medium supplemented with 20% heat-inactivated FBS and 10
ng/ml IL-3 (R&D Systems, Minneapolis, Minn.). Cells were
maintained at a density of 2.times.10.sup.5 to 1.times.10.sup.6/ml
and RPMI-1640 medium with 0.1% BSA was used as migration buffer.
Before the migration assay, cells were serum-starved for 30 minutes
in migration buffer and loaded with 8 .mu.g/ml calcein AM
(Invitrogen, Carlsbad, Calif.). Cells were spun down at 200 g for 5
minutes without brake and washed once with migration buffer to
remove free calcein AM. Cell density was adjusted to
1.times.10.sup.7/ml and 100 .mu.l were added to 6.5 mm transwell
8.0 .mu.m pore filter inserts (Costar, Cambridge, Mass.). 600 .mu.l
migration buffer containing either PBS, a control chemokine, or the
tyrosyl-tRNA synthetase polypeptides were added to the lower
chamber and cells were allowed to migrate for 4 to 16 hours (for
the 16-hour migration time, cells were stained after migration).
Cells that migrated to the lower chamber were collected and
resuspended in 100 .mu.l PBS, transferred into 384-well opaque
Greiner plate and counted by fluorescence in a plate reader. FIG.
21 shows that the tyrosyl-tRNA synthetase polypeptides are to
stimulate migration of the MO7e megakaryoblasts.
Example 6
Tyrosyl-tRNA Synthetase Polypeptides Promote Cell Adhesion to
Endothelial Monolayers by Stimulating Expression of VCAM-1
[0263] The ability of YRS polypeptides to stimulate adhesion of
THP-1 cells to endothelial monolayers of HUVEC-2 cells was tested.
HUVEC-2 cells (BD Biosciences, San Jose, Calif.) were cultured in
EGM-2 medium (Lonza, Allendale, N.J.) and used before they reached
10 passages. THP-1 cells (ATCC, Manassas, Va.) were cultured in
RPMI-1640 medium supplemented with 10% heat-inactivated FBS and
maintained at a density of 2-4.times.10.sup.5/ml. Cells were seeded
at approximately 1.times.10.sup.4 cells/well into
fibronectin-coated (10 .mu.g/ml, 2 hours at 37.degree. C.), opaque
96-well plates.
[0264] HUVEC-2 cells were grown until a monolayer was formed and
then stimulated overnight in EGM-2 medium with either PBS,
IL-1.beta. or the tyrosyl-tRNA synthetase polypeptides. THP-1 cells
were collected and incubated for 30 minutes in RPMI-1640 serum-free
medium containing 0.1% BSA and calcein AM (6 .mu.l/ml). The cells
were then washed in RPMI-1640 serum-free medium containing 0.1% BSA
and resuspended at a density of 1.5.times.10.sup.5 cells/ml in RPMI
medium containing 10% FBS. 100 .mu.l THP-cells were added to the
HUVEC monolayer and incubated for 15 minutes. Unbound THP-1 cells
were washed with PBS twice and the remaining cells were fixed with
2% formaldehyde and counted by fluorescence in a plate reader.
[0265] FIG. 22 shows adhesion of THP-1 fluorescent cells to an
endothelial monolayer that has been treated with the tyrosyl-tRNA
synthetase polypeptides
[0266] Adhesion molecule expression in endothelial monolayers was
measured following exposure to tyrosyl-tRNA synthetase
polypeptides. 1.times.10.sup.4 HUVEC-2 cells were seeded into a
96-well plate and grown for 48 hours as described in the previous
paragraph. Tyrosyl-tRNA synthetase polypeptides, diluted in growth
media, were added to the wells and incubated for 16 hours. The
culture medium was removed and cells were fixed with 50 .mu.l of Z
fix (Anatech Ltd, Battle Creek, Mich.) for 15 minutes at room
temperature. Wells were subsequently blocked with 50 .mu.l of
casein for 1 hour followed by multiple 200 .mu.l washes with PBS.
All subsequent reagents were diluted in casein and all steps were
performed at room temperature. Antibodies directed against VCAM-1
and E-selectin (Santa Cruz Biotech, Santa Cruz, Calif.) were added
for 1 hour. Wells were then washed as above and an HRP-labeled
secondary antibody (Jackson Immunoresearch, West Grove, Pa.) was
added for 1 hour. Wells were washed and the substrate for HRP was
added. 15 minutes later, an equal volume of 2 M sulfuric acid was
added and absorbance determined at 450 nm.
[0267] FIG. 23 shows an increase in VCAM-1 expression following
stimulation of the endothelial cells with the tyrosyl-tRNA
synthetase polypeptides.
Example 7
Tyrosyl-tRNA Synthetase Polypeptides Stimulate Migration of 293 and
CHO Cell Lines Transfected with the CXCR-2 Receptor
[0268] The effects of tyrosyl-tRNA synthetase polypeptides on
CXCR-2 signaling was tested by measuring the migration of CXCR-2
expressing cells in response to said polypeptides. 293/CXCR-2 cells
were maintained in DMEM medium supplemented with 10%
heat-inactivated FBS, 1% Penicillin-Streptomycin and 800 .mu.g/ml
Geneticin, all purchased from Invitrogen, Carlsbad, Calif. DMEM
medium with 0.1% BSA was used as migration buffer. Prior to
migration assay, cells were serum-starved for 30 minutes in
migration buffer, centrifuged at 200 g for 5 minutes and
resuspended in migration buffer at a final density of
1.times.10.sup.6 cells/ml. 100 .mu.l were added to 6.5 mm transwell
filter inserts (Costar, Cambridge, Mass.) and 600 .mu.l migration
buffer containing a control chemokine, the tyrosyl-tRNA synthetase
polypeptides or buffer only were added to the plate lower chambers.
Cells were allowed to migrate for 4 hours and the remaining cells
in the upper chamber (transwell filter inserts) were removed with a
cotton swap. The filter inserts were then transferred to a new
24-well plate containing 500 .mu.l cell dissociation buffer
(Invitrogen, Carlsbad, Calif.) and 12 .mu.g/ml Calcein AM
(Invitrogen, Carlsbad, Calif.). After 1 hour incubation at
37.degree. C., cells were collected and resuspended in 100 .mu.l
PBS, transferred into a 384-well opaque Greiner plate, and counted
by fluorescence in a plate reader.
[0269] CHO-K1/CXCR-2 cells were maintained in F12 medium
supplemented with 10% heat-inactivated FBS, 1%
Penicillin-Streptomycin-Glutamine and 800 .mu.g/ml Geneticin. F12
medium with 0.5% BSA was used as migration buffer. Prior to
migration, cells were serum-starved for 30 minutes in migration
buffer, collected by using cell dissociation buffer, spun down at
200 g for 5 minutes and resuspended in migration buffer at the
final density of 1.times.10.sup.6 cells/ml. 100 .mu.l were added to
6.5 mm transwell filter inserts and 600 .mu.l migration buffer
containing a control chemokine, the tyrosyl-tRNA synthetase
polypeptides or buffer only were added to the plate lower chambers.
Cells were allowed to migrate for 3 hours and the remaining cells
in the upper chamber (transwell filter inserts) were removed with a
cotton swap. The filter inserts were then transferred to a new
24-well plate containing 500 .mu.l PBS and 12 .mu.g/ml Calcein AM.
After 30 minutes incubation at 37.degree. C., filters were
transferred again into a new 24-well plate containing 500 .mu.l
phenol/red-free trypsin. After 2 to 5 minutes incubation, detached
cells were collected and resuspended in 100 .mu.l PBS, transferred
into a 384 well opaque Greiner plate and counted by fluorescence in
a plate reader.
[0270] FIG. 24 demonstrates the ability of the tyrosyl-tRNA
synthetase polypeptides to induce migration of CXCR-2 transfected
cells.
Example 8
Tyrosyl-tRNA Synthetase Polypeptides Stimulate Polymorphonuclear
(PMN) Cell Migration
[0271] To test the effects of YRS polypeptides on PMN cell
migration, human granulocyte cells were purified from fresh human
peripheral blood using RosetteSep.RTM. Human Granulocyte Enrichment
Kit (StemCell Technologies, Vancouver, BC) according to the
manufacturer's instructions. Serum-free RPMI medium supplemented
with 0.5% FBS was used as migration buffer. 4.times.10.sup.7 cells
were resuspended in 1 ml migration buffer and incubated for 30
minutes with 8 .mu.l of a 1 mg/ml Calcein AM solution (Invitrogen,
Carlsbad, Calif.). Cells were collected, spun down at 200 g for 5
minutes without brake, washed once with migration buffer and
resuspended in the same buffer at a final density of
1.times.10.sup.7/ml.
[0272] 100 .mu.l were added to 6.5 mm transwell filter inserts
(Costar, Cambridge, Mass.) and 600 .mu.l migration buffer
containing a control chemokine, the tyrosyl-tRNA synthetase
polypeptides or buffer only were added to the plate lower chambers.
Cells were allowed to migrate for 45 minutes in the incubator and
cells that migrated to the lower chamber were collected,
resuspended in 100 .mu.l PBS, transferred into a 384-well opaque
Greiner plate and counted by fluorescence in a plate reader.
[0273] FIG. 25 shows the bell-shaped migration curve typically
observed with chemokines. The tyrosyl-tRNA synthetase polypeptides
induced a biphasic migration of PMN both at low pM and at higher
.mu.M concentrations.
Example 9
Tyrosyl-tRNA Synthetase Polypeptides Prevent Neutrophil
Infiltration Into the Lungs after Lipopolysaccharide (LPS)
Challenge
Prophetic Example
[0274] Neutrophil migration from the circulatory system to the
lungs is implicated in chronic pulmonary obstructive disease (COPD)
(see, e.g., R. A. Stockley, Chest 121:151 S-155S, 2002). CXCR-2
expression can play a role in neutrophil migration (see, e.g.,
Rios-Santos et al., American Journal of Respiratory and Critical
Care Medicine 175:490-497, 2007). An animal model is developed to
test the role of tyrosyl-tRNA synthetase polypeptides in COPD. The
tyrosyl-tRNA synthetase polypeptides are administered to animals
intravenously at a concentration and at a frequency necessary to
achieve desensitization of circulating neutrophils prior to, and
during allergen challenge (e.g., between 100 ng/kg and 5 mg/kg and,
e.g., at 12 hours, 1 hour pre-LPS administration and 4 hours
post-LPS administration). The animals are then subjected to
allergen challenge (e.g., LPS instillation into the lungs via the
intranasal route of administration). After 4-8 hours, the animals
are euthanized and a tracheal catheter is inserted to collect
bronchoalveolar lavage (BAL) samples by flushing the lungs with
isotonic saline solution. BAL fluid is analyzed for total cell
counts and differential cell enumeration.
[0275] In this example, the tyrosyl-tRNA synthetase polypeptides
are capable of preventing neutrophil migration to the lung in
response to LPS challenge.
Example 10
Tyrosyl-tRNA Synthetase Polypeptides Impact Megakaryocyte
Progenitor Cells in Bone Marrow Cell Cultures
[0276] To test the effects of YRS polypeptides on megakaryocyte
progenitor cells in bone marrow cell cultures, clonogenic
progenitors of the megakaryocyte (CFU-Mk; Colony Forming
Unit--Megakaryocyte) lineage were assessed in serum-free,
collagen-based media MegaCult-C.RTM. 4950 supplemented with
proprietary concentrations of cytokines (StemCell Technologies,
Vancouver, BC). Normal human bone marrow light density cells
(Lonza, Allendale, N.J.) were stored at -152.degree. C. until
required for the assay. On the day of the experiment, cells were
thawed rapidly at 37.degree. C., the contents of the vial were
diluted in 10 mL of Iscove's modified Dulbecco's medium (IMDM)
containing 2% fetal bovine serum (FBS) and washed by centrifugation
(1200 rpm for 10 minutes, room temperature). The supernatant was
discarded and the cell pellet resuspended in a known volume of IMDM
containing 2% FBS. A cell count (3% glacial acetic acid) and
viability assessment (trypan blue exclusion test) were
performed.
[0277] The tyrosyl-tRNA synthetase polypeptides (stored in 50%
glycerol/0.5.times.PBS/2 mM DTT) were dialyzed in 0.5.times.PBS/2
mM DTT for a total of 5 hours, with one change of buffer after 3
hours in order to remove glycerol. After dialysis, proteins and
buffer sample were sterile filtered and concentration was adjusted
to compensate for the increase in volume.
[0278] Test proteins (YRS polypeptides) were added to tubes of
serum-free, collagen-based media MegaCult-C.RTM. 4950 supplemented
with cytokines (rhTpo, rhIL-3, and rhIL-6). Standard control
cultures (containing no test protein) and solvent control cultures
(containing no test protein but equivalent concentrations of
buffer) were also initiated. Bone marrow cells were then added to
each tube of media to give a final concentration of
1.times.10.sup.5 cells per slide. Bovine collagen was then added,
tubes were vortexed, and contents dispensed into triplicate double
chamber slides. All cultures were incubated for 10-12 days at
37.degree. C., 5% CO.sub.2.
[0279] Following incubation, cultures were assessed microscopically
for colony formation prior to dehydration and fixation of the
slide. Using an antibody staining protocol to detect GPIIa/IIIb
(CD41) expression, the colonies on the slide were stained using an
alkaline phosphatase detection system as described in the StemCell
Technical Manual, "Assays for the Quantitation of Human and Murine
Megakaryocytic Progenitors", Section 7, herein incorporated by
reference in its entirety. Colony numbers were scored and assessed
by trained StemCell personnel. The colonies were divided into the
following categories, based on size and morphology: i) CFU-Mk
(2-20)--the small megakaryocytic colony derived from this more
mature progenitor cell contains 2-20 cells; ii) CFU-Mk--the medium
megakaryocytic colony derived from this more primitive progenitor
cell contains 21-49 cells and; iii) CFU-Mk (>50)--the large
megakaryocytic colony derived from this most primitive
lineage-restricted progenitor cell contains >50 cells.
[0280] FIG. 26 shows the impact of the tyrosyl-tRNA synthetase
polypeptides on the most primitive lineage-restricted progenitors
(stimulation) (FIG. 26(A)), and on the more mature progenitors
(inhibition) (FIGS. 26 (A) and (B)).
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 15 <210> SEQ ID NO 1 <211> LENGTH: 528 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
1 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu His Leu Ile Thr Arg 1
5 10 15 Asn Leu Gln Glu Val Leu Gly Glu Glu Lys Leu Lys Glu Ile Leu
Lys 20 25 30 Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr
Gly Lys Pro 35 40 45 His Val Ala Tyr Phe Val Pro Met Ser Lys Ile
Ala Asp Phe Leu Lys 50 55 60 Ala Gly Cys Glu Val Thr Ile Leu Phe
Ala Asp Leu His Ala Tyr Leu 65 70 75 80 Asp Asn Met Lys Ala Pro Trp
Glu Leu Leu Glu Leu Arg Val Ser Tyr 85 90 95 Tyr Glu Asn Val Ile
Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu 100 105 110 Glu Lys Leu
Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser Lys Glu 115 120 125 Tyr
Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln His Asp 130 135
140 Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His Pro Leu
145 150 155 160 Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp
Glu Glu Tyr 165 170 175 Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp
Gln Arg Lys Ile Phe 180 185 190 Thr Phe Ala Glu Lys Tyr Leu Pro Ala
Leu Gly Tyr Ser Lys Arg Val 195 200 205 His Leu Met Asn Pro Met Val
Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215 220 Ser Ser Glu Glu Glu
Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp 225 230 235 240 Val Lys
Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu 245 250 255
Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys 260
265 270 Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys
Thr 275 280 285 Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala
Glu Val Val 290 295 300 His Pro Gly Asp Leu Lys Asn Ser Val Glu Val
Ala Leu Asn Lys Leu 305 310 315 320 Leu Asp Pro Ile Arg Glu Lys Phe
Asn Thr Pro Ala Leu Lys Lys Leu 325 330 335 Ala Ser Ala Ala Tyr Pro
Asp Pro Ser Lys Gln Lys Pro Met Ala Lys 340 345 350 Gly Pro Ala Lys
Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg Leu 355 360 365 Asp Ile
Arg Val Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala 370 375 380
Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg 385
390 395 400 Thr Val Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu Glu
Leu Gln 405 410 415 Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro
Gln Lys Met Arg 420 425 430 Gly Val Glu Ser Gln Gly Met Leu Leu Cys
Ala Ser Ile Glu Gly Ile 435 440 445 Asn Arg Gln Val Glu Pro Leu Asp
Pro Pro Ala Gly Ser Ala Pro Gly 450 455 460 Glu His Val Phe Val Lys
Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu 465 470 475 480 Leu Lys Pro
Lys Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys 485 490 495 Ile
Ser Glu Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr 500 505
510 Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser
515 520 525 <210> SEQ ID NO 2 <211> LENGTH: 528
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu
His Leu Ile Thr Arg 1 5 10 15 Asn Leu Gln Glu Val Leu Gly Glu Glu
Lys Leu Lys Glu Ile Leu Lys 20 25 30 Glu Arg Glu Leu Lys Ile Tyr
Trp Gly Thr Ala Thr Thr Gly Lys Pro 35 40 45 His Val Ala Tyr Phe
Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys 50 55 60 Ala Gly Cys
Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu 65 70 75 80 Asp
Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr 85 90
95 Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu
100 105 110 Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser
Lys Glu 115 120 125 Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val
Thr Gln His Asp 130 135 140 Ser Lys Lys Ala Gly Ala Glu Val Val Lys
Gln Val Glu His Pro Leu 145 150 155 160 Leu Ser Gly Leu Leu Tyr Pro
Gly Leu Gln Ala Leu Asp Glu Glu Tyr 165 170 175 Leu Lys Val Asp Ala
Gln Phe Gly Gly Ile Asp Gln Arg Lys Ile Phe 180 185 190 Thr Phe Ala
Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val 195 200 205 His
Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215
220 Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp
225 230 235 240 Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly
Asn Val Glu 245 250 255 Asn Asn Gly Val Leu Ser Phe Ile Lys His Val
Leu Phe Pro Leu Lys 260 265 270 Ser Glu Phe Val Ile Leu Arg Asp Glu
Lys Trp Gly Gly Asn Lys Thr 275 280 285 Tyr Thr Ala Tyr Val Asp Leu
Glu Lys Asp Phe Ala Ala Glu Val Val 290 295 300 His Pro Gly Asp Leu
Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu 305 310 315 320 Leu Asp
Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu 325 330 335
Ala Ser Ala Ala Ala Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys 340
345 350 Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg
Leu 355 360 365 Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys His
Pro Asp Ala 370 375 380 Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly
Glu Ala Glu Pro Arg 385 390 395 400 Thr Val Val Ser Gly Leu Val Gln
Phe Val Pro Lys Glu Glu Leu Gln 405 410 415 Asp Arg Leu Val Val Val
Leu Cys Asn Leu Lys Pro Gln Lys Met Arg 420 425 430 Gly Val Glu Ser
Gln Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile 435 440 445 Asn Arg
Gln Val Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly 450 455 460
Glu His Val Phe Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu 465
470 475 480 Leu Lys Pro Lys Lys Lys Val Phe Glu Lys Leu Gln Ala Asp
Phe Lys 485 490 495 Ile Ser Glu Glu Cys Ile Ala Gln Trp Lys Gln Thr
Asn Phe Met Thr 500 505 510 Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu
Lys Gly Gly Asn Ile Ser 515 520 525 <210> SEQ ID NO 3
<211> LENGTH: 364 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 3 Met Gly Asp Ala Pro Ser Pro
Glu Glu Lys Leu His Leu Ile Thr Arg 1 5 10 15 Asn Leu Gln Glu Val
Leu Gly Glu Glu Lys Leu Lys Glu Ile Leu Lys 20 25 30 Glu Arg Glu
Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro 35 40 45 His
Val Ala Tyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys 50 55
60 Ala Gly Cys Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu
65 70 75 80 Asp Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val
Ser Tyr 85 90 95 Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile
Gly Val Pro Leu 100 105 110 Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp
Tyr Gln Leu Ser Lys Glu 115 120 125 Tyr Thr Leu Asp Val Tyr Arg Leu
Ser Ser Val Val Thr Gln His Asp 130 135 140 Ser Lys Lys Ala Gly Ala
Glu Val Val Lys Gln Val Glu His Pro Leu 145 150 155 160 Leu Ser Gly
Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu Glu Tyr 165 170 175 Leu
Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Arg Lys Ile Phe 180 185
190 Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val
195 200 205 His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys
Met Ser 210 215 220 Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp
Arg Lys Glu Asp 225 230 235 240 Val Lys Lys Lys Leu Lys Lys Ala Phe
Cys Glu Pro Gly Asn Val Glu 245 250 255 Asn Asn Gly Val Leu Ser Phe
Ile Lys His Val Leu Phe Pro Leu Lys 260 265 270 Ser Glu Phe Val Ile
Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys Thr 275 280 285 Tyr Thr Ala
Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu Val Val 290 295 300 His
Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu 305 310
315 320 Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys
Leu 325 330 335 Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro
Met Ala Lys 340 345 350 Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val
Ile 355 360 <210> SEQ ID NO 4 <211> LENGTH: 1683
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 4 atgggggacg ctcccagccc tgaagagaaa ctgcacctta
tcacccggaa cctgcaggag 60 gttctggggg aagagaagct gaaggagata
ctgaaggagc gggaacttaa aatttactgg 120 ggaacggcaa ccacgggcaa
accacatgtg gcttactttg tgcccatgtc aaagattgca 180 gacttcttaa
aggcagggtg tgaggtaaca attctgtttg cggacctcca cgcatacctg 240
gataacatga aagccccatg ggaacttcta gaactccgag tcagttacta tgagaatgtg
300 atcaaagcaa tgctggagag cattggtgtg cccttggaga agctcaagtt
catcaaaggc 360 actgattacc agctcagcaa agagtacaca ctagatgtgt
acagactctc ctccgtggtc 420 acacagcacg attccaagaa ggctggagct
gaggtggtaa agcaggtgga gcaccctttg 480 ctgagtggcc tcttataccc
cggactgcag gctttggatg aagagtattt aaaagtagat 540 gcccaatttg
gaggcattga tcagagaaag attttcacct ttgcagagaa gtacctccct 600
gcacttggct attcaaaacg ggtccatctg atgaatccta tggttccagg attaacaggc
660 agcaaaatga gctcttcaga agaggagtcc aagattgatc tccttgatcg
gaaggaggat 720 gtgaagaaaa aactgaagaa ggccttctgt gagccaggaa
atgtggagaa caatggggtt 780 ctgtccttca tcaagcatgt cctttttccc
cttaagtccg agtttgtgat cctacgagat 840 gagaaatggg gtggaaacaa
aacctacaca gcttacgtgg acctggaaaa ggactttgct 900 gctgaggttg
tacatcctgg agacctgaag aattctgttg aagtcgcact gaacaagttg 960
ctggatccaa tccgggaaaa gtttaatacc cctgccctga aaaaactggc cagcgctgcc
1020 tacccagatc cctcaaagca gaagccaatg gccaaaggcc ctgccaagaa
ttcagaacca 1080 gaggaggtca tcccatcccg gctggatatc cgtgtgggga
aaatcatcac tgtggagaag 1140 cacccagatg cagacagcct gtatgtagag
aagattgacg tgggggaagc tgaaccacgg 1200 actgtggtga gcggcctggt
acagttcgtg cccaaggagg aactgcagga caggctggta 1260 gtggtgctgt
gcaacctgaa accccagaag atgagaggag tcgagtccca aggcatgctt 1320
ctgtgtgctt ctatagaagg gataaaccgc caggttgaac ctctggaccc tccggcaggc
1380 tctgctcctg gtgagcacgt gtttgtgaag ggctatgaaa agggccaacc
agatgaggag 1440 ctcaagccca agaagaaagt cttcgagaag ttgcaggctg
acttcaaaat ttctgaggag 1500 tgcatcgcac agtggaagca aaccaacttc
atgaccaagc tgggctccat ttcctgtaaa 1560 tcgctgaaag gggggaacat
tagctagcca gcccagcatc ttcccccctt cttccaccac 1620 tgagtcatct
gctgtctctt cagtctgctc catccatcac ccatttaccc atctctcagg 1680 aca
1683 <210> SEQ ID NO 5 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: C-terminal tag <400>
SEQUENCE: 5 Leu Glu His His His His His His 1 5 <210> SEQ ID
NO 6 <211> LENGTH: 348 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
VARIANT <222> LOCATION: 1, 2, 3, 4, 5, 6, 7, 8, 9 <223>
OTHER INFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 6 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Ile Phe Thr Phe Ala Glu 1 5 10
15 Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val His Leu Met Asn
20 25 30 Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser Ser Ser
Glu Glu 35 40 45 Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp
Val Lys Lys Lys 50 55 60 Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn
Val Glu Asn Asn Gly Val 65 70 75 80 Leu Ser Phe Ile Lys His Val Leu
Phe Pro Leu Lys Ser Glu Phe Val 85 90 95 Ile Leu Arg Asp Glu Lys
Trp Gly Gly Asn Lys Thr Tyr Thr Ala Tyr 100 105 110 Val Asp Leu Glu
Lys Asp Phe Ala Ala Glu Val Val His Pro Gly Asp 115 120 125 Leu Lys
Asn Ser Val Glu Val Ala Leu Asn Lys Leu Leu Asp Pro Ile 130 135 140
Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu Ala Ser Ala Ala 145
150 155 160 Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys Gly Pro
Ala Lys 165 170 175 Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg Leu
Asp Ile Arg Val 180 185 190 Gly Lys Ile Ile Thr Val Glu Lys His Pro
Asp Ala Asp Ser Leu Tyr 195 200 205 Val Glu Lys Ile Asp Val Gly Glu
Ala Glu Pro Arg Thr Val Val Ser 210 215 220 Gly Leu Val Gln Phe Val
Pro Lys Glu Glu Leu Gln Asp Arg Leu Val 225 230 235 240 Val Val Leu
Cys Asn Leu Lys Pro Gln Lys Met Arg Gly Val Glu Ser 245 250 255 Gln
Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile Asn Arg Gln Val 260 265
270 Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly Glu His Val Phe
275 280 285 Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu Leu Lys
Pro Lys 290 295 300 Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys
Ile Ser Glu Glu 305 310 315 320 Cys Ile Ala Gln Trp Lys Gln Thr Asn
Phe Met Thr Lys Leu Gly Ser 325 330 335 Ile Ser Cys Lys Ser Leu Lys
Gly Gly Asn Ile Ser 340 345 <210> SEQ ID NO 7 <211>
LENGTH: 2178 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 7 ttcagaaagt ggtggaggga agacttcctt
tttcccagag acagaaggtt atgcacccag 60 tggcctggga ccattgttct
gggctttttt tcccttcgac atggatttgc ttctcactgt 120 gtaccccaac
caccaaaacc accctgagat caatgctggt gctcctgcat cagatggctt 180
agagatcctt ccacctctta acacaagcat ctaggtccac tttactcaaa tctggcctca
240 gttgagagca gagtatacca tcagagccca ttctcctgtc tgctgtctgg
gacgtggaaa 300 gaaagttagc tctagggggt ctttccaggg gcctctgtaa
ggactggatg ctcctttccg 360 gaatccaaga gttcaccagg ctgcttctct
aatggacgat gatcctcttc ctcctgacgt 420 ctctccctgg cagcacccag
atgcagacag cctgtatgta gagaagattg acgtggggga 480 agctgaacca
cggactgtgg tgagcggcct ggtacagttc gtgcccaagg aggaactgca 540
ggacaggctg gtagtggtgc tgtgcaacct gaaaccccag aagatgagag gagtcgagtc
600 ccaaggcatg cttctgtgtg cttctatgtg agtgaggact tggagtgggg
cacaggacct 660 ggggaggcca ggaagagtag ggaatcagcc catatgatgt
ccttccacac accaggtgga 720 agctctgaga acacgtgcct cttccttgct
gatgccaaaa gttgatgcat gaaggactta 780 tcgtacaagt actgttaatg
aagcatttta cctacagtta attttgttaa aatagaaatg 840 gagggctcaa
accagtacat acccaagtct tactactagt aaggagtgga gcagggattc 900
aaatcccagt tttgatgtct ataaagtcct cgctacgtta ttttatactt cctcccctag
960 aaacacagat tttggtatct tgacacacaa ttttggtata gcctgggtta
atgtaaccct 1020 ggtgatatgc agggatgtag caagataaga ggacctcctg
gggctctggt actgaggatg 1080 ccctaaatcc catcagggcc cctgtgtaaa
ggcccggatt gctttggcct ccacagtcac 1140 tggaacccat ccatagcctc
actcttctct tgtcctgtgt cttcccagag aagggataaa 1200 ccgccaggtt
gaacctctgg accctccggc aggctctgct cctggtgagc acgtgtttgt 1260
gaagggctat gaaaagggcc aaccagatga ggagctcaag cccaagagga aagtcttcga
1320 gaagttgcag gctgacttca aaatttctga ggagtgcatc gcacagtgga
agcaaaccaa 1380 cttcatgacc aagctgggct ccatttcctg taaatcgctg
aaagggggga acattagcta 1440 gccagcccag catcttcccc ccttcttcca
ccactgagtc atctgctgtc tcttcagtct 1500 gctccaccca tcacccattt
acccatctct caggacacgg aagcagcggg tttggactct 1560 ttattcggtg
cagaactcgg caaggggcag cttaccctcc ccagaaccca ggatcatcct 1620
gtctggctgc agtgagagac caacccctaa caagggctgg gccacagcag ggagtccagc
1680 cctaccttct tcccttggca gctggagaaa tctggtttca atataactca
tttaaaaatt 1740 tatgccacag tccttataat tggaaaaata ctggtgccca
ggttttcttg gagttatcca 1800 agcagctgcg cccctagctg ggatctggta
cctggactag gctaattaca gcttctcccc 1860 aacaggaaac tgtgggattt
gaaaaggaaa gggaagggaa aacagagaac ctagtggtct 1920 accaagtggt
tggcaacttt cccaatgtct gcttactctg aggcttggca ctgggggcca 1980
gggcctgccc cagggctcct ggaatttccc ttgatccagc taggctggga cactccctaa
2040 atcagctgcg tgttgttagc atcaggcaga atgaatggca gagagtgatt
ctgtcttcat 2100 agagggtggg gtacttctcc ataaggcatc tcagtcaaat
ccccatcact gtcataaatt 2160 caaataaaat gtctgaac 2178 <210> SEQ
ID NO 8 <211> LENGTH: 388 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
VARIANT <222> LOCATION: 354, 355, 356, 357, 358, 359, 360,
361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,
374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,
387, 388 <223> OTHER INFORMATION: Xaa = Any Amino Acid
<400> SEQUENCE: 8 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu
His Leu Ile Thr Arg 1 5 10 15 Asn Leu Gln Glu Val Leu Gly Glu Glu
Lys Leu Lys Glu Ile Leu Lys 20 25 30 Glu Arg Glu Leu Lys Ile Tyr
Trp Gly Thr Ala Thr Thr Gly Lys Pro 35 40 45 His Val Ala Tyr Phe
Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys 50 55 60 Ala Gly Cys
Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu 65 70 75 80 Asp
Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr 85 90
95 Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu
100 105 110 Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser
Lys Glu 115 120 125 Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val
Thr Gln His Asp 130 135 140 Ser Lys Lys Ala Gly Ala Glu Val Val Lys
Gln Val Glu His Pro Leu 145 150 155 160 Leu Ser Gly Leu Leu Tyr Pro
Gly Leu Gln Ala Leu Asp Glu Glu Tyr 165 170 175 Leu Lys Val Asp Ala
Gln Phe Gly Gly Ile Asp Gln Arg Lys Ile Phe 180 185 190 Thr Phe Ala
Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val 195 200 205 His
Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215
220 Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp
225 230 235 240 Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly
Asn Val Glu 245 250 255 Asn Asn Gly Val Leu Ser Phe Ile Lys His Val
Leu Phe Pro Leu Lys 260 265 270 Ser Glu Phe Val Ile Leu Arg Asp Glu
Lys Trp Gly Gly Asn Lys Thr 275 280 285 Tyr Thr Ala Tyr Val Asp Leu
Glu Lys Asp Phe Ala Ala Glu Val Val 290 295 300 His Pro Gly Asp Leu
Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu 305 310 315 320 Leu Asp
Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu 325 330 335
Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys 340
345 350 Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa 385 <210> SEQ ID NO 9
<211> LENGTH: 1167 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 9 atgggggacg
ctcccagccc tgaagagaaa ctgcacctta tcacccggaa cctgcaggag 60
gttctggggg aagagaagct gaaggagata ctgaaggagc gggaacttaa aatttactgg
120 ggaacggcaa ccacgggcaa accacatgtg gcttactttg tgcccatgtc
aaagattgca 180 gacttcttaa aggcagggtg tgaggtaaca attctgtttg
cggacctcca cgcatacctg 240 gataacatga aagccccatg ggaacttcta
gaactccgag tcagttacta tgagaatgtg 300 atcaaagcaa tgctggagag
cattggtgtg cccttggaga agctcaagtt catcaaaggc 360 actgattacc
agctcagcaa agagtacaca ctagatgtgt acagactctc ctccgtggtc 420
acacagcacg attccaagaa ggctggagct gaggtggtaa agcaggtgga gcaccctttg
480 ctgagtggcc tcttataccc cggactgcag gctttggatg aagagtattt
aaaagtagat 540 gcccaatttg gaggcattga tcagagaaag attttcacct
ttgcagagaa gtacctccct 600 gcacttggct attcaaaacg ggtccatctg
atgaatccta tggttccagg attaacaggc 660 agcaaaatga gctcttcaga
agaggagtcc aagattgatc tccttgatcg gaaggaggat 720 gtgaagaaaa
aactgaagaa ggccttctgt gagccaggaa atgtggagaa caatggggtt 780
ctgtccttca tcaagcatgt cctttttccc cttaagtccg agtttgtgat cctacgagat
840 gagaaatggg gtggaaacaa aacctacaca gcttacgtgg acctggaaaa
ggactttgct 900 gctgaggttg tacatcctgg agacctgaag aattctgttg
aagtcgcact gaacaagttg 960 ctggatccaa tccgggaaaa gtttaatacc
cctgccctga aaaaactggc cagcgctgcc 1020 tacccagatc cctcaaagca
gaagccaatg gccaaaggcc tgccaagaat tcagaaccag 1080 aggaggtcat
cccatcccgg ctggatatcc gtgtggggaa aatcatcact gtggagaagc 1140
acccagatgc agacagcctg tatgtag 1167 <210> SEQ ID NO 10
<211> LENGTH: 318 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 10 Met Asn Pro Met Val Pro Gly
Leu Thr Gly Ser Lys Met Ser Ser Ser 1 5 10 15 Glu Glu Glu Ser Lys
Ile Asp Leu Leu Asp Arg Lys Glu Asp Val Lys 20 25 30 Lys Lys Leu
Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu Asn Asn 35 40 45 Gly
Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys Ser Glu 50 55
60 Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys Thr Tyr Thr
65 70 75 80 Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu Val Val
His Pro 85 90 95 Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn
Lys Leu Leu Asp 100 105 110 Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala
Leu Lys Lys Leu Ala Ser 115 120 125 Ala Ala Tyr Pro Asp Pro Ser Lys
Gln Lys Pro Met Ala Lys Gly Pro 130 135 140 Ala Lys Asn Ser Glu Pro
Glu Glu Val Ile Pro Ser Arg Leu Asp Ile 145 150 155 160 Arg Val Gly
Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala Asp Ser 165 170 175 Leu
Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg Thr Val 180 185
190 Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu Glu Leu Gln Asp Arg
195 200 205 Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln Lys Met Arg
Gly Val 210 215 220 Glu Ser Gln Gly Met Leu Leu Cys Ala Ser Ile Glu
Gly Ile Asn Arg 225 230 235 240 Gln Val Glu Pro Leu Asp Pro Pro Ala
Gly Ser Ala Pro Gly Glu His 245 250 255 Val Phe Val Lys Gly Tyr Glu
Lys Gly Gln Pro Asp Glu Glu Leu Lys 260 265 270 Pro Lys Lys Lys Val
Phe Glu Lys Leu Gln Ala Asp Phe Lys Ile Ser 275 280 285 Glu Glu Cys
Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr Lys Leu 290 295 300 Gly
Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser 305 310 315
<210> SEQ ID NO 11 <211> LENGTH: 1736 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 11
gaaagatttt cacctttgca gagaagtacc tccctgcact tggctattca aaacgggtcc
60 atctgatgaa tcctatggtt ccaggattaa caggcagcaa aatgagctct
tcagaagagg 120 agtccaagat tgatctcctt gatcggaagg aggatgtgaa
gaaaaaactg aagaaggcct 180 tctgtgagcc aggaaatgtg gagaacaatg
gggttctgtc cttcatcaag catgtccttt 240 ttccccttaa gtccgagttt
gtgatcctac gagatgagaa atggggtgga aacaaaacct 300 acacagctta
cgtggacctg gaaaaggact ttgctgctga ggttgtacat cctggagacc 360
tgaagaattc tgttgaagtc gcactgaaca agttgctgga tccaatccgg gaaaagttta
420 atacccctgc cctgaaaaaa ctggccagcg ctgcctaccc agatccctca
aagcagaagc 480 caatggccaa aggccctgcc aagaattcag aaccagagga
ggtcatccca tcccggctgg 540 atatccgtgt ggggaaaatc atcactgtgg
agaagcaccc agatgcagac agcctgtatg 600 tagagaagat tgacgtgggg
gaagctgaac cacggactgt ggtgagcggc ctggtacagt 660 tcgtgcccaa
ggaggaactg caggacaggc tggtagtggt gctgtgcaac ctgaaacccc 720
agaagatgag aggagtcgag tcccaaggca tgcttctgtg tgcttctata gaagggataa
780 accgccaggt tgaacctctg gaccctccgg caggctctgc tcctggtgag
cacgtgtttg 840 tgaagggcta tgaaaagggc caaccagatg aggagctcaa
gcccaagaag aaagtcttcg 900 agaagttgca ggctgacttc aaaatttctg
aggagtgcat cgcacagtgg aagcaaacca 960 acttcatgac caagctgggc
tccatttcct gtaaatcgct gaaagggggg aacattagct 1020 agccagccca
gcatcttccc cccttcttcc accactgagt catctgctgt ctcttcagtc 1080
tgctccatcc atcacccatt tacccatctc tcaggacacg gaagcagcgg gtttggactc
1140 tttattcggt gcagaactcg gcaaggggca gcttaccctc cccagaaccc
aggatcatcc 1200 tgtctggctg cagtgagaga ccaaccccta acaagggctg
ggccacagca gggagtccag 1260 ccctaccttc ttcccttggc agctggagaa
atctggtttc aatataactc atttaaaaat 1320 ttatgccaca gtccttataa
ttggaaaaat actggtgccc aggttttctt ggagttatcc 1380 aagcagctgc
gcccctagct gggatctggt acctggacta ggctaattac agcttctccc 1440
caacaggaaa ctgtgggatt tgaaaaggaa agggaaggga aaacagagaa cctagtggtc
1500 taccaagtgg ttggcaactt tcccaatgtc tgcttactct gaggcttggc
actgggggcc 1560 agggcctgcc ccagggctcc tggaatttcc cttgatccag
ctaggctggg acactcccta 1620 aatcagctgc gtgttgttag catcaggcag
aatgaatggc agagagtgat tctgtcttca 1680 tagagggtgg ggtacttctc
cataaggcat ctcagtcaaa tccccatcac tgtcat 1736 <210> SEQ ID NO
12 <211> LENGTH: 179 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 12 Met Ala Lys Gly Pro
Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Pro 1 5 10 15 Ser Arg Leu
Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys His 20 25 30 Pro
Asp Ala Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu Ala 35 40
45 Glu Pro Arg Thr Val Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu
50 55 60 Glu Leu Gln Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys
Pro Gln 65 70 75 80 Lys Met Arg Gly Val Glu Ser Gln Gly Met Leu Leu
Cys Ala Ser Ile 85 90 95 Glu Gly Ile Asn Arg Gln Val Glu Pro Leu
Asp Pro Pro Ala Gly Ser 100 105 110 Ala Pro Gly Glu His Val Phe Val
Lys Gly Tyr Glu Lys Gly Gln Pro 115 120 125 Asp Glu Glu Leu Lys Pro
Lys Lys Lys Val Phe Glu Lys Leu Gln Ala 130 135 140 Asp Phe Lys Ile
Ser Glu Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn 145 150 155 160 Phe
Met Thr Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly 165 170
175 Asn Ile Ser <210> SEQ ID NO 13 <211> LENGTH: 1167
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 13 atgggggacg ctcccagccc tgaagagaaa
ctgcacctta tcacccggaa cctgcaggag 60 gttctggggg aagagaagct
gaaggagata ctgaaggagc gggaacttaa aatttactgg 120 ggaacggcaa
ccacgggcaa accacatgtg gcttactttg tgcccatgtc aaagattgca 180
gacttcttaa aggcagggtg tgaggtaaca attctgtttg cggacctcca cgcatacctg
240 gataacatga aagccccatg ggaacttcta gaactccgag tcagttacta
tgagaatgtg 300 atcaaagcaa tgctggagag cattggtgtg cccttggaga
agctcaagtt catcaaaggc 360 actgattacc agctcagcaa agagtacaca
ctagatgtgt acagactctc ctccgtggtc 420 acacagcacg attccaagaa
ggctggagct gaggtggtaa agcaggtgga gcaccctttg 480 ctgagtggcc
tcttataccc cggactgcag gctttggatg aagagtattt aaaagtagat 540
gcccaatttg gaggcattga tcagagaaag attttcacct ttgcagagaa gtacctccct
600 gcacttggct attcaaaacg ggtccatctg atgaatccta tggttccagg
attaacaggc 660 agcaaaatga gctcttcaga agaggagtcc aagattgatc
tccttgatcg gaaggaggat 720 gtgaagaaaa aactgaagaa ggccttctgt
gagccaggaa atgtggagaa caatggggtt 780 ctgtccttca tcaagcatgt
cctttttccc cttaagtccg agtttgtgat cctacgagat 840 gagaaatggg
gtggaaacaa aacctacaca gcttacgtgg acctggaaaa ggactttgct 900
gctgaggttg tacatcctgg agacctgaag aattctgttg aagtcgcact gaacaagttg
960 ctggatccaa tccgggaaaa gtttaatacc cctgccctga aaaaactggc
cagcgctgcc 1020 tacccagatc cctcaaagca gaagccaatg gccaaaggcc
tgccaagaat tcagaaccag 1080 aggaggtcat cccatcccgg ctggatatcc
gtgtggggaa aatcatcact gtggagaagc 1140 acccagatgc agacagcctg tatgtag
1167 <210> SEQ ID NO 14 <211> LENGTH: 188 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: VARIANT <222> LOCATION: 1, 2, 3, 4, 5,
6, 7, 8 <223> OTHER INFORMATION: Xaa = Any Amino Acid
<400> SEQUENCE: 14 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Met
Ala Lys Gly Pro Ala Lys 1 5 10 15 Asn Ser Glu Pro Glu Glu Val Ile
Pro Ser Arg Leu Asp Ile Arg Val 20 25 30 Gly Lys Ile Ile Thr Val
Glu Lys His Pro Asp Ala Asp Ser Leu Tyr 35 40 45 Val Glu Lys Ile
Asp Val Gly Glu Ala Glu Pro Arg Thr Val Val Ser 50 55 60 Gly Leu
Val Gln Phe Val Pro Lys Glu Glu Leu Gln Asp Arg Leu Val 65 70 75 80
Val Val Leu Cys Asn Leu Lys Pro Gln Lys Met Arg Gly Val Glu Ser 85
90 95 Gln Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile Asn Arg Gln
Val 100 105 110 Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly Glu
His Val Phe 115 120 125 Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu
Glu Leu Lys Pro Lys 130 135 140 Lys Lys Val Phe Glu Lys Leu Gln Ala
Asp Phe Lys Ile Ser Glu Glu 145 150 155 160 Cys Ile Ala Gln Trp Lys
Gln Thr Asn Phe Met Thr Lys Leu Gly Ser 165 170 175 Ile Ser Cys Lys
Ser Leu Lys Gly Gly Asn Ile Ser 180 185 <210> SEQ ID NO 15
<211> LENGTH: 2262 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 15 gccagacaca
gtggctcaca cctgtaatct taacactttg gaaggctgag gcaggcggat 60
cacttgagcc caaaagttag agaccaaaac ccagtctcta cccaaaaaaa aaaaaaaaaa
120 aaaaattagc caggcatagt agcacatgcc tgtagtccca gctacttggg
aggctgaggt 180 gagaggatca cctgagcatg gggaagttga gactgcagtg
agccatgatc gcaccactgc 240 actccagcct gggcaacaga gtgagactct
atgtctcaaa aaaagaaaaa tgatagaaat 300 tagattagac ctattatacc
caaccggtat atagggtatc gatagtttct tacacagctg 360 ttgggcagag
cctgcagagc ttagagaagc ttatctttag attctcccag tttccttcta 420
tgtgcatggg cctggctctt agttggccat ccacttgtgc gtaatgctaa gatattggca
480 ttgatagctt tgtgcgaccc ttccagaaaa aaactcagta actcagtaaa
attttttttt 540 ttttttctaa aagagacaga gtctggctct gttgcccagc
ctggtcttga agtcctgggc 600 ttaagcaatc ctcccgtctc agcctcccaa
agtgctagaa ttacaggtgt gagctaccac 660 acctggccaa gactcagtaa
attctatgtg gaatgcatga atggaaatac ctaaaggagg 720 caaagctact
actgctccct ccccgctagt ctaataattg agggagagaa cagatgaaaa 780
tcaggtatgt catgtctgaa aggttgccaa cccagtatta aagaagttac aactcagtgt
840 ttagactctg gggattctac actaaatctt acctaatctc agtgtcttaa
cgtggtggga 900 tcagcagctg acctgccaca gggaagaatt ctacctcatg
gggttcttct cattcccaga 960 gccaatggcc aaaggccctg ccaagaattc
agaaccagag gaggtcatcc catcccggct 1020 ggatatccgt gtggggaaaa
tcatcactgt ggagaagcac ccagatgcag acagcctgta 1080 tgtagagaag
attgacgtgg gggaagctga accacggact gtggtgagcg gcctggtaca 1140
gttcgtgccc aaggaggaac tgcaggacag gctggtagtg gtgctgtgca acctgaaacc
1200 ccagaagatg agaggagtcg agtcccaagg catgcttctg tgtgcttcta
tagaagggat 1260 aaaccgccag gttgaacctc tggaccctcc ggcaggctct
gctcctggtg agcacgtgtt 1320 tgtgaagggc tatgaaaagg gccaaccaga
tgaggagctc aagcccaaga agaaagtctt 1380 cgagaagttg caggctgact
tcaaaatttc tgaggagtgc atcgcacagt ggaagcaaac 1440 caacttcatg
accaagctgg gctccatttc ctgtaaatcg ctgaaagggg ggaacattag 1500
ctagccagcc cagcatcttc cccccttctt ccaccactga gtcatctgct gtctcttcag
1560 tctgctccat ccatcaccca tttacccatc tctcaggaca cggaagcagc
gggtttggac 1620 tctttattcg gtgcagaact cggcaagggg cagcttaccc
tccccagaac ccaggatcat 1680 cctgtctggc tgcagtgaga gaccaacccc
taacaagggc tgggccacag cagggagtcc 1740 agccctacct tcttcccttg
gcagctggag aaatctggtt tcaatataac tcatttaaaa 1800 atttatgcca
cagtccttat aattggaaaa atactggtgc ccaggttttc ttggagttat 1860
ccaagcagct gcgcccctag ctgggatctg gtacctggac taggctaatt acagcttctc
1920 cccaacagga aactgtggga tttgaaaagg aaagggaagg gaaaacagag
aacctagtgg 1980 tctaccaagt ggttggcaac tttcccaatg tctgcttact
ctgaggcttg gcactggggg 2040 ccagggcctg ccccagggct cctggaattt
cccttgatcc agctaggctg ggacactccc 2100 taaatcagct gcgtgttgtt
agcatcaggc agaatgaatg gcagagagtg attctgtctt 2160 catagagggt
ggggtacttc tccataaggc atctcagtca aatccccatc actgtcataa 2220
attcaaataa aatgtctgaa caagggaaaa aaaaaaaaaa aa 2262
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 15 <210>
SEQ ID NO 1 <211> LENGTH: 528 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 1 Met Gly
Asp Ala Pro Ser Pro Glu Glu Lys Leu His Leu Ile Thr Arg 1 5 10 15
Asn Leu Gln Glu Val Leu Gly Glu Glu Lys Leu Lys Glu Ile Leu Lys 20
25 30 Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys
Pro 35 40 45 His Val Ala Tyr Phe Val Pro Met Ser Lys Ile Ala Asp
Phe Leu Lys 50 55 60 Ala Gly Cys Glu Val Thr Ile Leu Phe Ala Asp
Leu His Ala Tyr Leu 65 70 75 80 Asp Asn Met Lys Ala Pro Trp Glu Leu
Leu Glu Leu Arg Val Ser Tyr 85 90 95 Tyr Glu Asn Val Ile Lys Ala
Met Leu Glu Ser Ile Gly Val Pro Leu 100 105 110 Glu Lys Leu Lys Phe
Ile Lys Gly Thr Asp Tyr Gln Leu Ser Lys Glu 115 120 125 Tyr Thr Leu
Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln His Asp 130 135 140 Ser
Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His Pro Leu 145 150
155 160 Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu Glu
Tyr 165 170 175 Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Arg
Lys Ile Phe 180 185 190 Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly
Tyr Ser Lys Arg Val 195 200 205 His Leu Met Asn Pro Met Val Pro Gly
Leu Thr Gly Ser Lys Met Ser 210 215 220 Ser Ser Glu Glu Glu Ser Lys
Ile Asp Leu Leu Asp Arg Lys Glu Asp 225 230 235 240 Val Lys Lys Lys
Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu 245 250 255 Asn Asn
Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys 260 265 270
Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys Thr 275
280 285 Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu Val
Val 290 295 300 His Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu
Asn Lys Leu 305 310 315 320 Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr
Pro Ala Leu Lys Lys Leu 325 330 335 Ala Ser Ala Ala Tyr Pro Asp Pro
Ser Lys Gln Lys Pro Met Ala Lys 340 345 350 Gly Pro Ala Lys Asn Ser
Glu Pro Glu Glu Val Ile Pro Ser Arg Leu 355 360 365 Asp Ile Arg Val
Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala 370 375 380 Asp Ser
Leu Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg 385 390 395
400 Thr Val Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu Glu Leu Gln
405 410 415 Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln Lys
Met Arg 420 425 430 Gly Val Glu Ser Gln Gly Met Leu Leu Cys Ala Ser
Ile Glu Gly Ile 435 440 445 Asn Arg Gln Val Glu Pro Leu Asp Pro Pro
Ala Gly Ser Ala Pro Gly 450 455 460 Glu His Val Phe Val Lys Gly Tyr
Glu Lys Gly Gln Pro Asp Glu Glu 465 470 475 480 Leu Lys Pro Lys Lys
Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys 485 490 495 Ile Ser Glu
Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr 500 505 510 Lys
Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser 515 520
525 <210> SEQ ID NO 2 <211> LENGTH: 528 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
2 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu His Leu Ile Thr Arg 1
5 10 15 Asn Leu Gln Glu Val Leu Gly Glu Glu Lys Leu Lys Glu Ile Leu
Lys 20 25 30 Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr
Gly Lys Pro 35 40 45 His Val Ala Tyr Phe Val Pro Met Ser Lys Ile
Ala Asp Phe Leu Lys 50 55 60 Ala Gly Cys Glu Val Thr Ile Leu Phe
Ala Asp Leu His Ala Tyr Leu 65 70 75 80 Asp Asn Met Lys Ala Pro Trp
Glu Leu Leu Glu Leu Arg Val Ser Tyr 85 90 95 Tyr Glu Asn Val Ile
Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu 100 105 110 Glu Lys Leu
Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser Lys Glu 115 120 125 Tyr
Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln His Asp 130 135
140 Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His Pro Leu
145 150 155 160 Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp
Glu Glu Tyr 165 170 175 Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp
Gln Arg Lys Ile Phe 180 185 190 Thr Phe Ala Glu Lys Tyr Leu Pro Ala
Leu Gly Tyr Ser Lys Arg Val 195 200 205 His Leu Met Asn Pro Met Val
Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215 220 Ser Ser Glu Glu Glu
Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp 225 230 235 240 Val Lys
Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu 245 250 255
Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys 260
265 270 Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys
Thr 275 280 285 Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala
Glu Val Val 290 295 300 His Pro Gly Asp Leu Lys Asn Ser Val Glu Val
Ala Leu Asn Lys Leu 305 310 315 320 Leu Asp Pro Ile Arg Glu Lys Phe
Asn Thr Pro Ala Leu Lys Lys Leu 325 330 335 Ala Ser Ala Ala Ala Pro
Asp Pro Ser Lys Gln Lys Pro Met Ala Lys 340 345 350 Gly Pro Ala Lys
Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg Leu 355 360 365 Asp Ile
Arg Val Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala 370 375 380
Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg 385
390 395 400 Thr Val Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu Glu
Leu Gln 405 410 415 Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro
Gln Lys Met Arg 420 425 430 Gly Val Glu Ser Gln Gly Met Leu Leu Cys
Ala Ser Ile Glu Gly Ile 435 440 445 Asn Arg Gln Val Glu Pro Leu Asp
Pro Pro Ala Gly Ser Ala Pro Gly 450 455 460 Glu His Val Phe Val Lys
Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu 465 470 475 480 Leu Lys Pro
Lys Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys 485 490 495 Ile
Ser Glu Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr 500 505
510 Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser
515 520 525 <210> SEQ ID NO 3 <211> LENGTH: 364
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 3 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu
His Leu Ile Thr Arg 1 5 10 15 Asn Leu Gln Glu Val Leu Gly Glu Glu
Lys Leu Lys Glu Ile Leu Lys 20 25 30 Glu Arg Glu Leu Lys Ile Tyr
Trp Gly Thr Ala Thr Thr Gly Lys Pro 35 40 45 His Val Ala Tyr Phe
Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys 50 55 60 Ala Gly Cys
Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu 65 70 75 80 Asp
Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr 85 90
95 Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu
100 105 110 Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser
Lys Glu 115 120 125
Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln His Asp 130
135 140 Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His Pro
Leu 145 150 155 160 Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu
Asp Glu Glu Tyr 165 170 175 Leu Lys Val Asp Ala Gln Phe Gly Gly Ile
Asp Gln Arg Lys Ile Phe 180 185 190 Thr Phe Ala Glu Lys Tyr Leu Pro
Ala Leu Gly Tyr Ser Lys Arg Val 195 200 205 His Leu Met Asn Pro Met
Val Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215 220 Ser Ser Glu Glu
Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp 225 230 235 240 Val
Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu 245 250
255 Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys
260 265 270 Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn
Lys Thr 275 280 285 Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala
Ala Glu Val Val 290 295 300 His Pro Gly Asp Leu Lys Asn Ser Val Glu
Val Ala Leu Asn Lys Leu 305 310 315 320 Leu Asp Pro Ile Arg Glu Lys
Phe Asn Thr Pro Ala Leu Lys Lys Leu 325 330 335 Ala Ser Ala Ala Tyr
Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys 340 345 350 Gly Pro Ala
Lys Asn Ser Glu Pro Glu Glu Val Ile 355 360 <210> SEQ ID NO 4
<211> LENGTH: 1683 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 4 atgggggacg
ctcccagccc tgaagagaaa ctgcacctta tcacccggaa cctgcaggag 60
gttctggggg aagagaagct gaaggagata ctgaaggagc gggaacttaa aatttactgg
120 ggaacggcaa ccacgggcaa accacatgtg gcttactttg tgcccatgtc
aaagattgca 180 gacttcttaa aggcagggtg tgaggtaaca attctgtttg
cggacctcca cgcatacctg 240 gataacatga aagccccatg ggaacttcta
gaactccgag tcagttacta tgagaatgtg 300 atcaaagcaa tgctggagag
cattggtgtg cccttggaga agctcaagtt catcaaaggc 360 actgattacc
agctcagcaa agagtacaca ctagatgtgt acagactctc ctccgtggtc 420
acacagcacg attccaagaa ggctggagct gaggtggtaa agcaggtgga gcaccctttg
480 ctgagtggcc tcttataccc cggactgcag gctttggatg aagagtattt
aaaagtagat 540 gcccaatttg gaggcattga tcagagaaag attttcacct
ttgcagagaa gtacctccct 600 gcacttggct attcaaaacg ggtccatctg
atgaatccta tggttccagg attaacaggc 660 agcaaaatga gctcttcaga
agaggagtcc aagattgatc tccttgatcg gaaggaggat 720 gtgaagaaaa
aactgaagaa ggccttctgt gagccaggaa atgtggagaa caatggggtt 780
ctgtccttca tcaagcatgt cctttttccc cttaagtccg agtttgtgat cctacgagat
840 gagaaatggg gtggaaacaa aacctacaca gcttacgtgg acctggaaaa
ggactttgct 900 gctgaggttg tacatcctgg agacctgaag aattctgttg
aagtcgcact gaacaagttg 960 ctggatccaa tccgggaaaa gtttaatacc
cctgccctga aaaaactggc cagcgctgcc 1020 tacccagatc cctcaaagca
gaagccaatg gccaaaggcc ctgccaagaa ttcagaacca 1080 gaggaggtca
tcccatcccg gctggatatc cgtgtgggga aaatcatcac tgtggagaag 1140
cacccagatg cagacagcct gtatgtagag aagattgacg tgggggaagc tgaaccacgg
1200 actgtggtga gcggcctggt acagttcgtg cccaaggagg aactgcagga
caggctggta 1260 gtggtgctgt gcaacctgaa accccagaag atgagaggag
tcgagtccca aggcatgctt 1320 ctgtgtgctt ctatagaagg gataaaccgc
caggttgaac ctctggaccc tccggcaggc 1380 tctgctcctg gtgagcacgt
gtttgtgaag ggctatgaaa agggccaacc agatgaggag 1440 ctcaagccca
agaagaaagt cttcgagaag ttgcaggctg acttcaaaat ttctgaggag 1500
tgcatcgcac agtggaagca aaccaacttc atgaccaagc tgggctccat ttcctgtaaa
1560 tcgctgaaag gggggaacat tagctagcca gcccagcatc ttcccccctt
cttccaccac 1620 tgagtcatct gctgtctctt cagtctgctc catccatcac
ccatttaccc atctctcagg 1680 aca 1683 <210> SEQ ID NO 5
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: C-terminal tag <400> SEQUENCE: 5 Leu Glu His His
His His His His 1 5 <210> SEQ ID NO 6 <211> LENGTH: 348
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: VARIANT <222>
LOCATION: 1, 2, 3, 4, 5, 6, 7, 8, 9 <223> OTHER INFORMATION:
Xaa = Any Amino Acid <400> SEQUENCE: 6 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Lys Ile Phe Thr Phe Ala Glu 1 5 10 15 Lys Tyr Leu
Pro Ala Leu Gly Tyr Ser Lys Arg Val His Leu Met Asn 20 25 30 Pro
Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser Ser Ser Glu Glu 35 40
45 Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp Val Lys Lys Lys
50 55 60 Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu Asn Asn
Gly Val 65 70 75 80 Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys
Ser Glu Phe Val 85 90 95 Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn
Lys Thr Tyr Thr Ala Tyr 100 105 110 Val Asp Leu Glu Lys Asp Phe Ala
Ala Glu Val Val His Pro Gly Asp 115 120 125 Leu Lys Asn Ser Val Glu
Val Ala Leu Asn Lys Leu Leu Asp Pro Ile 130 135 140 Arg Glu Lys Phe
Asn Thr Pro Ala Leu Lys Lys Leu Ala Ser Ala Ala 145 150 155 160 Tyr
Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys Gly Pro Ala Lys 165 170
175 Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg Leu Asp Ile Arg Val
180 185 190 Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala Asp Ser
Leu Tyr 195 200 205 Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg
Thr Val Val Ser 210 215 220 Gly Leu Val Gln Phe Val Pro Lys Glu Glu
Leu Gln Asp Arg Leu Val 225 230 235 240 Val Val Leu Cys Asn Leu Lys
Pro Gln Lys Met Arg Gly Val Glu Ser 245 250 255 Gln Gly Met Leu Leu
Cys Ala Ser Ile Glu Gly Ile Asn Arg Gln Val 260 265 270 Glu Pro Leu
Asp Pro Pro Ala Gly Ser Ala Pro Gly Glu His Val Phe 275 280 285 Val
Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu Leu Lys Pro Lys 290 295
300 Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys Ile Ser Glu Glu
305 310 315 320 Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr Lys
Leu Gly Ser 325 330 335 Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile
Ser 340 345 <210> SEQ ID NO 7 <211> LENGTH: 2178
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 7 ttcagaaagt ggtggaggga agacttcctt tttcccagag
acagaaggtt atgcacccag 60 tggcctggga ccattgttct gggctttttt
tcccttcgac atggatttgc ttctcactgt 120 gtaccccaac caccaaaacc
accctgagat caatgctggt gctcctgcat cagatggctt 180 agagatcctt
ccacctctta acacaagcat ctaggtccac tttactcaaa tctggcctca 240
gttgagagca gagtatacca tcagagccca ttctcctgtc tgctgtctgg gacgtggaaa
300 gaaagttagc tctagggggt ctttccaggg gcctctgtaa ggactggatg
ctcctttccg 360 gaatccaaga gttcaccagg ctgcttctct aatggacgat
gatcctcttc ctcctgacgt 420 ctctccctgg cagcacccag atgcagacag
cctgtatgta gagaagattg acgtggggga 480 agctgaacca cggactgtgg
tgagcggcct ggtacagttc gtgcccaagg aggaactgca 540 ggacaggctg
gtagtggtgc tgtgcaacct gaaaccccag aagatgagag gagtcgagtc 600
ccaaggcatg cttctgtgtg cttctatgtg agtgaggact tggagtgggg cacaggacct
660 ggggaggcca ggaagagtag ggaatcagcc catatgatgt ccttccacac
accaggtgga 720 agctctgaga acacgtgcct cttccttgct gatgccaaaa
gttgatgcat gaaggactta 780 tcgtacaagt actgttaatg aagcatttta
cctacagtta attttgttaa aatagaaatg 840 gagggctcaa accagtacat
acccaagtct tactactagt aaggagtgga gcagggattc 900 aaatcccagt
tttgatgtct ataaagtcct cgctacgtta ttttatactt cctcccctag 960
aaacacagat tttggtatct tgacacacaa ttttggtata gcctgggtta atgtaaccct
1020 ggtgatatgc agggatgtag caagataaga ggacctcctg gggctctggt
actgaggatg 1080 ccctaaatcc catcagggcc cctgtgtaaa ggcccggatt
gctttggcct ccacagtcac 1140 tggaacccat ccatagcctc actcttctct
tgtcctgtgt cttcccagag aagggataaa 1200
ccgccaggtt gaacctctgg accctccggc aggctctgct cctggtgagc acgtgtttgt
1260 gaagggctat gaaaagggcc aaccagatga ggagctcaag cccaagagga
aagtcttcga 1320 gaagttgcag gctgacttca aaatttctga ggagtgcatc
gcacagtgga agcaaaccaa 1380 cttcatgacc aagctgggct ccatttcctg
taaatcgctg aaagggggga acattagcta 1440 gccagcccag catcttcccc
ccttcttcca ccactgagtc atctgctgtc tcttcagtct 1500 gctccaccca
tcacccattt acccatctct caggacacgg aagcagcggg tttggactct 1560
ttattcggtg cagaactcgg caaggggcag cttaccctcc ccagaaccca ggatcatcct
1620 gtctggctgc agtgagagac caacccctaa caagggctgg gccacagcag
ggagtccagc 1680 cctaccttct tcccttggca gctggagaaa tctggtttca
atataactca tttaaaaatt 1740 tatgccacag tccttataat tggaaaaata
ctggtgccca ggttttcttg gagttatcca 1800 agcagctgcg cccctagctg
ggatctggta cctggactag gctaattaca gcttctcccc 1860 aacaggaaac
tgtgggattt gaaaaggaaa gggaagggaa aacagagaac ctagtggtct 1920
accaagtggt tggcaacttt cccaatgtct gcttactctg aggcttggca ctgggggcca
1980 gggcctgccc cagggctcct ggaatttccc ttgatccagc taggctggga
cactccctaa 2040 atcagctgcg tgttgttagc atcaggcaga atgaatggca
gagagtgatt ctgtcttcat 2100 agagggtggg gtacttctcc ataaggcatc
tcagtcaaat ccccatcact gtcataaatt 2160 caaataaaat gtctgaac 2178
<210> SEQ ID NO 8 <211> LENGTH: 388 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: VARIANT <222> LOCATION: 354, 355, 356,
357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,
370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382,
383, 384, 385, 386, 387, 388 <223> OTHER INFORMATION: Xaa =
Any Amino Acid <400> SEQUENCE: 8 Met Gly Asp Ala Pro Ser Pro
Glu Glu Lys Leu His Leu Ile Thr Arg 1 5 10 15 Asn Leu Gln Glu Val
Leu Gly Glu Glu Lys Leu Lys Glu Ile Leu Lys 20 25 30 Glu Arg Glu
Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro 35 40 45 His
Val Ala Tyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys 50 55
60 Ala Gly Cys Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu
65 70 75 80 Asp Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val
Ser Tyr 85 90 95 Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile
Gly Val Pro Leu 100 105 110 Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp
Tyr Gln Leu Ser Lys Glu 115 120 125 Tyr Thr Leu Asp Val Tyr Arg Leu
Ser Ser Val Val Thr Gln His Asp 130 135 140 Ser Lys Lys Ala Gly Ala
Glu Val Val Lys Gln Val Glu His Pro Leu 145 150 155 160 Leu Ser Gly
Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu Glu Tyr 165 170 175 Leu
Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Arg Lys Ile Phe 180 185
190 Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val
195 200 205 His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys
Met Ser 210 215 220 Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp
Arg Lys Glu Asp 225 230 235 240 Val Lys Lys Lys Leu Lys Lys Ala Phe
Cys Glu Pro Gly Asn Val Glu 245 250 255 Asn Asn Gly Val Leu Ser Phe
Ile Lys His Val Leu Phe Pro Leu Lys 260 265 270 Ser Glu Phe Val Ile
Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys Thr 275 280 285 Tyr Thr Ala
Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu Val Val 290 295 300 His
Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu 305 310
315 320 Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys
Leu 325 330 335 Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro
Met Ala Lys 340 345 350 Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa 385
<210> SEQ ID NO 9 <211> LENGTH: 1167 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 9
atgggggacg ctcccagccc tgaagagaaa ctgcacctta tcacccggaa cctgcaggag
60 gttctggggg aagagaagct gaaggagata ctgaaggagc gggaacttaa
aatttactgg 120 ggaacggcaa ccacgggcaa accacatgtg gcttactttg
tgcccatgtc aaagattgca 180 gacttcttaa aggcagggtg tgaggtaaca
attctgtttg cggacctcca cgcatacctg 240 gataacatga aagccccatg
ggaacttcta gaactccgag tcagttacta tgagaatgtg 300 atcaaagcaa
tgctggagag cattggtgtg cccttggaga agctcaagtt catcaaaggc 360
actgattacc agctcagcaa agagtacaca ctagatgtgt acagactctc ctccgtggtc
420 acacagcacg attccaagaa ggctggagct gaggtggtaa agcaggtgga
gcaccctttg 480 ctgagtggcc tcttataccc cggactgcag gctttggatg
aagagtattt aaaagtagat 540 gcccaatttg gaggcattga tcagagaaag
attttcacct ttgcagagaa gtacctccct 600 gcacttggct attcaaaacg
ggtccatctg atgaatccta tggttccagg attaacaggc 660 agcaaaatga
gctcttcaga agaggagtcc aagattgatc tccttgatcg gaaggaggat 720
gtgaagaaaa aactgaagaa ggccttctgt gagccaggaa atgtggagaa caatggggtt
780 ctgtccttca tcaagcatgt cctttttccc cttaagtccg agtttgtgat
cctacgagat 840 gagaaatggg gtggaaacaa aacctacaca gcttacgtgg
acctggaaaa ggactttgct 900 gctgaggttg tacatcctgg agacctgaag
aattctgttg aagtcgcact gaacaagttg 960 ctggatccaa tccgggaaaa
gtttaatacc cctgccctga aaaaactggc cagcgctgcc 1020 tacccagatc
cctcaaagca gaagccaatg gccaaaggcc tgccaagaat tcagaaccag 1080
aggaggtcat cccatcccgg ctggatatcc gtgtggggaa aatcatcact gtggagaagc
1140 acccagatgc agacagcctg tatgtag 1167 <210> SEQ ID NO 10
<211> LENGTH: 318 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 10 Met Asn Pro Met Val Pro Gly
Leu Thr Gly Ser Lys Met Ser Ser Ser 1 5 10 15 Glu Glu Glu Ser Lys
Ile Asp Leu Leu Asp Arg Lys Glu Asp Val Lys 20 25 30 Lys Lys Leu
Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu Asn Asn 35 40 45 Gly
Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys Ser Glu 50 55
60 Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys Thr Tyr Thr
65 70 75 80 Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu Val Val
His Pro 85 90 95 Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn
Lys Leu Leu Asp 100 105 110 Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala
Leu Lys Lys Leu Ala Ser 115 120 125 Ala Ala Tyr Pro Asp Pro Ser Lys
Gln Lys Pro Met Ala Lys Gly Pro 130 135 140 Ala Lys Asn Ser Glu Pro
Glu Glu Val Ile Pro Ser Arg Leu Asp Ile 145 150 155 160 Arg Val Gly
Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala Asp Ser 165 170 175 Leu
Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg Thr Val 180 185
190 Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu Glu Leu Gln Asp Arg
195 200 205 Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln Lys Met Arg
Gly Val 210 215 220 Glu Ser Gln Gly Met Leu Leu Cys Ala Ser Ile Glu
Gly Ile Asn Arg 225 230 235 240 Gln Val Glu Pro Leu Asp Pro Pro Ala
Gly Ser Ala Pro Gly Glu His 245 250 255 Val Phe Val Lys Gly Tyr Glu
Lys Gly Gln Pro Asp Glu Glu Leu Lys 260 265 270 Pro Lys Lys Lys Val
Phe Glu Lys Leu Gln Ala Asp Phe Lys Ile Ser 275 280 285 Glu Glu Cys
Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr Lys Leu 290 295 300 Gly
Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser 305 310 315
<210> SEQ ID NO 11 <211> LENGTH: 1736 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 11
gaaagatttt cacctttgca gagaagtacc tccctgcact tggctattca aaacgggtcc
60 atctgatgaa tcctatggtt ccaggattaa caggcagcaa aatgagctct
tcagaagagg 120
agtccaagat tgatctcctt gatcggaagg aggatgtgaa gaaaaaactg aagaaggcct
180 tctgtgagcc aggaaatgtg gagaacaatg gggttctgtc cttcatcaag
catgtccttt 240 ttccccttaa gtccgagttt gtgatcctac gagatgagaa
atggggtgga aacaaaacct 300 acacagctta cgtggacctg gaaaaggact
ttgctgctga ggttgtacat cctggagacc 360 tgaagaattc tgttgaagtc
gcactgaaca agttgctgga tccaatccgg gaaaagttta 420 atacccctgc
cctgaaaaaa ctggccagcg ctgcctaccc agatccctca aagcagaagc 480
caatggccaa aggccctgcc aagaattcag aaccagagga ggtcatccca tcccggctgg
540 atatccgtgt ggggaaaatc atcactgtgg agaagcaccc agatgcagac
agcctgtatg 600 tagagaagat tgacgtgggg gaagctgaac cacggactgt
ggtgagcggc ctggtacagt 660 tcgtgcccaa ggaggaactg caggacaggc
tggtagtggt gctgtgcaac ctgaaacccc 720 agaagatgag aggagtcgag
tcccaaggca tgcttctgtg tgcttctata gaagggataa 780 accgccaggt
tgaacctctg gaccctccgg caggctctgc tcctggtgag cacgtgtttg 840
tgaagggcta tgaaaagggc caaccagatg aggagctcaa gcccaagaag aaagtcttcg
900 agaagttgca ggctgacttc aaaatttctg aggagtgcat cgcacagtgg
aagcaaacca 960 acttcatgac caagctgggc tccatttcct gtaaatcgct
gaaagggggg aacattagct 1020 agccagccca gcatcttccc cccttcttcc
accactgagt catctgctgt ctcttcagtc 1080 tgctccatcc atcacccatt
tacccatctc tcaggacacg gaagcagcgg gtttggactc 1140 tttattcggt
gcagaactcg gcaaggggca gcttaccctc cccagaaccc aggatcatcc 1200
tgtctggctg cagtgagaga ccaaccccta acaagggctg ggccacagca gggagtccag
1260 ccctaccttc ttcccttggc agctggagaa atctggtttc aatataactc
atttaaaaat 1320 ttatgccaca gtccttataa ttggaaaaat actggtgccc
aggttttctt ggagttatcc 1380 aagcagctgc gcccctagct gggatctggt
acctggacta ggctaattac agcttctccc 1440 caacaggaaa ctgtgggatt
tgaaaaggaa agggaaggga aaacagagaa cctagtggtc 1500 taccaagtgg
ttggcaactt tcccaatgtc tgcttactct gaggcttggc actgggggcc 1560
agggcctgcc ccagggctcc tggaatttcc cttgatccag ctaggctggg acactcccta
1620 aatcagctgc gtgttgttag catcaggcag aatgaatggc agagagtgat
tctgtcttca 1680 tagagggtgg ggtacttctc cataaggcat ctcagtcaaa
tccccatcac tgtcat 1736 <210> SEQ ID NO 12 <211> LENGTH:
179 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 Met Ala Lys Gly Pro Ala Lys Asn Ser Glu
Pro Glu Glu Val Ile Pro 1 5 10 15 Ser Arg Leu Asp Ile Arg Val Gly
Lys Ile Ile Thr Val Glu Lys His 20 25 30 Pro Asp Ala Asp Ser Leu
Tyr Val Glu Lys Ile Asp Val Gly Glu Ala 35 40 45 Glu Pro Arg Thr
Val Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu 50 55 60 Glu Leu
Gln Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln 65 70 75 80
Lys Met Arg Gly Val Glu Ser Gln Gly Met Leu Leu Cys Ala Ser Ile 85
90 95 Glu Gly Ile Asn Arg Gln Val Glu Pro Leu Asp Pro Pro Ala Gly
Ser 100 105 110 Ala Pro Gly Glu His Val Phe Val Lys Gly Tyr Glu Lys
Gly Gln Pro 115 120 125 Asp Glu Glu Leu Lys Pro Lys Lys Lys Val Phe
Glu Lys Leu Gln Ala 130 135 140 Asp Phe Lys Ile Ser Glu Glu Cys Ile
Ala Gln Trp Lys Gln Thr Asn 145 150 155 160 Phe Met Thr Lys Leu Gly
Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly 165 170 175 Asn Ile Ser
<210> SEQ ID NO 13 <211> LENGTH: 1167 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 13
atgggggacg ctcccagccc tgaagagaaa ctgcacctta tcacccggaa cctgcaggag
60 gttctggggg aagagaagct gaaggagata ctgaaggagc gggaacttaa
aatttactgg 120 ggaacggcaa ccacgggcaa accacatgtg gcttactttg
tgcccatgtc aaagattgca 180 gacttcttaa aggcagggtg tgaggtaaca
attctgtttg cggacctcca cgcatacctg 240 gataacatga aagccccatg
ggaacttcta gaactccgag tcagttacta tgagaatgtg 300 atcaaagcaa
tgctggagag cattggtgtg cccttggaga agctcaagtt catcaaaggc 360
actgattacc agctcagcaa agagtacaca ctagatgtgt acagactctc ctccgtggtc
420 acacagcacg attccaagaa ggctggagct gaggtggtaa agcaggtgga
gcaccctttg 480 ctgagtggcc tcttataccc cggactgcag gctttggatg
aagagtattt aaaagtagat 540 gcccaatttg gaggcattga tcagagaaag
attttcacct ttgcagagaa gtacctccct 600 gcacttggct attcaaaacg
ggtccatctg atgaatccta tggttccagg attaacaggc 660 agcaaaatga
gctcttcaga agaggagtcc aagattgatc tccttgatcg gaaggaggat 720
gtgaagaaaa aactgaagaa ggccttctgt gagccaggaa atgtggagaa caatggggtt
780 ctgtccttca tcaagcatgt cctttttccc cttaagtccg agtttgtgat
cctacgagat 840 gagaaatggg gtggaaacaa aacctacaca gcttacgtgg
acctggaaaa ggactttgct 900 gctgaggttg tacatcctgg agacctgaag
aattctgttg aagtcgcact gaacaagttg 960 ctggatccaa tccgggaaaa
gtttaatacc cctgccctga aaaaactggc cagcgctgcc 1020 tacccagatc
cctcaaagca gaagccaatg gccaaaggcc tgccaagaat tcagaaccag 1080
aggaggtcat cccatcccgg ctggatatcc gtgtggggaa aatcatcact gtggagaagc
1140 acccagatgc agacagcctg tatgtag 1167 <210> SEQ ID NO 14
<211> LENGTH: 188 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY: VARIANT
<222> LOCATION: 1, 2, 3, 4, 5, 6, 7, 8 <223> OTHER
INFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 14 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Pro Met Ala Lys Gly Pro Ala Lys 1 5 10 15
Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg Leu Asp Ile Arg Val 20
25 30 Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala Asp Ser Leu
Tyr 35 40 45 Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg Thr
Val Val Ser 50 55 60 Gly Leu Val Gln Phe Val Pro Lys Glu Glu Leu
Gln Asp Arg Leu Val 65 70 75 80 Val Val Leu Cys Asn Leu Lys Pro Gln
Lys Met Arg Gly Val Glu Ser 85 90 95 Gln Gly Met Leu Leu Cys Ala
Ser Ile Glu Gly Ile Asn Arg Gln Val 100 105 110 Glu Pro Leu Asp Pro
Pro Ala Gly Ser Ala Pro Gly Glu His Val Phe 115 120 125 Val Lys Gly
Tyr Glu Lys Gly Gln Pro Asp Glu Glu Leu Lys Pro Lys 130 135 140 Lys
Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys Ile Ser Glu Glu 145 150
155 160 Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr Lys Leu Gly
Ser 165 170 175 Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser 180
185 <210> SEQ ID NO 15 <211> LENGTH: 2262 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
15 gccagacaca gtggctcaca cctgtaatct taacactttg gaaggctgag
gcaggcggat 60 cacttgagcc caaaagttag agaccaaaac ccagtctcta
cccaaaaaaa aaaaaaaaaa 120 aaaaattagc caggcatagt agcacatgcc
tgtagtccca gctacttggg aggctgaggt 180 gagaggatca cctgagcatg
gggaagttga gactgcagtg agccatgatc gcaccactgc 240 actccagcct
gggcaacaga gtgagactct atgtctcaaa aaaagaaaaa tgatagaaat 300
tagattagac ctattatacc caaccggtat atagggtatc gatagtttct tacacagctg
360 ttgggcagag cctgcagagc ttagagaagc ttatctttag attctcccag
tttccttcta 420 tgtgcatggg cctggctctt agttggccat ccacttgtgc
gtaatgctaa gatattggca 480 ttgatagctt tgtgcgaccc ttccagaaaa
aaactcagta actcagtaaa attttttttt 540 ttttttctaa aagagacaga
gtctggctct gttgcccagc ctggtcttga agtcctgggc 600 ttaagcaatc
ctcccgtctc agcctcccaa agtgctagaa ttacaggtgt gagctaccac 660
acctggccaa gactcagtaa attctatgtg gaatgcatga atggaaatac ctaaaggagg
720 caaagctact actgctccct ccccgctagt ctaataattg agggagagaa
cagatgaaaa 780 tcaggtatgt catgtctgaa aggttgccaa cccagtatta
aagaagttac aactcagtgt 840 ttagactctg gggattctac actaaatctt
acctaatctc agtgtcttaa cgtggtggga 900 tcagcagctg acctgccaca
gggaagaatt ctacctcatg gggttcttct cattcccaga 960 gccaatggcc
aaaggccctg ccaagaattc agaaccagag gaggtcatcc catcccggct 1020
ggatatccgt gtggggaaaa tcatcactgt ggagaagcac ccagatgcag acagcctgta
1080 tgtagagaag attgacgtgg gggaagctga accacggact gtggtgagcg
gcctggtaca 1140 gttcgtgccc aaggaggaac tgcaggacag gctggtagtg
gtgctgtgca acctgaaacc 1200 ccagaagatg agaggagtcg agtcccaagg
catgcttctg tgtgcttcta tagaagggat 1260 aaaccgccag gttgaacctc
tggaccctcc ggcaggctct gctcctggtg agcacgtgtt 1320 tgtgaagggc
tatgaaaagg gccaaccaga tgaggagctc aagcccaaga agaaagtctt 1380
cgagaagttg caggctgact tcaaaatttc tgaggagtgc atcgcacagt ggaagcaaac
1440 caacttcatg accaagctgg gctccatttc ctgtaaatcg ctgaaagggg
ggaacattag 1500 ctagccagcc cagcatcttc cccccttctt ccaccactga
gtcatctgct gtctcttcag 1560
tctgctccat ccatcaccca tttacccatc tctcaggaca cggaagcagc gggtttggac
1620 tctttattcg gtgcagaact cggcaagggg cagcttaccc tccccagaac
ccaggatcat 1680 cctgtctggc tgcagtgaga gaccaacccc taacaagggc
tgggccacag cagggagtcc 1740 agccctacct tcttcccttg gcagctggag
aaatctggtt tcaatataac tcatttaaaa 1800 atttatgcca cagtccttat
aattggaaaa atactggtgc ccaggttttc ttggagttat 1860 ccaagcagct
gcgcccctag ctgggatctg gtacctggac taggctaatt acagcttctc 1920
cccaacagga aactgtggga tttgaaaagg aaagggaagg gaaaacagag aacctagtgg
1980 tctaccaagt ggttggcaac tttcccaatg tctgcttact ctgaggcttg
gcactggggg 2040 ccagggcctg ccccagggct cctggaattt cccttgatcc
agctaggctg ggacactccc 2100 taaatcagct gcgtgttgtt agcatcaggc
agaatgaatg gcagagagtg attctgtctt 2160 catagagggt ggggtacttc
tccataaggc atctcagtca aatccccatc actgtcataa 2220 attcaaataa
aatgtctgaa caagggaaaa aaaaaaaaaa aa 2262
* * * * *
References