U.S. patent application number 11/332472 was filed with the patent office on 2007-05-17 for methods and compositions targeting viral and cellular itam motifs, and use of same in identifying compounds with therapeutic activity.
Invention is credited to Elad Katz, John G. Monroe, Ramachandran Murali.
Application Number | 20070110760 11/332472 |
Document ID | / |
Family ID | 38041092 |
Filed Date | 2007-05-17 |
United States Patent
Application |
20070110760 |
Kind Code |
A1 |
Monroe; John G. ; et
al. |
May 17, 2007 |
Methods and compositions targeting viral and cellular ITAM motifs,
and use of same in identifying compounds with therapeutic
activity
Abstract
This invention provides methods of treating, reducing the
incidence of, and inhibiting metastasis formation of carcinomas,
sarcomas, Epstein-Barr virus-induced malignancies, B cell
proliferative disorders, and mast cell activation disorders,
comprising administering to a subject a compound that inhibits an
interaction of a first protein and an immunoreceptor tyrosine-based
activation motif (ITAM) of a second protein, and screening methods
for identifying ITAM-inhibitory compounds and peptides. This
invention also provides peptides that inhibit signaling by
ITAMs.
Inventors: |
Monroe; John G.;
(Philadelphia, PA) ; Katz; Elad; (Manchester,
GB) ; Murali; Ramachandran; (Swarthmore, PA) |
Correspondence
Address: |
Pearl Cohen Zedek Latzer, LLP;Suite 1001
10 Rockefeller Plaza
New York
NY
10020
US
|
Family ID: |
38041092 |
Appl. No.: |
11/332472 |
Filed: |
January 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60643906 |
Jan 14, 2005 |
|
|
|
60649900 |
Feb 4, 2005 |
|
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Current U.S.
Class: |
424/185.1 ;
514/19.4; 514/19.8; 514/3.7 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61P 35/04 20180101 |
Class at
Publication: |
424/185.1 ;
514/012 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 38/17 20060101 A61K038/17 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The invention described herein was supported in whole or in
part by grants from The National Institutes of Health (Grant
Numbers P01-CA093615, R01-A143620, R01-CA087609, R01-CA073746,
RO1-A132592, and CA09140) and the Department of Defense (Grant
Numbers DMAD17-00-1-0249 and W81WH-04-1-0435). The government has
certain rights in the invention.
Claims
1. A method of treating a breast cell carcinoma in a subject,
comprising administering to said subject a compound that inhibits
an interaction of a first protein and an immunoreceptor
tyrosine-based activation motif (ITAM) of a second protein, thereby
treating a breast cell carcinoma in a subject.
2. The method of claim 1, wherein said compound comprises a peptide
homologous to said ITAM.
3. The method of claim 2, wherein said peptide comprises a
modification selected from hydroxylation, amidation,
esterification, formylation, gamma-carboxyglutamic acid
hydroxylation, methylation, phosphorylation, sulfation,
glycosylation, reduction, oxidation, disulfide modification,
introduction of a thioether bond, introduction of a thiolester
bond, a backbone condensation, or a biotinylation.
4. The method of claim 2, wherein said peptide comprises an amino
acid selected from a D amino acid; pyrrolidone carboxylic acid;
2-aminoadipic acid; 3-aminoadipic acid; beta-alanine;
beta-aminoproprionic acid; 2-aminobutyric acid; 4-aminobutyric
acid; piperidinic acid; 6-aminocaproic acid; 6-aminoheptanoic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4 diaminobutyric acid; desmosine; 2,2
diaminopimelic acid; 2,3 diaminopropionic acid; N-ethylglycine;
N-ethylasparagine; hydroxylysine; allo-hydroxylysine;
3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;
N-methylglycine; sarcosine; methylisoleucine; methyllysine;
methylvaline; norvaline; norleucine; 6-aminohexanoic acid;
citrulline; cysteic acid; cyclohexylalanine; alpha-amino isobutyric
acid; t-butylglycine; t-butylalanine; phenylglycine; an
N-alpha-methyl amino acid, a C-alpha-methyl amino acid, a
beta-methyl amino acid, or orthinine.
5. The method of claim 1, wherein said second protein is a viral
protein.
6. A method of reducing an incidence of a breast cell carcinoma in
a subject, comprising administering to said subject a compound that
inhibits an interaction of a first protein and an immunoreceptor
tyrosine-based activation motif (ITAM) of a second protein, thereby
reducing an incidence of a breast cell carcinoma in a subject.
7. The method of claim 6, wherein said compound comprises a peptide
homologous to said ITAM.
8. The method of claim 7, wherein said peptide comprises a
modification selected from hydroxylation, amidation,
esterification, formylation, gamma-carboxyglutamic acid
hydroxylation, methylation, phosphorylation, sulfation,
glycosylation, reduction, oxidation, disulfide modification,
introduction of a thioether bond, introduction of a thiolester
bond, a backbone condensation, or a biotinylation.
9. The method of claim 7, wherein said peptide comprises an amino
acid selected from a D amino acid; pyrrolidone carboxylic acid;
2-aminoadipic acid; 3-aminoadipic acid; beta-alanine;
beta-aminoproprionic acid; 2-aminobutyric acid; 4-aminobutyric
acid; piperidinic acid; 6-aminocaproic acid; 6-aminoheptanoic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4 diaminobutyric acid; desmosine; 2,2
diaminopimelic acid; 2,3 diaminopropionic acid; N-ethylglycine;
N-ethylasparagine; hydroxylysine; allo-hydroxylysine;
3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;
N-methylglycine; sarcosine; methylisoleucine; methyllysine;
methylvaline; norvaline; norleucine; 6-aminohexanoic acid;
citrulline; cysteic acid; cyclohexylalanine; alpha-amino isobutyric
acid; t-butylglycine; t-butylalanine; phenylglycine; an
N-alpha-methyl amino acid, a C-alpha-methyl amino acid, a
beta-methyl amino acid, or orthinine.
10. The method of claim 6, wherein said second protein is a viral
protein.
11. A method of inhibiting a formation of a metastasis of a breast
cell carcinoma in a subject, comprising administering to said
subject a compound that inhibits an interaction of a first protein
and an immunoreceptor tyrosine-based activation motif (ITAM) of a
second protein, thereby inhibiting a formation of a metastasis of a
breast cell carcinoma in a subject.
12. The method of claim 11, wherein said compound comprises a
peptide homologous to said ITAM.
13. The method of claim 12, wherein said peptide comprises a
modification selected from hydroxylation, amidation,
esterification, formylation, gamma-carboxyglutamic acid
hydroxylation, methylation, phosphorylation, sulfation,
glycosylation, reduction, oxidation, disulfide modification,
introduction of a thioether bond, introduction of a thiolester
bond, a backbone condensation, or a biotinylation.
14. The method of claim 12, wherein said peptide comprises an amino
acid selected from a D amino acid; pyrrolidone carboxylic acid;
2-aminoadipic acid; 3-aminoadipic acid; beta-alanine;
beta-aminoproprionic acid; 2-aminobutyric acid; 4-aminobutyric
acid; piperidinic acid; 6-aminocaproic acid; 6-aminoheptanoic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4 diaminobutyric acid; desmosine; 2,2
diaminopimelic acid; 2,3 diaminopropionic acid; N-ethylglycine;
N-ethylasparagine; hydroxylysine; allo-hydroxylysine;
3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;
N-methylglycine; sarcosine; methylisoleucine; methyllysine;
methylvaline; norvaline; norleucine; 6-aminohexanoic acid;
citrulline; cysteic acid; cyclohexylalanine; alpha-amino isobutyric
acid; t-butylglycine; t-butylalanine; phenylglycine; an
N-alpha-methyl amino acid, a C-alpha-methyl amino acid, a
beta-methyl amino acid, or orthinine.
15. The method of claim 11, wherein said second protein is a viral
protein.
16. A method of treating a fibrosarcoma or Kaposi's sarcoma in a
subject, comprising administering to said subject a compound that
inhibits an interaction of a first protein and an immunoreceptor
tyrosine-based activation motif (ITAM) of a second protein, thereby
treating a fibrosarcoma or Kaposi's sarcoma in a subject.
17. The method of claim 16, wherein said compound comprises a
peptide homologous to said ITAM.
18. The method of claim 17, wherein said peptide comprises a
modification selected from hydroxylation, amidation,
esterification, formylation, gamma-carboxyglutamic acid
hydroxylation, methylation, phosphorylation, sulfation,
glycosylation, reduction, oxidation, disulfide modification,
introduction of a thioether bond, introduction of a thiolester
bond, a backbone condensation, or a biotinylation.
19. The method of claim 17, wherein said peptide comprises an amino
acid selected from a D amino acid; pyrrolidone carboxylic acid;
2-aminoadipic acid; 3-aminoadipic acid; beta-alanine;
beta-aminoproprionic acid; 2-aminobutyric acid; 4-aminobutyric
acid; piperidinic acid; 6-aminocaproic acid; 6-aminoheptanoic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4 diaminobutyric acid; desmosine; 2,2
diaminopimelic acid; 2,3 diaminopropionic acid; N-ethylglycine;
N-ethylasparagine; hydroxylysine; allo-hydroxylysine;
3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;
N-methylglycine; sarcosine; methylisoleucine; methyllysine;
methylvaline; norvaline; norleucine; 6-aminohexanoic acid;
citrulline; cysteic acid; cyclohexylalanine; alpha-amino isobutyric
acid; t-butylglycine; t-butylalanine; phenylglycine; an
N-alpha-methyl amino acid, a C-alpha-methyl amino acid, a
beta-methyl amino acid, or orthinine.
20. The method of claim 16, wherein said second protein is a viral
protein.
21. A method of reducing an incidence of a fibrosarcoma or Kaposi's
sarcoma in a subject, comprising administering to said subject a
compound that inhibits an interaction of a first protein and an
immunoreceptor tyrosine-based activation motif (ITAM) of a second
protein, thereby reducing an incidence of a fibrosarcoma or
Kaposi's sarcoma in a subject.
22. The method of claim 21, wherein said compound comprises a
peptide homologous to said ITAM.
23. The method of claim 22, wherein said peptide comprises a
modification selected from hydroxylation, amidation,
esterification, formylation, gamma-carboxyglutamic acid
hydroxylation, methylation, phosphorylation, sulfation,
glycosylation, reduction, oxidation, disulfide modification,
introduction of a thioether bond, introduction of a thiolester
bond, a backbone condensation, or a biotinylation.
24. The method of claim 22, wherein said peptide comprises an amino
acid selected from a D amino acid; pyrrolidone carboxylic acid;
2-aminoadipic acid; 3-aminoadipic acid; beta-alanine;
beta-aminoproprionic acid; 2-aminobutyric acid; 4-aminobutyric
acid; piperidinic acid; 6-aminocaproic acid; 6-aminoheptanoic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4 diaminobutyric acid; desmosine; 2,2
diaminopimelic acid; 2,3 diaminopropionic acid; N-ethylglycine;
N-ethylasparagine; hydroxylysine; allo-hydroxylysine;
3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;
N-methylglycine; sarcosine; methylisoleucine; methyllysine;
methylvaline; norvaline; norleucine; 6-aminohexanoic acid;
citrulline; cysteic acid; cyclohexylalanine; alpha-amino isobutyric
acid; t-butylglycine; t-butylalanine; phenylglycine; an
N-alpha-methyl amino acid, a C-alpha-methyl amino acid, a
beta-methyl amino acid, or orthinine.
25. The method of claim 21, wherein said second protein is a viral
protein.
26. A method of inhibiting a formation of a metastasis of a
fibrosarcoma or Kaposi's sarcoma in a subject, comprising
administering to said subject a compound that inhibits an
interaction of a first protein and an immunoreceptor tyrosine-based
activation motif (ITAM) of a second protein, thereby inhibiting a
formation of a metastasis of a fibrosarcoma or Kaposi's sarcoma in
a subject.
27. The method of claim 26, wherein said compound comprises a
peptide homologous to said ITAM.
28. The method of claim 27, wherein said peptide comprises a
modification selected from hydroxylation, amidation,
esterification, formylation, gamma-carboxyglutamic acid
hydroxylation, methylation, phosphorylation, sulfation,
glycosylation, reduction, oxidation, disulfide modification,
introduction of a thioether bond, introduction of a thiolester
bond, a backbone condensation, or a biotinylation.
29. The method of claim 27, wherein said peptide comprises an amino
acid selected from a D amino acid; pyrrolidone carboxylic acid;
2-aminoadipic acid; 3-aminoadipic acid; beta-alanine;
beta-aminoproprionic acid; 2-aminobutyric acid; 4-aminobutyric
acid; piperidinic acid; 6-aminocaproic acid; 6-aminoheptanoic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4 diaminobutyric acid; desmosine; 2,2
diaminopimelic acid; 2,3 diaminopropionic acid; N-ethylglycine;
N-ethylasparagine; hydroxylysine; allo-hydroxylysine;
3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;
N-methylglycine; sarcosine; methylisoleucine; methyllysine;
methylvaline; norvaline; norleucine; 6-aminohexanoic acid;
citrulline; cysteic acid; cyclohexylalanine; alpha-amino isobutyric
acid; t-butylglycine; t-butylalanine; phenylglycine; an
N-alpha-methyl amino acid, a C-alpha-methyl amino acid, a
beta-methyl amino acid, or orthinine.
30. The method of claim 26, wherein said second protein is a viral
protein.
31. A method of treating a Burkitt's lymphoma, a Hodgkin's disease,
or a nasopharyngeal carcinoma (NPC) in a subject, comprising
administering to said subject a compound that inhibits an
interaction of a first protein and an immunoreceptor tyrosine-based
activation motif (ITAM) of a second protein, thereby treating a
Burkitt's lymphoma, a Hodgkin's disease, or a NPC in a subject.
32. The method of claim 31, wherein said compound comprises a
peptide homologous to said ITAM.
33. The method of claim 32, wherein said peptide comprises a
modification selected from hydroxylation, amidation,
esterification, formylation, gamma-carboxyglutamic acid
hydroxylation, methylation, phosphorylation, sulfation,
glycosylation, reduction, oxidation, disulfide modification,
introduction of a thioether bond, introduction of a thiolester
bond, a backbone condensation, or a biotinylation.
34. The method of claim 32, wherein said peptide comprises an amino
acid selected from a D amino acid; pyrrolidone carboxylic acid;
2-aminoadipic acid; 3-aminoadipic acid; beta-alanine;
beta-aminoproprionic acid; 2-aminobutyric acid; 4-aminobutyric
acid; piperidinic acid; 6-aminocaproic acid; 6-aminoheptanoic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4 diaminobutyric acid; desmosine; 2,2
diaminopimelic acid; 2,3 diaminopropionic acid; N-ethylglycine;
N-ethylasparagine; hydroxylysine; allo-hydroxylysine;
3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;
N-methylglycine; sarcosine; methylisoleucine; methyllysine;
methylvaline; norvaline; norleucine; 6-aminohexanoic acid;
citrulline; cysteic acid; cyclohexylalanine; alpha-amino isobutyric
acid; t-butylglycine; t-butylalanine; phenylglycine; an
N-alpha-methyl amino acid, a C-alpha-methyl amino acid, a
beta-methyl amino acid, or orthinine.
35. The method of claim 31, wherein said second protein is a viral
protein.
36. A method of reducing an incidence of a Burkitt's lymphoma, a
Hodgkin's disease, or a nasopharyngeal carcinoma (NPC) in a
subject, comprising administering to said subject a compound that
inhibits an interaction of a first protein and an immunoreceptor
tyrosine-based activation motif (ITAM) of a second protein, thereby
reducing an incidence of a Burkitt's lymphoma, a Hodgkin's disease,
or NPC in a subject.
37. The method of claim 36, wherein said compound comprises a
peptide homologous to said ITAM.
38. The method of claim 37, wherein said peptide comprises a
modification selected from hydroxylation, amidation,
esterification, formylation, gamma-carboxyglutamic acid
hydroxylation, methylation, phosphorylation, sulfation,
glycosylation, reduction, oxidation, disulfide modification,
introduction of a thioether bond, introduction of a thiolester
bond, a backbone condensation, or a biotinylation.
39. The method of claim 37, wherein said peptide comprises an amino
acid selected from a D amino acid; pyrrolidone carboxylic acid;
2-aminoadipic acid; 3-aminoadipic acid; beta-alanine;
beta-aminoproprionic acid; 2-aminobutyric acid; 4-aminobutyric
acid; piperidinic acid; 6-aminocaproic acid; 6-aminoheptanoic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4 diaminobutyric acid; desmosine; 2,2
diaminopimelic acid; 2,3 diaminopropionic acid; N-ethylglycine;
N-ethylasparagine; hydroxylysine; allo-hydroxylysine;
3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;
N-methylglycine; sarcosine; methylisoleucine; methyllysine;
methylvaline; norvaline; nor-leucine; 6-aminohexanoic acid;
citrulline; cysteic acid; cyclohexylalanine; alpha-amino isobutyric
acid; t-butylglycine; t-butylalanine; phenylglycine; an
N-alpha-methyl amino acid, a C-alpha-methyl amino acid, a
beta-methyl amino acid, or orthinine.
40. The method of claim 36, wherein said second protein is a viral
protein.
41. A method of inhibiting a formation of a metastasis of a
Burkitt's lymphoma, a Hodgkin's disease, or a nasopharyngeal
carcinoma (NPC) in a subject, comprising administering to said
subject a compound that inhibits an interaction of a first protein
and an immunoreceptor tyrosine-based activation motif (ITAM) of a
second protein, thereby inhibiting a formation of a metastasis of a
Burkitt's lymphoma, a Hodgkin's disease, or a NPC in a subject.
42. The method of claim 41, wherein said compound comprises a
peptide homologous to said ITAM.
43. The method of claim 42, wherein said peptide comprises a
modification selected from hydroxylation, amidation,
esterification, formylation, gamma-carboxyglutamic acid
hydroxylation, methylation, phosphorylation, sulfation,
glycosylation, reduction, oxidation, disulfide modification,
introduction of a thioether bond, introduction of a thiolester
bond, a backbone condensation, or a biotinylation.
44. The method of claim 42, wherein said peptide comprises an amino
acid selected from a D amino acid; pyrrolidone carboxylic acid;
2-aminoadipic acid; 3-aminoadipic acid; beta-alanine;
beta-aminoproprionic acid; 2-aminobutyric acid; 4-aminobutyric
acid; piperidinic acid; 6-aminocaproic acid; 6-aminoheptanoic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4 diaminobutyric acid; desmosine; 2,2
diaminopimelic acid; 2,3 diaminopropionic acid; N-ethylglycine;
N-ethylasparagine; hydroxylysine; allo-hydroxylysine;
3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;
N-methylglycine; sarcosine; methylisoleucine; methyllysine;
methylvaline; norvaline; norleucine; 6-aminohexanoic acid;
citrulline; cysteic acid; cyclohexylalanine; alpha-amino isobutyric
acid; t-butylglycine; t-butylalanine; phenylglycine; an
N-alpha-methyl amino acid, a C-alpha-methyl amino acid, a
beta-methyl amino acid, or orthinine.
45. The method of claim 41, wherein said second protein is a viral
protein.
46. A method of treating a B cell proliferative disorder in a
subject, comprising administering to said subject a compound that
inhibits an interaction of a first protein and an immunoreceptor
tyrosine-based activation motif (ITAM) of a second protein, thereby
treating a B cell proliferative disorder in a subject.
47. A method of reducing an incidence of a B cell proliferative
disorder in a subject, comprising administering to said subject a
compound that inhibits an interaction of a first protein and an
immunoreceptor tyrosine-based activation motif (ITAM) of a second
protein, thereby reducing an incidence of a B cell proliferative
disorder in a subject.
48. A method of treating a mast cell activation disorder in a
subject, comprising administering to said subject a compound that
inhibits an interaction of a first protein and an immunoreceptor
tyrosine-based activation motif (ITAM) of a second protein, thereby
treating a mast cell activation disorder in a subject.
49. A method of reducing an incidence of a mast cell activation
disorder in a subject, comprising administering to said subject a
compound that inhibits an interaction of a first protein and an
immunoreceptor tyrosine-based activation motif (ITAM) of a second
protein, thereby reducing an incidence of a mast cell activation
disorder in a subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of U.S. Provisional
Application Ser. No. 60/643,906, filed Jan. 14, 2005, and U.S.
Provisional Application Ser. No. 60/649,900, filed Feb. 4, 2005.
These applications are hereby incorporated in their entirety by
reference herein.
FIELD OF INVENTION
[0003] This invention provides methods of treating, reducing the
incidence of, and inhibiting metastasis formation of carcinomas,
sarcomas, Epstein-Barr virus-induced malignancies, B cell
proliferative disorders, and mast cell activation disorders,
comprising administering to a subject a compound that inhibits an
interaction of a first protein and an immunoreceptor tyrosine-based
activation motif (ITAM) of a second protein, and screening methods
for identifying ITAM-inhibitory compounds and peptides. This
invention also provides peptides that inhibit signaling by
ITAMs.
BACKGROUND OF THE INVENTION
[0004] Oncogenic viruses such as Epstein-Barr virus (EBV) and
Kaposi sarcoma-associated herpesvirus (KSHV) are associated with a
number of malignancies, such as Kaposi sarcoma, Burkitt's lymphoma,
Hodgkin's lymphoma, post-transplant lymphoproliferative disease,
and the epithelial cell malignancy nasopharyngeal carcinoma (NPC).
Methods for treating these and similar malignancies are urgently
needed.
[0005] In addition, a variety of immune cell activation diseases
and disorders (e.g. B cell proliferative diseases and disorders and
mast cell activation diseases and disorders) cause significant
morbidity and mortality in the human population. Methods for
treating such diseases and disorders are also urgently needed.
SUMMARY OF THE INVENTION
[0006] This invention provides methods of treating, reducing the
incidence of, and inhibiting metastasis formation of carcinomas,
sarcomas, Epstein-Barr virus-induced malignancies, B cell
proliferative disorders, and mast cell activation disorders,
comprising administering to a subject a compound that inhibits an
interaction of a first protein and an immunoreceptor tyrosine-based
activation motif (ITAM) of a second protein, and screening methods
for identifying ITAM-inhibitory compounds and peptides. This
invention also provides peptides that inhibit signaling by
ITAMs.
[0007] In one embodiment, the present invention provides a method
of treating a carcinoma in a subject, comprising administering to
the subject a compound that inhibits an interaction of a first
protein and an ITAM of a second protein, thereby treating a
carcinoma in a subject.
[0008] In another embodiment, the present invention provides a
method of reducing an incidence of a carcinoma in a subject,
comprising administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby reducing an incidence of a carcinoma in a subject.
[0009] In another embodiment, the present invention provides a
method of inhibiting a formation of a metastasis of a carcinoma in
a subject, comprising administering to the subject a compound that
inhibits an interaction of a first protein and an ITAM of a second
protein, thereby inhibiting a formation of a metastasis of a
carcinoma in a subject.
[0010] In another embodiment, the present invention provides a
method of treating a sarcoma in a subject, comprising administering
to the subject a compound that inhibits an interaction of a first
protein and an ITAM of a second protein, thereby treating a sarcoma
in a subject.
[0011] In another embodiment, the present invention provides a
method of reducing an incidence of a sarcoma in a subject,
comprising administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby reducing an incidence of a sarcoma in a subject.
[0012] In another embodiment, the present invention provides a
method of inhibiting a formation of a metastasis of a sarcoma in a
subject, comprising administering to the subject a compound that
inhibits an interaction of a first protein and an ITAM of a second
protein, thereby inhibiting a formation of a metastasis of a
sarcoma in a subject.
[0013] In another embodiment, the present invention provides a
method of treating an Epstein-Barr virus-induced malignancy in a
subject, comprising administering to the subject a compound that
inhibits an interaction of a first protein and an ITAM of a second
protein, thereby treating an Epstein-Barr virus-induced malignancy
in a subject.
[0014] In another embodiment, the present invention provides a
method of reducing an incidence of an Epstein-Barr virus-induced
malignancy in a subject, comprising administering to the subject a
compound that inhibits an interaction of a first protein and an
ITAM of a second protein, thereby reducing an incidence of an
Epstein-Barr virus-induced malignancy in a subject.
[0015] In another embodiment, the present invention provides a
method of inhibiting a formation of a metastasis of an Epstein-Barr
virus-induced malignancy in a subject, comprising administering to
the subject a compound that inhibits an interaction of a first
protein and an ITAM of a second protein, thereby inhibiting a
formation of a metastasis of an Epstein-Barr virus-induced
malignancy in a subject.
[0016] In another embodiment, the present invention provides a
method of treating a B cell proliferative disorder in a subject,
comprising administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby treating a B cell proliferative disorder in a subject.
[0017] In another embodiment, the present invention provides a
method of reducing an incidence of a B cell proliferative disorder
in a subject, comprising administering to the subject a compound
that inhibits an interaction of a first protein and an ITAM of a
second protein, thereby reducing an incidence of a pathological
immune cell activation in a subject.
[0018] In another embodiment, the present invention provides a
method of treating a mast cell activation disorder in a subject,
comprising administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby treating a mast cell activation disorder in a subject.
[0019] In another embodiment, the present invention provides a
method of reducing an incidence of a mast cell activation disorder
in a subject, comprising administering to the subject a compound
that inhibits an interaction of a first protein and an ITAM of a
second protein, thereby reducing an incidence of a mast cell
activation disorder in a subject.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1: Contribution of ITAM domain in MMTV Env to cell
transformation. (A) Schematic comparison of the ITAM in MMTV Env
and consensus ITAM sequence. Conserved residues are shaded. (B)
Development of mammary epithelial cell acinar structures on
Matrigel. (C) Left panel: Representative images of
three-dimensional cultures of un-transfected ("wild-type"), mutated
envelope-transfected ("Env.sup.2xY>F"), and unmutated
envelope-transfected ("Env.sup.+") NMuMG cells at day 6 of culture.
For WT and Env.sup.2xY>F, images are also depicted at a
magnification of 4. Note polarized structures with hollow lumen in
both cell types. Bars, 50 .mu.m. Right panel: Mean fluorescence
intensity of Env expression in the transfected cells. "Clone 1"
refers to the MMTV+NMuMG cells. (D) Quantification of structure
size in a representative experiment. (E) Representative images of
three-dimensional cultures of un-transfected ("wild-type"),
unmutated envelope-transfected ("Env.sup.+"), and MMTV infected
NMuMG cells at day 6 of culture. Black bars represent the median
for each culture. Surface expression of MMTV SU (gp52) is depicted
for mutated envelope-transfected and un-mutated
envelope-transfected NMuMG cells. Normal goat IgG was used as the
control antibody.
[0021] FIG. 2: Involvement of Src and Syk kinases in MMTV
Env-induced cell transformation. (A) MMTV SU co-immunoprecipitated
with Syk in a tyrosine-dependent manner. Mutated
envelope-transfected ("Env.sup.2xY>F") and unmutated
envelope-transfected ("Env.sup.+") two-dimensional cultures were
treated with sodium pervanadate, lysed, normalized for total
protein content, and subjected to MMTV SU or Syk IP. A Western blot
for MMTV SU is depicted. Syk co-immunoprecipitated only with Env,
but not with Env.sup.2xY>F. (B) un-transfected ("wild-type") and
unmutated envelope-transfected cultures were treated on day 3 three
dimensionally with either normal assay media, PP2 (500 ng/ml), or
Piceatannol (500 ng/ml), then imaged on day 6. Bars, 50 .mu.m. (C)
Quantification of structure size in a representative experiment.
Black bars represent the median for each culture.
[0022] FIG. 3. Expression of MMTV Env in murine mammary epithelial
cells is sufficient for cell transformation. (A) Representative
images of three-dimensional cultures, on Matrigel.RTM. cushions, of
un-transfected ("wild-type") and unmutated envelope-transfected
("Env.sup.+") NMuMG cells at day 6 of culture are depicted. Bars,
50 .mu.m. (B) Left panel: Keratin-18 staining of un-transfected and
unmutated envelope-transfected NMuMG cells at day 6. Keratin-18 is
in the bright area shaded in diagonal lines; nuclear staining
(DAPI) is in the unmarked, bright area. Right panel: E-cadherin
surface expression, as quantified by flow cytometry, is reduced in
two-dimensional cultures of unmutated envelope-transfected and
MMTV+ ("clone 1") NMuMG cells. (C) Un-transfected ("wild-type") and
unmutated envelope-transfected ("Env.sup.+") cultures were treated
17 h before imaging with either normal assay media, 1 .mu.g/ml
TRAIL, or 100 nM TNF. Induction of apoptosis was confirmed by TUNEL
assays.
[0023] FIG. 4. MMTV Env also transforms human mammary epithelial
cells. (A) Left panel: Colony formation assay for un-transfected
("wild-type"), mutated envelope-transfected ("Env.sup.2xY>F"),
and unmutated envelope-transfected ("Env.sup.+") MCF-10F cells.
Colonies were defined as cell clusters >60 .mu.m in size. Right
panel: The percentage of colonies formed in five agar-methocel
cultures in an experiment representative of three independent
experiments. (B) Representative images from collagen-matrix
cultures of un-transfected, mutated envelope-transfected, and
unmutated envelope-transfected MCF-10F cell lines (experiment
representative of three independent experiments). Ductal structures
are visible in wild-type cultures. Complete loss of ductal
structure was observed in Env.sup.+ cultures, whereas
Env.sup.2xY>F cells exhibited a mixed phenotype. (C)
Representative images from invasion assays of un-transfected
("wild-type"), Env.sup.2xY>F, and Env.sup.+ MCF-10F cells. Left
panel: Stained mesh after the completion of the invasion assays.
Invading cells were visualized by stain. The right panel depicts
total invading cell counts from a representative experiment (in
duplicate) out of three independent experiments.
[0024] FIG. 5. MMTV envelope sequence, domains, and topological
models. (A) Schematic representation of MMTV Env protein and its
domains. Potential transmembrane domains are numbered and depicted
in diagonally striped boxes. The ITAM region is marked in a shaded
box. (B) Models for the surface expression of mature MMTV Env on
the host cell surface. The domains are marked as in A.
Hydrophobicity scores for the transmembrane domains were as
follows: 1: 1,489; 2: 699; 3: 2,658; and 4: 3,134.
[0025] FIG. 6. Interaction of ITAM peptide with Syk kinase. (A)
Human 293T cells were transiently transfected with empty dsRed
construct (row i), dsRed.YVLL (row ii), or dsRed.KRPPYVLL (row iii)
fusion proteins, along with Syk-GFP. Single channel images show the
expression of either dsRed (left panels) or GFP (middle panels)
constructs. The right panels of rows i-iii show a merged image of
these constructs. The extreme right panel in row iii is enhanced
view of the dsRed channel, which was weak for the dsRed.KRPPYVLL
construct. Co-localization of Syk and the peptides is quantified in
B-C. (D) Bal-17 B cell lyases were used for quantification of ITAM
peptide interactions. Equivalent amounts of cell lysates were mixed
with either biotin-KRPPAVLL (control peptide) or biotin-KRPPYVLL
(ITAM peptide) in concentration between 10-50 .mu.M. Pull-downs
were detected with anti-Syk antibody (upper panel) and quantified
(lower panel).
[0026] FIG. 7. MMTV Env-induced mammary epithelial transformation
in 3D cultures depends on ITAM:Syk interaction. Wild-type or
Env-expressing NmuMG cells were incubated on Matrigel.RTM. for six
days, in the presence or absence of 10 .mu.M ITAM peptide or the
Syk inhibitors piceatannol (1 .mu.g/ml) and SI-31 (100 nM).
Three-dimensional structures >50 .mu.m were considered enlarged.
(A) The percentage of enlarged structures is shown for each
treatment. (B) Pictures of enlarged (top) and normal size 3D
structures (bottom).
[0027] FIG. 8. Reversion of MMTV-induced transformed phenotype of
mammary epithelial cells by ITAM peptide. Representative pictures
from three-dimensional cultures: MMTV+Mm5MT tumor cells were
incubated on Matrigel.RTM. (for six days, in the presence or
absence of ITAM peptide (5-20 .mu.M) or the Syk inhibitor SI-31
(100 nM). Reversion to round 3D phenotype was readily apparent in
cultures treated with 20 .mu.M peptide or SI-31.
[0028] FIG. 9. Reversion of ITAM-induced transformed phenotype of
fibroblasts by ITAM peptide. (A) Colony formation assay for NIH3T3
cells transduced with the ITAM-containing chimera, MAHB. Colonies
were defined as viable cell clusters greater than 75 .mu.m in size
and scored on day 21. ITAM peptide (0-20 .mu.M) was added with the
feeding media throughout the experiment and lead to reduction in
both size and number of colonies formed.
[0029] FIG. 10. Effect of ITAM peptide on B lymphocyte stimulation.
Proliferation of mouse splenic B cells: (A) Cells were incubated
for 48 h in the presence or absence of 20 .mu.M peptide and
stimulated with 10 .mu.g/ml anti-BCR antibodies. [.sup.3H]
Thymidine incorporation was measured in the last 4 h of the
experiment. Inhibition of the B cell proliferation by the ITAM
peptide was highly significant (p=0.0017). (B) Similar stimulations
were performed with the non-ITAM containing ligands, LPS (20
.mu.g/ml) and anti-CD40 (5 .mu.g/ml). Concominant addition of the
ITAM peptide had no effect on these stimuli. (C) Inhibition of
tyrosine phosphorylation was confirmed by Western blotting. Splenic
B cells were pre-incubated with 0-100 .mu.M ITAM peptide for 3 h
and then stimulated with anti-BCR antibodies as above for further 5
min. Lysates were detected with anti-phospho-tyrosine antibody.
[0030] FIG. 11. Fc.epsilon.RI-mediated degranulation of RBL-2H3
cells. The effect of ITAM peptide on mast cell function was
evaluated by degranulation induced by Fc.epsilon.RI cross-linking.
RBL-2H3 cells were incubated overnight with anti-DNP IgE (1
.mu.g/ml), in the presence or absence of ITAM peptide (25 .mu.M).
Subsequently, the cells were cross-linked with DNP-HSA (10 ng/ml)
to induce degranulation, as detected by hexosaminidase activity.
Degranulation is shown as percent of maximal activity obtained from
whole cell lysates. ITAM peptide treatment caused a significant
reduction (p=0.093) in Fc.epsilon.RI-induced degranulation.
[0031] FIG. 12. MAHB: a membrane-bound, nonviral ITAM-containing
protein. (a) Illustration of the MAHB fusion protein, which encodes
the cytoplasmic domains of Ig.alpha. and Ig.beta. separated by an
HA tag (white). The protein is targeted to the membrane by the
myristoylation/palmitoylation sequence of Lck. (b) GFP expression
of NMuMG cells transduced with the empty vector (MIGR), MAHB, and
ITAM-mutant as assessed by flow cytometry and controlled by
nontransduced WT cells. (c) Expression of MAHB and ITAM-mutant in
NMuMG cells grown in 2D culture as identified by HA staining in
(unmarked, bright areas), with nuclear staining (DAPI; bright areas
shaded in diagonal lines). Bar, 50 .mu.m.
[0032] FIG. 13. Expression of a nonviral ITAM-containing protein
disrupts 3D acinar architecture of murine mammary epithelial cells
(a) On a scale of zero (normal) to five (most abnormal), acini were
scored based on their structure and given one point for each of the
abnormalities listed. Depicted are examples of normal and disrupted
acini with their corresponding score. Bar, 50 .mu.m. (b) 3D
cultures of MIGR, MAHB, and ITAM-mutant-expressing NMuMG cells at
day 6 of culture on a Matrigel cushion. Representative acini
depicted are GFP.sup.+. The average score was calculated by scoring
50 acini from each cell type. ***: P<0.0001 (one-way ANOVA
analysis). Bar, 50 .mu.m. (c) The morphological phenotypes of acini
scored in (B). The percentage of 3D acini with each morphological
characteristic is shown. As acini can have multiple abnormalities,
the percentages do not add up to 100%.
[0033] FIG. 14. Expression of MAHB in murine mammary epithelial
cells leads to anchorage independence. Colony formation assay for
NMuMG cells transduced with the empty vector (MIGR), MAHB, and
ITAM-mutant. Colonies were scored on day 28 and defined as viable
cell clusters greater than 75 .mu.m in size. Representative bright
field images overlaid with GFP to indicate protein expression from
one of three independent experiments are shown. The average size
and total number of colonies formed by each cell type (out of 1
10.sup.4 cells seeded) in a representative experiment are also
indicated. ***: P<0.0001 (one-way ANOVA analysis). Bar, 75
.mu.m.
[0034] FIG. 15. MAHB expression in murine mammary epithelial cells
induces an EMT phenotype. E-cadherin and vimentin expression in
NMuMG cells transduced with MIGR, MAHB, and ITAM-mutant. (a) Top,
mean fluorescence intensity (MFI) of E-cadherin surface expression
as quantified by flow cytometry from 2D cultures. Data from one
representative experiment is depicted. Bottom, 3D cultures stained
for E-cadherin on day 6, Representative images are depicted with
nuclear staining (DAPI; unmarked, bright areas) and E-cadherin
(bright areas shaded in diagonal lines). (b) 3D cultures stained
for vimentin on day 9. Representative images are depicted with
nuclear staining (DAPI; unmarked, bright areas) in blue and
vimentin (bright areas shaded in diagonal lines). (a, b) All images
are depicted under the same magnification, and all structures are
GFP.sup.+.
[0035] FIG. 16. MAHB-expressing acini become sensitive to TRAIL and
TNF.alpha.. Sensitivity of MAHB- and ITAM-mutant-expressing NmuMG
acini to apoptotic-inducing agents. Acini were treated in 3D
cultures on day 8 for 20 h with either 1 mg/ml TRAIL, or 100 nM
TNF.alpha.. Cultures were then stained for activated caspase-3 to
assess apoptosis induction Representative images of the same
magnification are depicted with nuclear staining (DAPI; unmarked,
bright areas) and activated caspase-3 (bright areas shaded in
diagonal lines); all structures are GFP.sup.+.
[0036] FIG. 17. Involvement of Src and Syk kinases in MAHB-induced
transformation. Phosphorylation status of MAHB and association with
phosphorylated Syk in 2D cultures of NMuMG cells. (a) Upper panel
depicts a representative Western blot of NMuMG whole-cell lysates
probed with an anti-phosphotyrosine antibody. Lower panel shows the
same blot stripped and re-probed with an HA antibody. (b) HA IP
from pervanadate-treated cells (representative of four
experiments). Upper panel: Western blot probed with an antibody
against phosphotyrosine. Lower panel: the same blot stripped and
re-probed with an antibody specific for Syk. Results are
representative of four independent experiments. (c) MAHB expressing
NMuMG acini were treated in 3D cultures for 3 days with either
vehicle (DMSO), PP2 (1 .mu.g/ml), Piceatannol (1 .mu.g/ml), or a
specific Syk inhibitor (0.25 .mu.M). Representative bright field
images taken on day 6 are depicted; structures are GFP.sup.+. Bar,
50 .mu.m. Average score of acinar structures was calculated by
scoring 50 acini for each treatment. ***: P<0.0001 (one-way
ANOVA analysis).
[0037] FIG. 18. MAHB transforms murine fibroblasts. (a) Colony
formation assay for NIH3T3 cells transduced with the empty vector
(MIGR), MAHB, and ITAM-mutant. Colonies were defined as viable cell
clusters greater than 75 .mu.m in size and scored on day 21.
Representative images from one of three independent experiments.
Original magnification, .times.10. (b) Average size of colonies
formed from each cell type in three independent colony formation
assays. Black bar and value represent the mean for the three
experiments. ***: P<0.0001 (one-way ANOVA analysis). (c) Focus
formation assay with NIH3T3 cells transduced with the empty vector
(MIGR), MAHB, and ITAM-mutant. Average number of foci formed in a 4
cm.sup.2 area from three independent experiments is shown; error
bars indicate standard deviation. *: P<0.05 (one-way ANOVA
analysis).
DETAILED DESCRIPTION OF THE INVENTION
[0038] This invention provides methods of treating, reducing the
incidence of, and inhibiting metastasis formation of carcinomas,
sarcomas, Epstein-Barr virus-induced malignancies, B cell
proliferative disorders, and mast cell activation disorders,
comprising administering to a subject a compound that inhibits an
interaction of a first protein and an immunoreceptor tyrosine-based
activation motif (ITAM) of a second protein, and screening methods
for identifying ITAM-inhibitory compounds and peptides. This
invention also provides peptides that inhibit signaling by
ITAMs.
[0039] In one embodiment, the present invention provides a method
of treating a carcinoma in a subject, comprising administering to
the subject a compound that inhibits an interaction of a first
protein and an ITAM of a second protein, thereby treating a
carcinoma in a subject. In one embodiment of methods and
compositions of the present invention, the interaction between the
first protein and the ITAM is an intracellular interaction.
[0040] In another embodiment, the present invention provides a
method of reducing an incidence of a carcinoma in a subject,
comprising administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby reducing an incidence of a carcinoma in a subject.
[0041] In another embodiment, the present invention provides a
method of inhibiting a formation of a metastasis of a carcinoma in
a subject, comprising administering to the subject a compound that
inhibits an interaction of a first protein and an ITAM of a second
protein, thereby inhibiting a formation of a metastasis of a
carcinoma in a subject.
[0042] In another embodiment, the present invention provides a
method of reducing an invasiveness of a metastasis of a carcinoma
in a subject, comprising administering to the subject a compound
that inhibits an interaction of a first protein and an ITAM of a
second protein, thereby reducing an invasiveness of a metastasis of
a carcinoma in a subject.
[0043] In another embodiment, the present invention provides a
method of reversing a malignant transformation of a carcinoma in a
subject, comprising administering to the subject a compound that
inhibits an interaction of a first protein and an ITAM of a second
protein, thereby reversing a malignant transformation of a
carcinoma in a subject.
[0044] As provided herein (Examples 1-4), findings of the present
invention show that expression of a protein containing a viral
ITAM, but not a mutant-ITAM-containing protein, is capable of
transformation of epithelial cells, as shown by multiple assays.
Activity of cellular signaling molecules that associated with the
ITAM (e.g. Syk kinase) was necessary for the transformation to
occur. These findings show that blocking association of a viral
ITAM with cellular molecules and blocking viral ITAM-mediated
signaling are effective strategies for preventing and reducing the
incidence of malignant transformation. In addition, testing of a
peptide of the present invention showed that ITAM
association-blocking peptides can not only prevent, but also
reverse transformation of epithelial cells, and thus can be used to
treat existing carcinomas (Examples 5-6).
[0045] In addition, the findings of the present invention show that
expression of a viral ITAM-containing protein increases
invasiveness of epithelial cells, an indication of metastatic
properties (Example 4). Thus, blocking association of an ITAM with
cellular molecules blocking ITAM-mediated signaling are effective
strategies for inhibiting metastases and reducing invasiveness of
carcinoma cells.
[0046] Expression of a protein containing a cellular ITAM is also
capable of transformation of epithelial cells (Examples 16-19).
Thus, blocking association of a cellular ITAM with cellular
molecules and blocking cellular ITAM-mediated signaling are
effective strategies for preventing and reducing the incidence of
carcinoma malignant transformation, treating carcinomas, inhibiting
metastases, and reducing invasiveness of carcinoma cells.
[0047] Moreover, the present invention provides methods of
modifying peptides of the present invention and identifying further
improved peptides using models of carcinoma, both primary tumors
and metastases (Examples 8-9); or, in another embodiment, using
3-dimensional culture assays of ITAM-containing protein-transfected
cells (e.g. as described in Example 21); or, in another embodiment,
using colony formation assays (e.g. as described in Example 22);
or, in another embodiment, using ITAM co-IP assays (e.g. as
described in Example 23); or, in another embodiment, using ability
to abrogate or reduce EMT (e.g. as described in Example 24); or, in
another embodiment, using ability to abrogate or reduce sensitivity
to apoptosis (e.g. as described in Example 25); or, in another
embodiment, using ability to abrogate or reduce phosphorylation of
an ITAM-containing protein (e.g. as described in Example 29). These
methods facilitate, in another embodiment, selection of further
improved ITAM-inhibitory peptides.
[0048] "ITAMs" are, in one embodiment, motifs found in immune cells
and some viral proteins, characterized by 2 YXXL/I (SEQ ID No: 11)
sequences. The ITAM-containing protein (i.e. the "second protein")
targeted by methods and compositions of the present invention is,
in another embodiment, a viral protein. In another embodiment, the
ITAM-containing protein is a cellular protein. In another
embodiment, the ITAM or ITAM motif is a viral ITAM or ITAM motif.
In another embodiment, the ITAM or ITAM motif is a cellular ITAM or
ITAM motif.
[0049] In another embodiment, "ITAM" refers to a protein motif that
functions as a docking site for SH2-containing signaling proteins
involved in linking receptor-initiated signals to downstream
cellular responses. In another embodiment, "ITAM" refers to a
protein motif that interacts with proteins from Syk/Zap-70 family
tyrosine kinases. In another embodiment, the ITAM motif interacts
with Src family tyrosine kinases. In another embodiment, the ITAM
motif interacts with both Syk/Zap-70 family tyrosine kinases and
Src family tyrosine kinases. In another embodiment, one or more
residues of the ITAM motif are phosphorylated by a Src family
kinase. In another embodiment, the phosphorylation leads to
Src-homology 2 (SH2)-mediated docking and activation of Syk family
kinases. In another embodiment, adaptor molecules other than Syk
kinases are recruited following phosphorylation of the ITAM motif.
Each possibility represents a separate embodiment of the present
invention.
[0050] "Viral ITAM" refers, in another embodiment, to an ITAM that
is present on a viral protein. In another embodiment, the term
refers to an ITAM that is expressed by a cell in response to a
viral infection. "Cellular ITAM" refers, in another embodiment, to
an ITAM that is present on a cellular protein. Each possibility
represents a separate embodiment of the present invention.
[0051] In another embodiment, the ITAM has the sequence
PAYDYAAIIVKRPPYVLLPVDIGD (SEQ ID No: 1). In another embodiment, the
ITAM is homologous to SEQ ID No: 1. In another embodiment, the ITAM
has the sequence
(D/E)X.sub.7(D/E)X.sub.2YX.sub.2LX.sub.7YX.sub.2(L/I), wherein X is
any amino acid (SEQ ID No: 3). In another embodiment, the ITAM has
the sequence (D/E)X.sub.8(D/E)X.sub.2YX.sub.2LX.sub.12YX.sub.2(L/I)
(SEQ ID No: 4). In another embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.2YX.sub.2LX.sub.7YX.sub.2(L/I) (SEQ ID No:
5). In another embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.2YX.sub.2LX.sub.12YX.sub.2(L/I) (SEQ ID
No: 6). In another embodiment, the ITAM has the sequence
(D/E)X.sub.7(D/E)X.sub.2YX.sub.2LX.sub.7-12YX.sub.2(L/I) (SEQ ID
No: 7). In another embodiment, the ITAM has the sequence
(D/E)X.sub.8(D/E)X.sub.2YX.sub.2LX.sub.7-12YX.sub.2(L/I) (SEQ ID
No: 8). In another embodiment, the ITAM has the sequence
YX.sub.2(I/L)(X.sub.6-8)YX.sub.2(I/L) (SEQ ID No: 9). In another
embodiment, the ITAM has the sequence
(D/E)X.sub.0-2YXX(L/I)X.sub.6-8YXX(L/I) (SEQ ID No: 10). In another
embodiment, the ITAM has the sequence
(D/E)X.sub.0-2YAAIX.sub.6-8YVLL (SEQ ID No: 12). In another
embodiment, the ITAM has the sequence (D/E)YAAIX.sub.6-8YVLL (SEQ
ID No: 13). In another embodiment, the ITAM has the sequence
(D/E)X.sub.0-2YAAIX.sub.6YVLL (SEQ ID No: 14). In another
embodiment, the ITAM has the sequence (D/E)YAAIX.sub.6YVLL (SEQ ID
No: 15). In another embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.0-2YXX(L/I)X.sub.6-8YXX(L/I) (SEQ ID No:
16). In another embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.0-2YXXLX.sub.6-8YXX(L/I) (SEQ ID No: 17).
In another embodiment, the ITAM has the sequence
DMPDDYEDENLYEGLNLDDCSMYEDI (SEQ ID No: 18). In another embodiment,
the ITAM is homologous to SEQ ID No: 18. In another embodiment, the
ITAM has the sequence (D/E)X.sub.7-8(D/E)X.sub.0-2YEGLX.sub.6-8YEDI
(SEQ ID No: 19). In another embodiment, the ITAM has the sequence
(D/E)X.sub.7(D/E)X.sub.2YEGLX.sub.6-8YEDI (SEQ ID No: 20). In
another embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.0-2YEGLX.sub.7YEDI (SEQ ID No: 21). In
another embodiment, the ITAM has the sequence
(D/E)X.sub.7(D/E)X.sub.2YEGLX.sub.7YEDI (SEQ ID No: 22). In another
embodiment, the ITAM has the sequence
EKFGVDMPDDYEDENLYEGLNLDDCSMYEDI (SEQ ID No: 23). In another
embodiment, the ITAM is homologous to SEQ ID No: 23. In another
embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.0-2YEDEX.sub.7-16YEDI (SEQ ID No: 24). In
another embodiment, the ITAM has the sequence
(D/E)X.sub.9-10(D/E)X.sub.0-1YEDEX.sub.13YEDI (SEQ ID No: 25). In
another embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.0-2YEDEX.sub.2YEGLX.sub.7YEDI (SEQ ID No:
26). In another embodiment, the ITAM has the sequence
(D/E)X.sub.9-10(D/E)X.sub.0-1YEDEX.sub.2YEGLX.sub.7YEDI (SEQ ID No:
27). In another embodiment, the ITAM has the sequence
DKDDGKAGMEEDHTYEGLNIDQTATYEDI (SEQ ID No: 28). In another
embodiment, the ITAM is homologous to SEQ ID No: 28. In another
embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.0-2YEGLX.sub.6-8YEDI (SEQ ID No: 29). In
another embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.2YEGLX.sub.6-8YEDI (SEQ ID No: 30). In
another embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.0-2YEGLX.sub.7YEDI (SEQ ID No: 31). In
another embodiment, the ITAM has the sequence
(D/E)X.sub.7-8(D/E)X.sub.2YEGLX.sub.7YEDI (SEQ ID No: 32). In
another embodiment, the ITAM has the sequence
SSCRLTNCLDSSAYVYAAIIVLMPPYVLL (SEQ ID No: 36). In another
embodiment, the ITAM is homologous to SEQ ID No: 36. In another
embodiment, the ITAM has the sequence (D/E)X.sub.5YAAIX.sub.6YVLL
(SEQ ID No: 37). In another embodiment, the ITAM has the sequence
RLTNCLDSSAYDYAAIIVKRPPYVLL (SEQ ID No: 38). In another embodiment,
the ITAM is homologous to SEQ ID No: 38. In another embodiment, the
ITAM has the sequence (D/E)X.sub.4DYAAIX.sub.6YVLL (SEQ ID No: 39).
In another embodiment, the ITAM has the sequence
DSSAYDYAAIIVKRPPYVLL (SEQ ID No: 40). In another embodiment, the
ITAM is homologous to SEQ ID No: 40. In another embodiment, the
ITAM has the sequence DX.sub.4DYAAX.sub.6YVLL (SEQ ID No: 41). In
another embodiment, the ITAM has the sequence
PYDAEDGGDGGPYQPLRGQDPNQLYARL (SEQ ID No: 90). In another
embodiment, the ITAM is homologous to SEQ ID No: 90. In another
embodiment, the ITAM has the sequence
GPYQPLRGQDPNQLYARLGGGGGNGTLPPPPYSPQRETSLHLYEEI (SEQ ID No: 91). In
another embodiment, the ITAM is homologous to SEQ ID No: 91. In
another embodiment, the ITAM has the sequence
EDPYWGNGDRHSDYQPLGTQDQSLYLGL (SEQ ID No: 92). In another
embodiment, the ITAM is homologous to SEQ ID No: 92. In another
embodiment, the ITAM has the sequence
PPYEDLDWGNGDRHSDYQPLGNQDPSLYLGL (SEQ ID No: 93). In another
embodiment, the ITAM is homologous to SEQ ID No: 93. In another
embodiment, the ITAM has the sequence
YDAPSHRPPSYGGSGGYATLGQQEPSLYAGL (SEQ ID No: 94). In another
embodiment, the ITAM is homologous to SEQ ID No: 94. In another
embodiment, the ITAM has the sequence
DRDGDPVPPDYDAPSHRPPSYGGSGGYATLGQQEPSLYAGL (SEQ ID No: 95). In
another embodiment, the ITAM is homologous to SEQ ID No: 95. In
another embodiment, the ITAM has the sequence
LSKLTALVAVATWFAILMTYLVLPSANNIIVLSLLVAAEGIQSIYLLV (SEQ ID No: 96).
In another embodiment, the ITAM is homologous to SEQ ID No: 96. In
another embodiment, the ITAM has the sequence
ESNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGL (SEQ ID No: 97). In another
embodiment, the ITAM is homologous to SEQ ID No: 97. In another
embodiment, the ITAM has the sequence EDSDWGNGDRHSDYQPLGNQDPSLYLGL
(SEQ ID No: 98). In another embodiment, the ITAM is homologous to
SEQ ID No: 98. In other embodiments, the ITAM has any of the ITAM
sequences found in GenBank Accession Numbers AAO86793-AAO86822,
inclusive, AAD53747-AAD53750, inclusive, Q9QGQ3-Q9QGQ6, inclusive,
AAL50731-AAL50751, inclusive, AAK94430-AAK94430, inclusive,
Q9WHI0-Q9WHI4, inclusive, Q9WHH8-Q9WHH9, inclusive, and
AAD30529-AAD30535, inclusive. In other embodiments, the ITAM is
homologous to any of the ITAM sequences found in the above GenBank
Accession Numbers. In other embodiments, the ITAM has any of the
ITAM sequences enumerated in the Examples herein. In another
embodiment, the ITAM has any other ITAM sequence known in the art.
Each possibility represents a separate embodiment of the present
invention.
[0052] In another embodiment, more than one of the above ITAM
sequences or ITAM motifs are targeted, inhibited, or blocked by a
method of the present invention. In another embodiment, two ITAM
sequences are targeted, inhibited, or blocked. In another
embodiment, three ITAM sequences are targeted, inhibited, or
blocked. In another embodiment, four ITAM sequences are targeted,
inhibited, or blocked. In another embodiment, more than four ITAM
sequences are targeted, inhibited, or blocked. Each possibility
represents a separate embodiment of the present invention.
[0053] In another embodiment of methods and compositions of the
present invention, the protein that interacts with the ITAM (i.e.
the "first protein") is a cellular protein. In another embodiment,
the protein is a signaling protein. In another embodiment, the
protein is a tyrosine kinase. In another embodiment, the protein is
a tyrosine kinase substrate. In another embodiment, the protein is
a signaling protein. In another embodiment, the protein is a viral
protein that acts as a signaling protein, tyrosine kinase, or
tyrosine kinase substrate in eukaryotic cells. In another
embodiment, the protein is any other type of protein known in the
art. Each possibility represents a separate embodiment of the
present invention.
[0054] The carcinoma that is the target of methods and compositions
of the present invention is, in another embodiment, a breast cell
carcinoma. In another embodiment, the carcinoma is an epithelial
cell malignancy. In another embodiment, the carcinoma is a mammary
epithelial cell malignancy. In other embodiments, the carcinoma is
a ductal carcinoma (e.g. infiltrating ductal carcinoma) squamous
cell carcinoma, squamous epithelial carcinoma, lobular carcinoma
(e.g. of the breast), adenocarcinoma (e.g. an endometrioid
adenocarcinoma), small cell carcinoma, carcinoma of the vulva,
renal cell carcinoma, non-small cell lung carcinoma, soft-tissue
carcinoma, basal cell carcinoma, buccal cell carcinoma,
thyroid/follicular carcinoma, sebaceous gland carcinoma, adrenal
carcinoma, transitional cell carcinoma, urothelial carcinoma,
fibrolamellar carcinoma, or hepatocellular carcinoma. In another
embodiment, the carcinoma is any other carcinoma or type of
carcinoma known in the art. Each possibility represents a separate
embodiment of the present invention.
[0055] In another embodiment, the present invention provides a
method of treating a sarcoma in a subject, comprising administering
to the subject a compound that inhibits an interaction of a first
protein and an ITAM of a second protein, thereby treating a sarcoma
in a subject.
[0056] In another embodiment, the present invention provides a
method of reducing an incidence of a sarcoma in a subject,
comprising administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby reducing an incidence of a sarcoma in a subject.
[0057] In another embodiment, the present invention provides a
method of inhibiting a formation of a metastasis of a sarcoma in a
subject, comprising administering to the subject a compound that
inhibits an interaction of a first protein and an ITAM of a second
protein, thereby inhibiting a formation of a metastasis of a
sarcoma in a subject.
[0058] In another embodiment, the present invention provides a
method of reducing an invasiveness of a metastasis of a sarcoma in
a subject, comprising administering to the subject a compound that
inhibits an interaction of a first protein and an ITAM of a second
protein, thereby reducing an invasiveness of a metastasis of a
sarcoma in a subject.
[0059] In another embodiment, the present invention provides a
method of reversing a malignant transformation of a sarcoma in a
subject, comprising administering to the subject a compound that
inhibits an interaction of a first protein and an ITAM of a second
protein, thereby reversing a malignant transformation of a sarcoma
in a subject.
[0060] As provided herein (Example 20), findings of the present
invention show that expression of a protein containing an ITAM, but
not a mutant-ITAM-containing protein, is capable of transformation
of connective tissue cells, as shown by multiple assays. These
findings show that blocking association of an ITAM with cellular
molecules and blocking ITAM-mediated signaling are effective
strategies for preventing and reducing the incidence of sarcoma
malignant transformation, treating sarcomas, inhibiting metastases,
and reducing invasiveness of sarcoma cells.
[0061] Moreover, the present invention provides methods of
modifying peptides of the present invention and identifying further
improved peptides using models of sarcoma, both primary tumors and
metastases (Examples 8, 11, and 31); or, in another embodiment,
using colony formation assays (e.g. as described in Example 22);
or, in another embodiment, using ITAM co-IP assays (e.g. as
described in Example 23); or, in another embodiment, using ability
to abrogate or reduce phosphorylation of an ITAM-containing protein
(e.g. as described in Example 29); or, in another embodiment, using
a focus formation assay (e.g. as described in Example 30) These
methods facilitate, in another embodiment, selection of further
improved ITAM-inhibitory peptides.
[0062] The sarcoma that is the target of methods of the present
invention is, in one embodiment, a fibrosarcoma. In another
embodiment, the sarcoma is a Kaposi's sarcoma. In another
embodiment, the sarcoma is a connective tissue cell malignancy. In
another embodiment, the sarcoma is a fibroblast-derived tumor. In
another embodiment, the sarcoma is a Ewing Sarcoma. In another
embodiment, the sarcoma is a neuroectodermal tumor (e.g. a
primitive neuroectodermal tumor). In another embodiment, the
sarcoma is a post-radiation sarcoma. In another embodiment, the
sarcoma is a synovial cell sarcoma. In another embodiment, the
sarcoma is a clear cell sarcoma. In another embodiment, the sarcoma
is a rhabdomyosarcoma. In another embodiment, the sarcoma is a
uterine sarcoma. In another embodiment, the sarcoma is an
endometrial stromal sarcoma. In another embodiment, the sarcoma is
an osteosarcoma. In another embodiment, the sarcoma is a
chondrosarcoma. In another embodiment, the sarcoma is a
leiomyosarcoma. In another embodiment, the sarcoma is an
endothelial sarcoma. In another embodiment, the sarcoma is any
other type of sarcoma known in the art. Each possibility represents
a separate embodiment of the present invention.
[0063] In another embodiment, the present invention provides a
method of treating an Epstein-Barr virus-induced malignancy in a
subject, comprising administering to the subject a compound that
inhibits an interaction of a first protein and an ITAM of a second
protein, thereby treating an Epstein-Barr virus-induced malignancy
in a subject.
[0064] In another embodiment, the present invention provides a
method of reducing an incidence of an Epstein-Barr virus-induced
malignancy in a subject, comprising administering to the subject a
compound that inhibits an interaction of a first protein and an
ITAM of a second protein, thereby reducing an incidence of an
Epstein-Barr virus-induced malignancy in a subject.
[0065] In another embodiment, the present invention provides a
method of inhibiting a formation of a metastasis of an Epstein-Barr
virus-induced malignancy in a subject, comprising administering to
the subject a compound that inhibits an interaction of a first
protein and an ITAM of a second protein, thereby inhibiting a
formation of a metastasis of an Epstein-Barr virus-induced
malignancy in a subject.
[0066] In another embodiment, the present invention provides a
method of reducing an invasiveness of a metastasis of a
Epstein-Barr virus-induced malignancy in a subject, comprising
administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby reducing an invasiveness of a metastasis of a Epstein-Barr
virus-induced malignancy in a subject.
[0067] In another embodiment, the present invention provides a
method of reversing a malignant transformation of an Epstein-Barr
virus-induced malignancy in a subject, comprising administering to
the subject a compound that inhibits an interaction of a first
protein and an ITAM of a second protein, thereby reversing a
malignant transformation of an Epstein-Barr virus-induced
malignancy in a subject.
[0068] As provided herein (Examples), findings of the present
invention show that expression of a protein containing an ITAM, but
not a mutant-ITAM-containing protein, is capable of transformation
of multiple cell types, including cell types relevant to
EBV-induced malignancies. Activity of cellular signaling molecules
that associated with the ITAM (e.g. Syk kinase) was necessary for
the transformation to occur. These findings show that blocking
association of an ITAM with cellular molecules and blocking
ITAM-mediated signaling are effective strategies for preventing and
reducing the incidence of malignant transformation. In addition,
testing of a peptide of the present invention showed that ITAM
association-blocking peptides can not only prevent, but also
reverse cell transformation. The LMP2A protein of EBV contains an
ITAM. The findings of the present invention thus show that blocking
association of the LMP2A ITAM with cellular molecules and blocking
LMP2A ITAM-mediated signaling are effective strategies for
preventing and reducing the incidence of malignant transformation,
and for treatment and prevention of metastases of EBV-induced
malignancies.
[0069] Moreover, the present invention provides methods of
modifying peptides of the present invention and identifying further
improved peptides using models of EBV-induced malignancies
(Examples 8 and 10); or, in another embodiment, using colony
formation assays (e.g. as described in Example 22); or, in another
embodiment, using ITAM co-IP assays (e.g. as described in Example
23); or, in another embodiment, using ability to abrogate or reduce
phosphorylation of an ITAM-containing protein (e.g. as described in
Example 29); or, in another embodiment, using a focus formation
assay (e.g. as described in Example 30). These methods facilitate,
in another embodiment, selection of further improved
ITAM-inhibitory peptides.
[0070] The Epstein-Barr virus-induced malignancy that is the target
of methods of the present invention is, in one embodiment, a
Burkitt's lymphoma. In another embodiment, the Epstein-Barr
virus-induced malignancy is Hodgkin's disease. In another
embodiment, the Epstein-Barr-virus-induced malignancy is a
nasopharyngeal carcinoma (NPC). In another embodiment, the
Epstein-Barr virus-induced malignancy is post-transplant
lymphoproliferative disease. In another embodiment, the
Epstein-Barr virus-induced malignancy is any other Epstein-Barr
virus-induced malignancy known in the art. Each possibility
represents a separate embodiment of the present invention.
[0071] In another embodiment, the present invention provides a
method of treating a B cell proliferative disorder in a subject,
comprising administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby treating a B cell proliferative disorder in a subject.
[0072] In another embodiment, the present invention provides a
method of reducing an incidence of a B cell proliferative disorder
in a subject, comprising administering to the subject a compound
that inhibits an interaction of a first protein and an ITAM of a
second protein, thereby reducing an incidence of a pathological
immune cell activation in a subject.
[0073] In another embodiment, the present invention provides a
method of treating a mast cell activation disorder in a subject,
comprising administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby treating a mast cell activation disorder in a subject.
[0074] In another embodiment, the present invention provides a
method of reducing an incidence of a mast cell activation disorder
in a subject, comprising administering to the subject a compound
that inhibits an interaction of a first protein and an ITAM of a
second protein, thereby reducing an incidence of a mast cell
activation disorder in a subject.
[0075] In another embodiment, the mast cell activation disorder is
a systemic mastocytosis. (in another embodiment, with a cutaneous
manifestation [e.g. Urticaria Pigmentosa]; in another embodiment,
without a cutaneous manifestation). In another embodiment, the mast
cell activation disorder is an aggressive mastocytosis. In another
embodiment, the mast cell activation disorder is an indolent
mastocytosis. In another embodiment, the mast cell activation
disorder is a mastocytosis with an associated hematologic disorder.
In another embodiment, the mast cell activation disorder is a mast
cell leukemia. In another embodiment, the mast cell activation
disorder is a cutaneous mastocytosis. In another embodiment, the
mast cell activation disorder is an urticaria pigmentosa. In
another embodiment, the mast cell activation disorder is a
telengiecstasia. In another embodiment, the mast cell activation
disorder is a macularis eruptive perstans. In another embodiment,
the mast cell activation disorder is a solitary mastocytoma. In
another embodiment, the mast cell activation disorder is an
urticaria pigmentosa. In another embodiment, the mast cell
activation disorder is a diffuse cutaneous mastocytosis. In another
embodiment, the mast cell activation disorder is any other mast
cell activation disorder known in the art. Each possibility
represents a separate embodiment of the present invention.
[0076] As provided herein (Example 7), findings of the present
invention show that ITAM association-blocking peptides prevent
BCR-induced proliferation, ITAM-based BCR signaling, and
degranulation of RBL-2H3 mast cells. Thus, blocking association of
an ITAM with cellular molecules and blocking ITAM-mediated
signaling are effective strategies for preventing, reducing the
incidence of, and treating B cell proliferative disorders and mast
cell activation disorders.
[0077] Moreover, the present invention provides methods of
modifying peptides of the present invention and identifying further
improved peptides using models of carcinoma, both primary tumors
and metastases (Examples 8 and 14); or, in another embodiment,
using ITAM co-IP assays (e.g. as described in Example 23); or, in
another embodiment, using ability to abrogate or reduce
ITAM-dependent B cell activation (e.g. as described in Example 26);
or, in another embodiment, using ability to abrogate or reduce
ITAM-dependent BCR signaling (e.g. as described in Example 27); or,
in another embodiment, using ability to abrogate or reduce
ITAM-dependent mast cell degranulation (e.g. as described in
Example 28); or, in another embodiment, using ability to abrogate
or reduce phosphorylation of an ITAM-containing protein (e.g. as
described in Example 29). These methods facilitate, in another
embodiment, selection of further improved ITAM-inhibitory
peptides.
[0078] The B cell proliferative disorder that is the target of
methods and compositions of the present invention is, in another
embodiment, a B cell lymphoma. In another embodiment, the B cell
proliferative disorder is a B cell leukemia. In another embodiment,
the B cell proliferative disorder is any other B cell proliferative
disorder known in the art. Each possibility represents a separate
embodiment of the present invention.
[0079] In another embodiment, the present invention provides a
method of treating a thrombosis disorder in a subject, comprising
administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby treating a thrombosis disorder in a subject. Example 13
provides methods to facilitate selection of further improved
ITAM-inhibitory peptides for this method.
[0080] In another embodiment, the present invention provides a
method of reducing an incidence of a thrombosis disorder in a
subject, comprising administering to the subject a compound that
inhibits an interaction of a first protein and an ITAM of a second
protein, thereby reducing an incidence of a thrombosis disorder in
a subject.
[0081] In another embodiment, the embodiment, the thrombosis
disorder is an autoimmune hemolytic anemia. In another embodiment,
the embodiment, the thrombosis disorder is an idiopathic
thrombocytopenic purpura. In another embodiment, the embodiment,
the thrombosis disorder is any other thrombosis disorder known in
the art. Each possibility represents a separate embodiment of the
present invention.
[0082] In another embodiment, the present invention provides a
method of treating a hantavirus pulmonary syndrome in a subject,
comprising administering to the subject a compound that inhibits an
interaction of a first protein and an ITAM of a second protein,
thereby treating a hantavirus pulmonary syndrome in a subject
Example 12 provides methods to facilitate selection of further
improved ITAM-inhibitory peptides for this method.
[0083] In another embodiment, the present invention provides a
method of reducing an incidence of a hantavirus pulmonary syndrome
in a subject, comprising administering to the subject a compound
that inhibits an interaction of a first protein and an ITAM of a
second protein, thereby reducing an incidence of a hantavirus
pulmonary syndrome in a subject.
[0084] As provided herein, findings of the present invention show
that, under the conditions utilized herein, the ITAM motif of MMTV
env is, in one embodiment, cytoplasmic. In another embodiment, all
the transmembrane domains depicted in FIG. 5B are utilized. In
another embodiment, transmembrane domains 3 and 4 are utilized
(alternative model 1 and alternative model 2, respectively). Each
possibility represents a separate embodiment of the present
invention.
[0085] The compound of methods and compositions of the present
invention is, in another embodiment, a peptide homologous to the
ITAM that is targeted. In another embodiment, the compound
comprises a peptide homologous to the ITAM. In another embodiment,
the compound consists of a peptide homologous to the ITAM. In
another embodiment, the compound is a derivative of a peptide
homologous to the ITAM. Each possibility represents a separate
embodiment of the present invention.
[0086] "Peptide" refers, in another embodiment, to a peptide
containing only naturally occurring amino acids. In another
embodiment, the term refers to a peptide that contains one or more
modified amino acids. In another embodiment, the term refers to a
peptide that contains one or more non-standard amino acids. In
another embodiment, the term refers to peptide that has been
chemically modified. In another embodiment, the modified or
non-standard amino acid is incorporated after the N-terminal
residue. In another embodiment, the modified or non-standard amino
acid is incorporated in place of the N-terminal residue In another
embodiment, the modified or non-standard amino acid is incorporated
after the C-terminal residue. In another embodiment, the modified
or non-standard amino acid is incorporated in place of the
C-terminal residue. In another embodiment, the modified or
non-standard amino acid is incorporated at an intermediate residue.
In another embodiment, the modified or non-standard amino acid is
incorporated at a combination of one of the above positions. Each
possibility represents a separate embodiment of the present
invention.
[0087] In another embodiment, "peptide" refers to an oligomer of
amino acid residues that are connected by peptide bonds. In another
embodiment, the term refers to an oligomer comprising both
naturally occurring and, optionally, modified amino acid residues
that are connected by peptide bonds. In another embodiment, one of
the above oligomers is chemically modified, in a manner enumerated
herein. Each possibility represents a separate embodiment of the
present invention.
[0088] In another embodiment, a peptide of the present invention is
modified to improve its water-solubility. In another embodiment,
the peptide is modified to improve its cell membrane-permeability.
In another embodiment, the peptide is modified to improve its
proteolytic stability. In another embodiment, the peptide is
modified to improve its bioavailability. In another embodiment, the
peptide is modified to improve its specificity for a particular
ITAM sequence. In another embodiment, the peptide is modified to
decrease its activity against one or more non-target sequences
(e.g. other ITAM sequences). Each possibility represents a separate
embodiment of the present invention.
[0089] In another embodiment, the peptide is modified by
acetylation (Example 6). In other embodiments, the acyl group is an
alkanoyl group, (e.g. acetyl), hexanoyl, octanoyl, an aroyl group,
(e.g. benzoyl), or a blocking group e.g. Fmoc
(fluorenylmethyl-O--CO--), carbobenzoxy (benzyl-O--CO--),
monomethoxysuccinyl, naphthyl-NH--CO--, acetylamino-caproyl, or
adamantyl-NH--CO--. In another embodiment, the modification is
hydroxylation (e.g. on the C-terminal end). In another embodiment,
the modification is amidation (in some embodiments, of the carboxyl
terminal or of another free carboxyl groups). In another
embodiment, the modification is formylation. In another embodiment,
the modification is gamma-carboxyglutamic acid hydroxylation. In
another embodiment, the modification is methylation. In another
embodiment, the modification is phosphorylation. In another
embodiment, the modification is sulfation. In another embodiment,
the modification is glycosylation. In another embodiment, the
modification is reduction. In another embodiment, the modification
is oxidation. In another embodiment, the modification is disulfide
modification. In another embodiment, the modification is
introduction of a thioether bond. In another embodiment, the
modification is introduction of a thiolester bond. In another
embodiment, the modification is a backbone condensation. In another
embodiment, the modification is biotinylation. In another
embodiment, the modification is an esterification of the carboxyl
terminal or of another free carboxyl or hydroxy group. In another
embodiment, the modification is conjugation to a lipophilic moiety
(e.g. caproyl, lauryl, or stearoyl). In another embodiment, the
modification is conjugation to an antibody or other biological
ligand. In another embodiment, the modification is any other
modification of a peptide that is known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0090] In another embodiment, a moiety that provides a net positive
charge is incorporated onto the N-terminus of the peptide. In other
embodiments, the moiety is a straight chain, branched, cyclic, or
heterocyclic alkyl group; a straight chain, branched, cyclic, or
heterocyclic alkanoyl group; or 1-15 additional amino acids
independently selected from L-configuration or D-configuration
amino acids, optionally substituted with a straight chain,
branched, cyclic or heterocyclic alkyl group; or a straight chain,
branched, cyclic or heterocyclic alkanoyl group.
[0091] In other embodiments, the C terminus of a peptide of the
present invention is modified to comprise a free hydroxyl, an
amide, an imide, a sugar, or 1-15 additional amino acids,
optionally substituted with a free hydroxyl, an amide, an imide or
a sugar. In another embodiment, the C-termini is modified in the
same manner as the modified N-termini, described above. In another
embodiment, the C terminus is modified with
2-acetamido-2-deoxyglucose. In another embodiment, the C terminus
is modified by addition of triacetyl 2-acetamido-2-deoxyglucose. In
another embodiment, the C terminus is modified by addition of a
.beta.-acetyl-2,3-diamino propionic acid group. Each possibility
represents a separate embodiment of the present invention.
[0092] In another embodiment, the peptides of this invention are
modified by addition of two adjacent amino acids that are resistant
to cleavage by endopeptidases. In another embodiment, conventional
inter-residue amide bonds are replaced by bonds resistant to
proteases, (in other embodiments, a thioamide bond or a reduced
amide bond).
[0093] The modified amino acid present in peptides of methods and
compositions of the present invention is, in various embodiments, a
D amino acid, pyrrolidone carboxylic acid, 2-aminoadipic acid,
3-aminoadipic acid, beta-alanine or beta-aminoproprionic acid,
2-aminobutyric acid, 4-aminobutyric acid or piperidinic acid,
6-aminocaproic acid, 6-aminoheptanoic acid, 2-aminoheptanoic acid,
2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic
acid, 2,4 diaminobutyric acid, desmosine, 2,2 diaminopimelic acid,
2,3 diaminopropionic acid, N-ethylglycine, N-ethylasparagine,
hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,
4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine or
sarcosine, methylisoleucine, methyllysine, methylvaline, norvaline,
norleucine, 6-aminohexanoic acid, citrulline, cysteic acid,
cyclohexylalanine, alpha-amino isobutyric acid, t-butylglycine,
t-butylalanine and phenylglycine an N-alpha-methyl amino acid, a
C-alpha-methyl amino acid, a beta-methyl amino acid, or orthinine.
In another embodiment, the modified amino acid is any other
modified amino acid known in the art. Each possibility represents a
separate embodiment of the present invention.
[0094] "Comprises a peptide" refers, in one embodiment, to a
molecule that contains one of the peptides enumerated above and a
non-amino acid moiety attached to one or both ends of the peptide.
The non-amino acid moiety is, in various embodiments, any suitable
chemical group known in the art. Each possibility represents a
separate embodiment of the present invention. In another
embodiment, the non-amino acid moiety is attached to one or more of
the intermediate peptide residues. In another embodiment, the
non-amino acid moiety is attached to the peptide via a peptide
bond. In another embodiment, the non-amino acid moiety is attached
to the peptide via any other type of bond known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0095] In another embodiment, the present invention provides a
peptide having the sequence Ac-KRPPYVLL-OH (SEQ ID No: 2). In
another embodiment, the peptide of methods and compositions of the
present invention has a sequence homologous to SEQ ID No: 2. In
another embodiment, the peptide has a sequence identical to
KRPPYVLLPVDIGD (SEQ ID No: 33) or a fragment thereof. In another
embodiment, the peptide has a sequence homologous to KRPPYVLLPVDIGD
or a fragment thereof. In another embodiment, the peptide has a
sequence identical to a fragment of PAYDYAAIIVKRPPYVLLPVDIGD (SEQ
ID No: 1). In another embodiment, the peptide has a sequence
homologous to a fragment of PAYDYAAIIVKRPPYVLLPVDIGD. In another
embodiment, the peptide has a sequence homologous to
DMPDDYEDENLYEGLNLDDCSMYEDI (SEQ ID No: 18) or a fragment thereof.
In another embodiment, the peptide has a sequence identical to
DMPDDYEDENLYEGLNLDDCSMYEDI or a fragment thereof. In another
embodiment, the peptide has a sequence identical to DCSMYEDI (SEQ
ID No: 34) or a fragment thereof. In another embodiment, the
peptide has a sequence homologous to DCSMYEDI or a fragment
thereof. In another embodiment, the peptide has a sequence
identical to a fragment of DKDDGKAGMEEDHTYEGLNIDQTATYEDI (SEQ ID
No: 28). In another embodiment, the peptide has a sequence
homologous to a fragment of DKDDGKAGMEEDHTYEGLNIDQTATYEDI. In
another embodiment, the peptide has a sequence identical to
QTATYEDI (SEQ ID No: 35) or a fragment thereof. In another
embodiment, the peptide has a sequence homologous to QTATYEDI or a
fragment thereof. In another embodiment, the peptide has the
sequence YVLL (SEQ ID No: 49). In another embodiment, the peptide
has a sequence identical to KRPPYLVV (SEQ ID No: 89) or a fragment
thereof. In another embodiment, the peptide has a sequence
homologous to KRPPYLVV or a fragment thereof. In another
embodiment, the peptide has a sequence identical to any other ITAM
sequence enumerated herein. In another embodiment, the peptide has
a sequence homologous to any other ITAM sequence enumerated herein.
In another embodiment, the peptide has a sequence identical to any
other ITAM motif enumerated herein. In another embodiment, the
peptide has a sequence homologous to any other ITAM motif
enumerated herein. Each possibility represents a separate
embodiment of the present invention. In another embodiment, the
peptide has a sequence identical to any other ITAM sequence known
in the art. In another embodiment, the peptide has a sequence
homologous to any other ITAM sequence known in the art. In another
embodiment, the peptide has a sequence identical to any other ITAM
motif known in the art. In another embodiment, the peptide has a
sequence homologous to any other ITAM motif known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0096] In another embodiment, the peptide of methods and
compositions of the present invention is an ITAM inhibitory
peptide. In other embodiments, the ITAM inhibited by the peptide of
the present invention has any of the ITAM sequences of the present
invention. Each possibility represents a separate embodiment of the
present invention.
[0097] In another embodiment of methods and compositions of the
present invention, the inhibitory compound or peptide blocks
interaction of an ITAM motif with Syk2, ZAP70, or a related or
similar cellular protein without blocking other cellular functions
of the cellular protein. In another embodiment, the peptide
preferentially inhibits signaling via a viral ITAM over one or more
cellular ITAMs. In another embodiment, the preferential inhibition
is due to a sequence difference between the viral ITAM and the
cellular ITAMs. Each possibility represents a separate embodiment
of the present invention.
[0098] In another embodiment, a peptide of the present invention is
homologous to a peptide of SEQ ID No: 1-98. The terms "homology,"
"homologous," etc, when in reference to any protein or peptide,
refer, in one embodiment, to a percentage of amino acid residues in
the candidate sequence that are identical with the residues of a
corresponding native polypeptide, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
homology, and not considering any conservative substitutions as
part of the sequence identity. Methods and computer programs for
the alignment are well known in the art.
[0099] "Homology" refers, in another embodiment, to identity to a
sequence selected from SEQ ID No: 1-98 of greater than 70%. In
another embodiment, "homology" refers to identity to a sequence
selected from SEQ ID No: 1-98 of greater than 72%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1-98
of greater than 75%. In another embodiment, "homology" refers to
identity to a sequence selected from SEQ ID No: 1-98 of greater
than 78% In another embodiment, "homology" refers to identity to
one of SEQ ID No: 1-98 of greater than 80%. In another embodiment,
"homology" refers to identity to one of SEQ ID No: 1-98 of greater
than 82%. In another embodiment, "homology" refers to identity to a
sequence selected from SEQ ID No: 1-98 of greater than 83%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 1-98 of greater than 85%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 1-98 of greater than 87%.
In another embodiment, "homology" refers to identity to a sequence
selected from SEQ ID No: 1-98 of greater than 88%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1-98
of greater than 90%. In another embodiment, "homology" refers to
identity to one of SEQ ID No: 1-98 of greater than 92%. In another
embodiment, "homology" refers to identity to a sequence selected
from SEQ ID No: 1-98 of greater than 93%. In another embodiment,
"homology" refers to identity to one of SEQ ID No: 1-98 of greater
than 95%. In another embodiment, "homology" refers to identity to a
sequence selected from SEQ ID No: 1-98 of greater than 96%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 1-98 of greater than 97%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 1-98 of greater than 98%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 1-98 of greater than 99%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 1-98 of 100%. Each
possibility represents a separate embodiment of the present
invention.
[0100] In another embodiment, homology is determined via
determination of candidate sequence hybridization, methods of which
are well described in the art (See, for example, "Nucleic Acid
Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985);
Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Press, N.Y.; and Ausubel et al., 1989, Current
Protocols in Molecular Biology, Green Publishing Associates and
Wiley Interscience, N.Y). For example methods of hybridization may
be carried out under moderate to stringent conditions, to the
complement of a DNA encoding a native caspase peptide.
Hybridization conditions being, for example, overnight incubation
at 42.degree. C. in a solution comprising: 10-20% formamide,
5.times.SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM-sodium
phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA.
[0101] Protein and/or peptide homology for any amino acid sequence
listed herein is determined, in another embodiment, by methods well
described in the art, including immunoblot analysis, or via
computer algorithm analysis of amino acid sequences, utilizing any
of a number of software packages available, via established
methods. Some of these packages may include the FASTA, BLAST,
MPsrch or Scanps packages, and may employ the use of the Smith and
Waterman algorithms, and/or global/local or BLOCKS alignments for
analysis, for example. Each method of determining homology
represents a separate embodiment of the present invention.
[0102] In other embodiments, the ITAM-inhibitory peptides of any of
the methods described above have any of the characteristics of an
ITAM-inhibitory peptide of the present invention. Each
characteristic represents a separate embodiment of the present
invention.
[0103] Methods for in vivo transformation assays are well known in
the art and are described in the Examples herein. Three-dimensional
recombinant basement membrane cultures provide, in another
embodiment, an in vivo model of the acinar architecture of mammary
epithelium. Mammary epithelial cells grown in three-dimensional
cultures recapitulate, in another embodiment, numerous features of
breast epithelium in vivo. These include the formation of
growth-arrested polarized acini with hollow lumen and deposition of
basement membrane components, such as collagen IV and laminin V.
Three-dimensional cultures provide, in another embodiment, the
appropriate structural and functional context for studying the
events involved in morphogenesis of glandular epithelium.
[0104] In another embodiment, the present invention provides a
method of screening inhibitors (in one embodiment, "peptide-based
inhibitors") of ITAM-dependent signaling, comprising one of the
assays described herein in the Examples. In another embodiment, the
present invention provides a method of screening inhibitors of
malignant transformation into a carcinoma cell, comprising one of
the assays described herein in the Examples. In another embodiment,
the present invention provides a method of screening inhibitors of
malignant transformation into a sarcoma cell, comprising one of the
assays described herein in the Examples. In another embodiment, the
present invention provides a method of screening inhibitors of
transformation into a Epstein-Barr virus-induced malignancy,
comprising one of the assays described herein in the Examples. In
another embodiment, the present invention provides a method of
screening inhibitors of cancer cell metastasis, comprising one of
the assays described herein in the Examples. In another embodiment,
the present invention provides a method of screening inhibitors of
a B cell proliferative disorder, comprising one of the assays
described herein in the Examples. In another embodiment, the
present invention provides a method of screening inhibitors of a
mast cell activation disorder, comprising one of the assays
described herein in the Examples. In another embodiment, the
present invention provides a method of screening inhibitors of a
thrombosis disorder, comprising one of the assays described herein
in the Examples. In another embodiment, the present invention
provides a method of screening inhibitors of a hantavirus pulmonary
syndrome, comprising one of the assays described herein in the
Examples. In another embodiment, the present invention provides a
method of screening inhibitors of a any other disease or disorder
that involves aberrant ITAM signaling, comprising one of the assays
described herein in the Examples. Each possibility represents a
separate embodiment of the present invention.
[0105] In one embodiment, the screening method is 3-dimensional
culture assays of ITAM-containing protein-transfected cells (e.g.
as described in Example 21); or, in another embodiment, colony
formation assays (e.g. as described in Example 22); or, in another
embodiment, ITAM co-IP assays (e.g. as described in Example 23);
or, in another embodiment, ability to abrogate or reduce EMT (e.g.
as described in Example 24); or, in another embodiment, ability to
abrogate or reduce sensitivity to apoptosis (e.g. as described in
Example 25); or, in another embodiment, ability to abrogate or
reduce ITAM-dependent B cell activation (e.g. as described in
Example 26); or, in another embodiment, ability to abrogate or
reduce ITAM-dependent BCR signaling (e.g. as described in Example
27); or, in another embodiment, ability to abrogate or reduce
ITAM-dependent mast cell degranulation (e.g. as described in
Example 28); or, in another embodiment, ability to abrogate or
reduce phosphorylation of an ITAM-containing protein (e.g. as
described in Example 29); or, in another embodiment, a focus
formation assay (e.g. as described in Example 30). Each possibility
represents a separate embodiment of the present invention.
[0106] Various embodiments of dosage ranges of compounds of the
present invention can be used in methods of the present invention.
In one embodiment, the dosage is in the range of 1-10 mg/day. In
another embodiment, the dosage is 2-10 mg/day. In another
embodiment, the dosage is 3-10 mg/day. In another embodiment, the
dosage is 5-10 mg/day. In another embodiment, the dosage is 2-20
mg/day. In another embodiment, the dosage is 3-20 mg/day. In
another embodiment, the dosage is 5-20 mg/day. In another
embodiment, the dosage is 10-20 mg/day. In another embodiment, the
dosage is 3-40 mg/day. In another embodiment, the dosage is 5-40
mg/day. In another embodiment, the dosage is 10-40 mg/day. In
another embodiment, the dosage is 20-40 mg/day. In another
embodiment, the dosage is 5-50 mg/day. In another embodiment, the
dosage is 10-50 mg/day. In another embodiment, the dosage is 20-50
mg/day. In one embodiment, the dosage is 1-100 mg/day. In another
embodiment, the dosage is 2-100 mg/day. In another embodiment, the
dosage is 3-100 mg/day. In another embodiment, the dosage is 5-100
mg/day. In another embodiment the dosage is 10-100 mg/day. In
another embodiment the dosage is 20-100 mg/day. In another
embodiment the dosage is 40-100 mg/day. In another embodiment the
dosage is 60-100 mg/day.
[0107] In another embodiment, the dosage is 0.1 mg/day. In another
embodiment, the dosage is 0.2 mg/day. In another embodiment, the
dosage is 0.3 mg/day. In another embodiment, the dosage is 0.5
mg/day. In another embodiment, the dosage is 1 mg/day. In another
embodiment, the dosage is 2 mg/day. In another embodiment, the
dosage is 3 mg/day. In another embodiment, the dosage is 5 mg/day.
In another embodiment, the dosage is 10 mg/day. In another
embodiment, the dosage is 15 mg/day. In another embodiment, the
dosage is 20 mg/day. In another embodiment, the dosage is 30
mg/day. In another embodiment, the dosage is 40 mg/day. In another
embodiment, the dosage is 60 mg/day. In another embodiment, the
dosage is 80 mg/day. In another embodiment, the dosage is 100
mg/day.
[0108] In another embodiment, the dosage is 10 .mu.g/dose. In
another embodiment, the dosage is 20 .mu.g/dose. In another
embodiment, the dosage is 30 .mu.g/dose. In another embodiment, the
dosage is 40 .mu.g/dose. In another embodiment, the dosage is 60
.mu.g/dose. In another embodiment, the dosage is 80 .mu.g/dose. In
another embodiment, the dosage is 100 .mu.g/dose. In another
embodiment, the dosage is 150 .mu.g/dose. In another embodiment,
the dosage is 200 .mu.g/dose. In another embodiment, the dosage is
300 .mu.g/dose. In another embodiment, the dosage is 400
.mu.g/dose. In another embodiment, the dosage is 600 .mu.g/dose. In
another embodiment, the dosage is 800 .mu.g/dose. In another
embodiment, the dosage is 1000 .mu.g/dose. In another embodiment,
the dosage is 1.5 mg/dose. In another embodiment, the dosage is 2
mg/dose. In another embodiment, the dosage is 3 mg/dose. In another
embodiment, the dosage is 5 mg/dose. In another embodiment, the
dosage is 10 mg/dose. In another embodiment, the dosage is 15
mg/dose. In another embodiment, the dosage is 20 mg/dose. In
another embodiment, the dosage is 30 mg/dose. In another
embodiment, the dosage is 50 mg/dose. In another embodiment, the
dosage is 80 mg/dose. In another embodiment, the dosage is 100
mg/dose.
[0109] In another embodiment, the dosage is 10-20 .mu.g/dose. In
another embodiment, the dosage is 20-30 .mu.g/dose. In another
embodiment, the dosage is 20-40 .mu.g/dose. In another embodiment,
the dosage is 30-60 .mu.g/dose. In another embodiment, the dosage
is 40-80 .mu.g/dose. In another embodiment, the dosage is 50-100
.mu.g/dose. In another embodiment, the dosage is 50-150 .mu.g/dose.
In another embodiment, the dosage is 100-200 .mu.g/dose. In another
embodiment, the dosage is 200-300 .mu.g/dose. In another
embodiment, the dosage is 300-400 .mu.g/dose. In another
embodiment, the dosage is 400-600 .mu.g/dose. In another
embodiment, the dosage is 500-800 .mu.g/dose. In another
embodiment, the dosage is 800-1000 .mu.g/dose. In another
embodiment, the dosage is 1000-1500 .mu.g/dose. In another
embodiment, the dosage is 1500-2000 .mu.g/dose. In another
embodiment, the dosage is 2-3 mg/dose. In another embodiment, the
dosage is 2-5 mg/dose. In another embodiment, the dosage is 2-10
mg/dose. In another embodiment, the dosage is 2-20 mg/dose. In
another embodiment, the dosage is 2-30 mg/dose. In another
embodiment, the dosage is 2-50 mg/dose. In another embodiment, the
dosage is 2-80 mg/dose. In another embodiment, the dosage is 2-100
mg/dose. In another embodiment, the dosage is 3-10 mg/dose. In
another embodiment, the dosage is 3-20 mg/dose. In another
embodiment, the dosage is 3-30 mg/dose. In another embodiment, the
dosage is 3-50 mg/dose. In another embodiment, the dosage is 3-80
mg/dose. In another embodiment, the dosage is 3-100 mg/dose. In
another embodiment, the dosage is 5-10 mg/dose. In another
embodiment, the dosage is 5-20 mg/dose. In another embodiment, the
dosage is 5-30 mg/dose. In another embodiment, the dosage is 5-50
mg/dose. In another embodiment, the dosage is 5-80 mg/dose. In
another embodiment, the dosage is 5-100 mg/dose. In another
embodiment, the dosage is 10-20 mg/dose. In another embodiment, the
dosage is 10-30 mg/dose. In another embodiment, the dosage is 10-50
mg/dose. In another embodiment, the dosage is 10-80 mg/dose. In
another embodiment, the dosage is 10-100 mg/dose.
[0110] In another embodiment, the dosage is 10 .mu.g/tablet. In
another embodiment, the dosage is 20 .mu.g/tablet. In another
embodiment, the dosage is 30 .mu.g/tablet. In another embodiment,
the dosage is 40 .mu.g/tablet. In another embodiment, the dosage is
60 .mu.g/tablet. In another embodiment, the dosage is 80
.mu.g/tablet. In another embodiment, the dosage is 100
.mu.g/tablet. In another embodiment, the dosage is 150
.mu.g/tablet. In another embodiment, the dosage is 200
.mu.g/tablet. In another embodiment, the dosage is 300
.mu.g/tablet. In another embodiment, the dosage is 400
.mu.g/tablet. In another embodiment, the dosage is 600
.mu.g/tablet. In another embodiment, the dosage is 800
.mu.g/tablet. In another embodiment, the dosage is 1000
.mu.g/tablet. In another embodiment, the dosage is 1.5 mg/tablet.
In another embodiment, the dosage is 2 mg/tablet. In another
embodiment, the dosage is 3 mg/tablet. In another embodiment, the
dosage is 5 mg/tablet. In another embodiment, the dosage is 10
mg/tablet. In another embodiment, the dosage is 15 mg/tablet. In
another embodiment, the dosage is 20 mg/tablet. In another
embodiment, the dosage is 30 mg/tablet. In another embodiment, the
dosage is 50 mg/tablet. In another embodiment, the dosage is 80
mg/tablet. In another embodiment, the dosage is 100 mg/tablet.
[0111] In another embodiment, the dosage is 10-20 .mu.g/tablet. In
another embodiment, the dosage is 20-30 .mu.g/tablet. In another
embodiment, the dosage is 20-40 .mu.g/tablet. In another
embodiment, the dosage is 30-60 .mu.g/tablet. In another
embodiment, the dosage is 40-80 .mu.g/tablet. In another
embodiment, the dosage is 50-100 .mu.g/tablet. In another
embodiment, the dosage is 50-150 .mu.g/tablet. In another
embodiment, the dosage is 100-200 .mu.g/tablet. In another
embodiment, the dosage is 200-300 .mu.g/tablet. In another
embodiment, the dosage is 300-400 .mu.g/tablet. In another
embodiment, the dosage is 400-600 .mu.g/tablet. In another
embodiment, the dosage is 500-800 .mu.g/tablet. In another
embodiment, the dosage is 800-1000 .mu.g/tablet. In another
embodiment, the dosage is 1000-1500 .mu.g/tablet. In another
embodiment, the dosage is 1500-2000 .mu.g/tablet. In another
embodiment, the dosage is 2-3 mg/tablet. In another embodiment, the
dosage is 2-5 mg/tablet. In another embodiment, the dosage is 2-10
mg/tablet. In another embodiment, the dosage is 2-20 mg/tablet. In
another embodiment, the dosage is 2-30 mg/tablet. In another
embodiment, the dosage is 2-50 mg/tablet. In another embodiment,
the dosage is 2-80 mg/tablet. In another embodiment, the dosage is
2-100 mg/tablet. In another embodiment, the dosage is 3-10
mg/tablet. In another embodiment, the dosage is 3-20 mg/tablet. In
another embodiment, the dosage is 3-30 mg/tablet. In another
embodiment, the dosage is 3-50 mg/tablet. In another embodiment,
the dosage is 3-80 mg/tablet. In another embodiment, the dosage is
3-100 mg/tablet. In another embodiment, the dosage is 5-10
mg/tablet. In another embodiment, the dosage is 5-20 mg/tablet. In
another embodiment, the dosage is 5-30 mg/tablet. In another
embodiment, the dosage is 5-50 mg/tablet. In another embodiment,
the dosage is 5-80 mg/tablet. In another embodiment, the dosage is
5-100 mg/tablet. In another embodiment, the dosage is 10-20
mg/tablet. In another embodiment, the dosage is 10-30 mg/tablet. In
another embodiment, the dosage is 10-50 mg/tablet. In another
embodiment, the dosage is 10-80 mg/tablet. In another embodiment,
the dosage is 10-100 mg/tablet.
[0112] In another embodiment, the present invention provides a kit
comprising a reagent utilized in performing a method of the present
invention. In another embodiment, the present invention provides a
kit comprising a composition, tool, or instrument of the present
invention.
[0113] In another embodiment, the present invention relates to the
use of an ITAM-inhibitor peptide and/or its analog, derivative,
isomer, metabolite, pharmaceutically acceptable salt,
pharmaceutical product, hydrate, N-oxide, or a combination thereof
for treating, preventing, suppressing, inhibiting or reducing the
incidence of the diseases and disorders enumerated herein. Thus, in
one embodiment, the methods of the present invention comprise
administering an analog of the peptide. In another embodiment, the
methods of the present invention comprise administering a
derivative of the peptide. In another embodiment, the methods of
the present invention comprise administering an isomer of the
peptide. In another embodiment, the methods of the present
invention comprise administering a metabolite of the peptide. In
another embodiment, the methods of the present invention comprise
administering a pharmaceutically acceptable salt of the peptide. In
another embodiment, the methods of the present invention comprise
administering a pharmaceutical product of the peptide. In another
embodiment, the methods of the present invention comprise
administering a hydrate of the peptide. In another embodiment, the
methods of the present invention comprise administering an N-oxide
of the peptide. In another embodiment, the methods of the present
invention comprise administering any combination of an analog,
derivative, isomer, metabolite, pharmaceutically acceptable salt,
pharmaceutical product, hydrate or N-oxide of the peptide.
[0114] In another embodiment, the term "isomer" includes, but, in
another embodiment, is not limited to, optical isomers and analogs,
structural isomers and analogs, conformational isomers and analogs,
and the like.
[0115] The pharmaceutical compositions comprising the compound of
the present invention can be, in another embodiment, administered
to a subject by any method known to a person skilled in the art,
such as parenterally, paracancerally, transmucosally,
transdermally, intramuscularly, intravenously, intra-dermally,
subcutaneously, intra-peritonealy, intra-ventricularly,
intra-cranially, intra-vaginally or intra-tumorally.
[0116] In another embodiment of methods and compositions of the
present invention, the pharmaceutical compositions are administered
orally, and are thus formulated in a form suitable for oral
administration, i.e. as a solid or a liquid preparation. Suitable
solid oral formulations include tablets, capsules, pills, granules,
pellets and the like. Suitable liquid oral formulations include
solutions, suspensions, dispersions, emulsions, oils and the like.
In another embodiment of the present invention, the active
ingredient is formulated in a capsule. In accordance with this
embodiment, the compositions of the present invention comprise, in
addition to the active compound and the inert carrier or diluent, a
hard gelating capsule.
[0117] In another embodiment, the pharmaceutical compositions are
administered by intravenous, intra-arterial, or intra-muscular
injection of a liquid preparation. Suitable liquid formulations
include solutions, suspensions, dispersions, emulsions, oils and
the like. In another embodiment, the pharmaceutical compositions
are administered intravenously and are thus formulated in a form
suitable for intravenous administration. In another embodiment, the
pharmaceutical compositions are administered intra-arterially and
are thus formulated in a form suitable for intra-arterial
administration. In another embodiment, the pharmaceutical
compositions are administered intra-muscularly and are thus
formulated in a form suitable for intra-muscular
administration.
[0118] In another embodiment, the pharmaceutical compositions are
administered topically to body surfaces and are thus formulated in
a form suitable for topical administration. Suitable topical
formulations include gels, ointments, creams, lotions, drops and
the like. For topical administration, the compositions such as
salts, esters, N-oxides, and the like are prepared and applied as
solutions, suspensions, or emulsions in a physiologically
acceptable diluent with or without a pharmaceutical carrier.
[0119] In another embodiment, the pharmaceutical composition is
administered as a suppository, for example a rectal suppository or
a urethral suppository. In another embodiment, the pharmaceutical
composition is administered by subcutaneous implantation of a
pellet. In another embodiment, the pellet provides for controlled
release of the active agent over a period of time.
[0120] In another embodiment, the active compound is delivered in a
vesicle, e.g. a liposome. Each possibility represents a separate
embodiment of the present invention.
EXPERIMENTAL DETAILS SECTION
Example 1
Expression of ITAM-Containing MMTV ENV Leads to Depolarization of
Mammary Epithelial Acinar Structures
Materials and Experimental Methods (Examples 1-4)
Cell Lines
[0121] NMuMG and MCF-10F cell lines were obtained from the American
Type Culture Collection. MMTV-transfected clones of the NMuMG cell
line were generated by transfecting NMuMG cells with MMTV. Clones
expressing high levels of MMTV virions were selected. All NMuMG
cell lines were maintained in DMEM containing 10% heat-inactivated
FBS, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin, and 10
.mu.g/ml insulin at 37.degree. C. and 5% CO.sub.2.
[0122] Cell transfections were accomplished using the
GenePorter.RTM. system (Gene Therapy Systems). The Q61 plasmid was
used for complete envelope expression. Y>F mutations in the MMTV
SU tyrosine residues were introduced using the Quickchange.RTM. XL
kit (Stratagene). Stable pools were generated and maintained by
sorting for SU.sup.hi expressing cells every 5-10 passages.
Flow Cytometry
[0123] Cells grown in two-dimensional cultures (10.sup.6) were
analyzed for flow cytometry on FACSCalibur (BD Biosciences). Goat
polyclonal anti-SU (Dzuris J L et al, Expression of mouse mammary
tumor virus envelope protein does not prevent superinfection in
vivo or in vitro. Virology. 1999 Oct. 25; 263 (2):418-26) or rat
anti-human E-cadherin (Sigma-Aldrich) were used as primary antibody
and donkey anti-goat IgG-FITC conjugated or goat anti-rat IgG-Cy5
conjugated (Jackson ImmunoResearch Laboratories) were used as
secondary antibody. Normal goat IgGs or rat IgGs were used as the
negative control.
Three-Dimensional Cultures
[0124] NMuMG cells (5.times.10.sup.3 cells per chamber) were
cultured on Matrigel.RTM. (BD Biosciences) cushions. Assay medium
(DMEM/F12 supplemented with 2% donor horse serum, 10 .mu.g/ml
insulin, 1 ng/ml cholera toxin, 100 .mu.g/ml hydrocortisone, 50
U/ml penicillin, and 50 .mu.g/ml streptomycin) containing 2%
Matrigel was replaced every 4 days. The structures were analyzed,
at a magnification of 20, on a Zeiss Axiovert 200M equipped with
PCO SensiCam video camera and Slidebook.RTM. software (Intelligent
Imaging Innovations). Cell staining was performed with rat
anti-human Keratin-18 (Lab Vision), rat anti-human E-cadherin and
goat anti-rat IgG-FITC conjugated antibodies (Jackson
ImmunoResearch Laboratories), or goat anti-rat IgG-Alexa-555
conjugated antibodies (Molecular Probes). Quantification of
structure size was performed using a 10.times.50-.mu.m grid
reticule (Fisher Scientific) and 20-100 structures were counted
from each chamber. The inhibitors PP2 and Piceatannol (EMD) were
added on day 3 of culture, and pictures were taken on day 6. In
apoptosis assays, TNF (R&D Systems) or TRAIL (BIOMOL Research
Laboratories, Inc) were added on day 5 of culture, and pictures
were taken on day 6. TUNEL assay was performed using a kit from
Roche Applied Science (Indianapolis, Ind.).
Cell Lysis, Immunoprecipitation, and Western Blotting
[0125] Cells grown in two-dimensional cultures to confluency were
stimulated for 2 min with 50 micromolar (.mu.M) sodium pervanadate
and harvested using cell lifters (Sigma-Aldrich). The cell pellet
was lysed using PhosphoSafe.RTM. (EMD), supplemented with protease
inhibitor cocktail (Roche Applied Science), and 0.5% weight per
volume (wt/vol) sodium azide. Equivalent protein loads were used
for immuno-precipitation (IP) as determined by Bichinchoninic Acid
assay (Sigma-Aldrich). The antibody used for IP was mouse anti-SU
black 86 monoclonal antibody (Burzyn D et al, Toll-like receptor
4-dependent activation of dendritic cells by a retrovirus. J.
Virol. 2004 January; 78(2):576-84). For Western blotting, goat
anti-SU or rabbit anti-Syk N-19 (Santa Cruz Biotechnology, Inc)
polyclonal antibodies were used as primary antibodies, and donkey
anti-goat IgG-alkaline phosphatase conjugated antibody or goat
anti-rat IgG-alkaline phosphatase conjugated antibody (Jackson
ImmunoResearch Laboratories) as secondary antibodies. For
development and quantification, an ECF substrate was used followed
by a scan using Storm 860 and analysis by ImageQuant 5.2 (all
obtained from Amersham Biosciences).
Model of Human Cell Transformation
[0126] Spontaneously immortalized MCF-10F cells. MCF-10F cells and
the transfected stable pools derived therefrom were maintained in
DMEM:F-12 [1:1] medium with a 1.05 mM Ca2.sup.+ concentration.
After transfection, cells were assayed for determination of
survival efficiency, colony efficiency, colony size, ductulogenic
capacity, and invasiveness in Boyden chambers.
Colony Formation in Agar-Methocel
[0127] This technique was used as an in vitro assay for
anchorage-independent growth, a parameter indicative of
transformation. Cell lines were suspended at a density of
2.times.10.sup.4 cells/ml in 2 ml of 0.8% methocel (Sigma-Aldrich)
dissolved in DMEM:F-12 (1:1) medium containing 20% horse serum.
Cells from each transfection group were plated in eight 24-well
chambers pre-coated with 0.5 ml 5% agar base in DMEM:F-12 medium.
Cells were fed with fresh feeding medium containing 0.8% methocel
twice per week. The top four wells were stained with neutral red
(1:300) 24 h after plating, and the total number of viable cells
was counted. The bottom four wells were maintained in culture for
21 d, after which these wells were stained with neutral red, and
colonies was counted. 10 colonies per well were measured by using a
graduated reticule under microscope at a magnification of 10.
Colony efficiency was determined by a count of the number of
colonies >63 .mu.m in diameter, and expressed as a percentage of
the original number of viable cells 24 h after plating.
Ductulogenesis in Collagen Matrix
[0128] Parental MCF-10F and transfected cells were suspended at a
final density of 2.times.10.sup.3 cells/ml in 89.3% Vitrogen
100.RTM. collagen matrix (Collagen) and plated in four 24-well
chambers precoated with 89.3% collagen. The cells were fed with
fresh feeding medium containing 20% horse serum twice per week and
were examined under an inverted microscope for a period of 21 d or
longer to determine whether they formed ductal structures or grew
as ball-like spherical masses. Structures were photographed, fixed
in 10% neutral buffered formalin, embedded in paraffin, sectioned,
and stained with hematoxylin-eosin for histological
examination.
Invasion Assay
[0129] Trypsinized cells (2.5.times.10.sup.4) were seeded in the
top chamber of BioCoat Matrigel Invasion Chambers.RTM. (BD
Biosciences) and incubated for 22 h at 37.degree. C. High calcium
medium with 20% horse serum was used as a chemo-attractant. The
filters were fixed, stained by DiffQuick.RTM. (Sigma-Aldrich), cut
out, and mounted onto glass slides. The total number of cells that
crossed the membrane was counted under a light microscope.
Results
[0130] The MMTV env gene encodes a type-1 membrane glycoprotein
that, after proteolytic cleavage, exists as two mature proteins,
the surface unit (SU or gp52) and the transmembrane unit (TM or
gp36). SU contains the sequence: 418-PAYDYAAIIVKRPPYVLLPVDIGD-441
(SEQ ID No: 1) (FIG. 1 A).
[0131] To test whether the MMTV Env and its ITAM participate in
MMTV-mediated transformation, three-dimensional cultures of NMuMG
murine mammary epithelial cells were used. NMuMG cells, a normal
murine mammary epithelial cell line, were stably transfected with
both the SU and TM subunits of the MMTV Env; the stably transfected
line is referred to as NMuMG.Q4. NMuMG.Q4 cells and
mock-transfected NMuMG cells were seeded in three-dimensional
cultures on a Matrigel.RTM. cushion. Within the first 6 days (d),
WT mock-transfected NMuMG cells were observed to form a polarized
disc structure (FIG. 1 C). By contrast, NMuMG.Q4 cells generated
depolarized acini in frequencies ranging between 30-90% of all
structures (nine independent experiments; FIG. 1 B). The
differences in depolarization are likely to reflect the variable
Env expression levels in the NMuMG.Q4 cell line. When grown in
two-dimensional cultures, the transfected cells did not appear
morphologically different from the parental NMuMG cell line and did
not exhibit a reproducible growth rate advantage.
[0132] To determine the contribution of the tyrosine residues in
the ITAM domain of SU, an additional stable transfected pool was
generated, NMuMG.F6, that expressed the MMTV Env with two Y>F
substitutions (Env2xY>F) in the ITAM (amino acids 422 and 432 in
MMTV [C3H] sequence). NMuMG.F6 was almost indistinguishable from
wild-type or mock-transfected cells (FIG. 1 C, left panel).
Enlarged structures were occasionally in three-dimensional cultures
of wild-type or NMuMG.F6 cells, with low frequency, similar to that
of mock-transfected NMuMG cells (six independent experiments; FIG.
1 D). Differences in surface expression levels of MMTV SU did not
account for the observed differences in transformation, as Env
expression in the NMuMG.Q4 and NMuMG.F6 lines was equivalent (FIG.
1 B, bottom). To show that the disruption in the three-dimensional
morphology observed was not an artifactual result of Env
over-expression, the Env expression levels in NMuMG.Q4 and NMuMG.F6
cells was compared to an MMTV-transfected clone (C1). The Env
expression in the MMTV-transfected clone was higher, ruling out an
artifactual effect (FIG. 1C, right panel).
[0133] Thus, in this in vitro model, MMTV Env expression induces
depolarization of mammary epithelial acinar structures, in a manner
dependent on tyrosine residues within the ITAM. These alterations
are similar to those induced by known breast oncogenes such as
ErbB2/HER2, showing that MMTV Env is capable of transformation of
breast cells in an ITAM-dependent manner.
Example 2
SRC and SYK Tyrosine Kinases Contribute to MMTV ENV-Induced Acinar
Depolarization
[0134] In lymphocytes, ITAM signaling is dependent, under the
conditions utilized herein, on activity of two tyrosine kinase
families, the Src family kinases and the smaller Syk/Zap-70 family
kinases. To directly show the role of the SU ITAM domain in
signaling, direct interaction between the MMTV SU and Syk kinase
was measured in the presence of tyrosine phosphatase inhibitors. Up
to 34% SU protein co-immunoprecipitated with Syk (three independent
experiments; FIG. 2 A). To confirm this finding, pharmacologic
inhibitors of either Src (PP2) or Syk/ZAP70 (piceatannol) were
shown to be sufficient to block morphological changes associated
with Env transformation (FIG. 2B; quantification for one
representative experiment depicted in FIG. 2C).
[0135] These findings provide further evidence that the ITAM was
responsible for the observed cell transformation.
Example 3
MMTV ENV-Expressing Cells Exhibit a Transformed Mammary Epithelial
Phenotype
[0136] NMuMG cells stably expressing infectious MMTV virus were
also evaluated. These cells (MMTV.sup.+ cells) exhibited
morphological features resembling mesenchymal cells in
three-dimensional cultures and a greater degree of depolarization
in comparison with NMuMG.Q4 cells (FIG. 3A). A higher level of
surface MMTV SU expression (FIG. 1C) or positional effects due to
virus integration and long-term culture likely accounts for the
differences between the MMTV.sup.+ cells and the NMuMG.Q4
cells.
[0137] NMuMG.Q4 and MMTV.sup.+ cells both exhibited down-regulation
of Keratin-18 and E-cadherin expression, indicators of
epithelial-mesenchymal transition, (FIG. 3 B, left panel), while
expression of these markers in NMuMG.F6 cells closely resembled
wild-type and mock-transfected cells. Down-regulation of E-cadherin
surface expression could also be detected in Env-expressing cells
maintained in two-dimensional cultures (FIG. 3 B, right panel).
[0138] Sensitivity to apoptosis induced by TNF-related
apoptosis-inducing ligand (TRAIL) and TNF marks transformation and
depolarization in many three-dimensional mammary epithelial culture
systems. Accordingly, sensitivity of the Env-expressing cells to
the pro-apoptotic effects of these agents was tested. As depicted
in FIG. 3C. Env-expressing but not WT or mock-transfected cells
were be sensitive to these agents, as determined by a marked
attenuation in growth and loss of the spreading, nonpolarized
structures exhibited in the absence of TNF or TRAIL.
[0139] Thus, MMTV Env expression in epithelial results in
epithelial-mesenchymal transition.
Example 4
Expression of MMTV ENV Leads to Human Mammary Epithelial Cell
Transformation
[0140] To determine whether Env expression leads to phenotypes
normally associated with breast malignancy in primary human mammary
epithelial cells, ability of MMTV Env to transform human mammary
epithelial cells was determined. Stable transfectants of the
primary human mammary epithelial line, MCF-10F, were generated,
expressing either WT Env (MCF-10F.Q400) or the Env2xY>F envelope
mutant (MCF-10F.Y1). Two assays were used as correlates of cell
transformation: colony formation in agar-methocel and
three-dimensional growth in a collagen matrix. Although both cell
lines produced colonies, colony formation in agar-methocel in the
Env-expressing cells was significantly and reproducibly more
efficient in MCF-10F-Q400 cells (55-90%) compared with MCF-10F.Y1
cells (2540%) (FIG. 4A, right panel). In addition, the MCF-10F.Q400
colonies were approximately twofold larger than those of MCF-10F.Y1
cells (FIG. 4A, left panel). These differences were more evident at
earlier time points, where identifiable Env-expressing colonies
were detected as early as 5 d in culture.
[0141] The transforming properties of the Env protein in human
cells were also detected using a collagen matrix assay, wherein
mammary epithelial cells form ductal structures resembling their
organization in the mammary gland. In MCF-10F.Q400 cells, notable
loss of ductal structure (only spherical structures were observed)
was observed in comparison with wild-type (MCF-10F.Q400) cells and
ITAM mutant (MCF-10F.Y1) cells (FIG. 4B).
[0142] In addition, Matrigel.RTM. invasion assays were conducted to
evaluate the invasive properties of Env-expressing human cells. As
depicted in FIG. 4C, MCF-10F.Y1 cells were only mildly invasive
(<100 cells/25,000 cells seeded), whereas MCF-10F.Q400 cells
were highly invasive, with more than twice as many cells
scored.
[0143] These findings demonstrate that expression of a viral
ITAM-containing protein confers transformation upon human mammary
epithelial cells.
Example 5
Inhibition of ITAM-SYK Interactions Using Synthetic ITAM
Analogues
Materials and Experimental Methods (Examples 5-7)
Cells and Peptides
[0144] The NMuMG, Mm5MT, HEK-293T, NIH3T3, Bal-17 and RBL-2H3 cell
lines were obtained from the American Type Culture Collection.
NMuMG and Mm5MT cell lines were maintained in DMEM containing 10%
heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml
penicillin-streptomycin, and 10 .mu.g/ml insulin at 37.degree. C.
and 10% CO.sub.2. HEK-293T and NIH3T3 cells were grown in DMEM
containing 10% heat-inactivated FBS, 2 mM L-glutamine, and 100 U/ml
penicillin-streptomycin at 37.degree. C. and 10% CO2. Bal-17 cells
were grown in RPMI containing 10% heat-inactivated FBS, 2 mM
L-glutamine, 100 U/ml penicillin-streptomycin and 50 mM
.beta.-mercapto-ethanol at 37.degree. C. and 5% CO.sub.2. RBL-2H3
cells were grown in RPMI containing 15% heat-inactivated FBS, 2 mM
L-glutamine and 100 U/ml penicillin-streptomycin at 37.degree. C.
and 5% CO.sub.2.
[0145] Peptide synthesis was performed commercially by Global
Peptide Services (purity .gtoreq.90%).
Florescent Constructs and Laser Scanning Microscopy
[0146] Peptide conjugates were produced by QuikChange XL.RTM. kit
from Stratagene. The dsRed2-C1 construct (BD Clontech) was modified
using the following primers: dsRed.YVLL:
5'-CCACCTGTTCCTGTATGTGCTGCTATGAAGATCTCGAGCTC-3' (SEQ ID No 42); and
5'-GGTGGACAAGGACATACACGACGATACTTCTAGAGCTCGAG-3 (SEQ ID No 43).
[0147] dsRed.KRPPYVLL:
5'-CCTGTTCCTGAAGAGGCCATATGTGCTGCTATAGAGATCTCGAG-3' (SEQ D No 44);
and 5'-CGACAAGGACTTCTCCGGCGGTATACACGACGATATCTCTAGAGCTC-3' (SEQ ID
No 45). The Syk-eGFP construct was a provided by Dr. Robert Geahlen
(Purdue University).
[0148] For laser scanning microscopy a Zeiss LSM510 META laser
scanning confocal module on a Zeiss Axiovert 200M inverted
microscope was used. Objective used: C-APO 40x/1.2 water DIC.
Lasers: 488 nm laser line from Argon laser (30 mW) for eGFP
excitation; 543 nm laser line from HeNe laser (1 mW) for dsRed
excitation. Analysis was performed on Zeiss LSM510 META v3.7
software.
Peptide: Syk Binding Assay
[0149] Whole cell lysates of Bal-17 B cells (3.times.10.sup.7) were
used for quantification of ITAM peptide interactions. Equivalent
cell lysates were mixed with either biotin-KRPPAVLL (control
peptide; SEQ ID No 46) or biotin-KRPPYVLL (ITAM peptide; SEQ ID No
47) in concentration between 10-50 .mu.M. Pull-downs were performed
with Immobilized Neutravidin Protein (Pierce), as previously
described. The resulting lysate pull-downs were run on SDS-PAGE
were detected by Western blotting with rabbit anti-Syk antibody
(N-19, Santa Cruz). Donkey anti-rabbit IgG-alkaline phosphatase
conjugated antibody was used as a secondary antibody (Jackson
ImmunoResearch Laboratories). For development and quantification,
an ECF substrate was used followed by a scan using Storm 860 and
analysis by ImageQuant 5.2 (all obtained from Amersham
Biosciences).
Three-Dimensional Acinar Cultures
[0150] NmuMG (wild-type or Env-expressing) or Mm5MT cells
(10.sup.4) were incubated on Matrigel (BD Labware) for six days, as
described previously. Cells were treated with or without ITAM
peptide (10 .mu.M) or the Syk inhibitors piceatannol (1 .mu.g/ml)
and SI-31 (100 nM) (EMD). The acinar structures were analyzed and
captured, at a magnification of 20.times., on a Zeiss Axiovert 200M
equipped with PCO SensiCam video camera and Slidebook v4 software
(Intelligent Imaging Innovations). Three-dimensional structures
>50 .mu.m were considered enlarged. Data shown is based on size
of 50 random acini.
Colony Formation Assay
[0151] NIH3 T3 cells expressing the ITAM-containing MAHB chimera
(.times.10.sup.4) were suspended in 0.3% agar in DMEM containing
10% FBS. Cells from each transduction group were plated in 6-well
plates pre-coated with 0.6% agar base in DMEM containing 10% FBS.
Cells were fed with fresh top agar (0.3% agar in DMEM with 10% FBS,
with or without ITAM peptide) every 5 days. Colonies were counted
and measured using a graduated reticule under microscope at
10.times. magnification on day 21.
B Lymphocyte Stimulation and Proliferation
[0152] Proliferation of mouse splenic B cells: cells
(5.times.10.sup.5) were incubated for 48 h in the presence or
absence of 20 .mu.M peptide and stimulated with 10 .mu.g/ml
anti-BCR antibodies (Jackson ImmunoResearch Laboratories), LPS
(Sigma) or anti-CD40 antibodies (BD Pharmingen). [.sup.3H]
Thymidine incorporation (1 .mu.Ci/well) was measured in the last 4
h of the experiment.
[0153] Inhibition of tyrosine phosphorylation was confirmed by
Western blotting. Splenic B cells (10.sup.7) were pre-incubated
with 0-100 .mu.M ITAM peptide for 3 h at 37.degree. C. and then
stimulated with anti-BCR antibodies as above for further 5 min.
Lysates (20 .mu.g/lane) were run on SDS-PAGE, blotted and detected
with anti-phosphotyrosine antibody (4G10-HRP; Upstate Biotech).
Molecular weights shown are based on the molecular weight
markers.
Fc.epsilon.RI-Mediated Degranulation of RBL-2H3 Cells
[0154] The effect of ITAM peptide on mast cell function was
evaluated by degranulation induced by Fc.epsilon.RI cross-linking.
RBL-2H3 cells (5.times.10.sup.4) were incubated overnight with
anti-DNP IgE (1 .mu.g/ml). Subsequently, degranulation was induced
with DNP-HSA (10 ng/ml, 2 h), in the presence or absence of ITAM
peptide (25 .mu.M). Degranulation, as detected by hexosaminidase
activity, is depicted as percent of maximal activity obtained from
whole cell lysates.
Results
[0155] A water-soluble fragment of the MMTV ITAM was identified:
429-KRPPYVLL-435 (SEQ ID No: 48). dsRed chimera proteins were
generated with 4 amino acids of this ITAM fragment or the entire
fragment (dsRed.YVLL (SEQ ID No: 49) and dsRed.KRPPYVLL,
respectively). Human 293T cells were transiently transfected with
the dsRed constructs and with a Syk-GFP fusion protein, and
co-localization of the florescent proteins was determined by
confocal microscopy (FIG. 6a). In cells expressing the empty dsRed
vector and Syk-GFP, the signal for both florescent proteins was
diffuse and did not co-localize. The 4 amino acid (AA) chimera
(dsRed.YVLL) was strongly expressed and co-localization was readily
observed, although some Syk-GFP could be detected independently of
the dsRed construct. The 8 AA chimera (dsRed.KRPPYVLL) was
expressed to a lesser extent, but co-localization was still
detected.
[0156] To confirm specific binding of the ITAM peptide to Syk,
pull-down assays of biotin-KRPPYVLL from whole cell lysates were
performed. When compared to a control peptide with a tyrosine to
alanine substitution (biotin-KRPPAVLL; SEQ ID No: 46), a
substantial amount of Syk could be detected at a concentration of
50 .mu.M (1.5- to 2-fold over the control, n=3). (FIG. 6B).
[0157] Thus, synthetic peptides of the present invention are
capable of stable interaction with ITAM-binding proteins.
Example 6
ITAM-Based Peptide is Comparable to Syk and Src Inhibition of
ITAM-Induced Transformation
[0158] An ITAM peptide of the present invention (Ac-KRPPYVLL-OH)
(SEQ ID No: 2), designed to block the interaction of Syk/Zap70 with
the MMTV Env ITAM motif, was then tested as an inhibitor of
transformation of mammary epithelial cells by MMTV Env expression.
In 3D cultures, untransformed mammary epithelial cells form round
hollow acinar structures. By contrast, cells expressing MMTV Env
exhibit enlarged 3D acinar structures and resemble the epithelial
to mesnchymal transition, as described hereinabove. Synthetic
peptide inhibitors of the present invention were compared to Src
and Syk kinase inhibitors for their ability to reverse the
transformed phenotype to normal round acini. The ITAM peptide at 10
.mu.M concentration was as effective in phenotypic reversal as the
Src inhibitor PP2 and Syk inhibitors Piceatannol and SI-31 (FIG.
7). By contrast, integrin-dependent polarization of the acini was
not affected by the peptides.
[0159] The MMTV.sup.+ carcinoma line, Mm5MT, expresses high levels
of MMTV Env. In 3D cultures, Mm5MT cells form structures that are
not round, but rather are branched and lack a hollow lumen. When
treated with ITAM peptide, Src or Syk inhibitors, Mm5MT acini
exhibited a more round phenotype (FIG. 8). This observation
confirms the finding that the effects of ITAM peptide treatment are
similar to those of Src or Syk inhibition. The round acini
resulting from such treatments did not have a hollow lumen.
[0160] Lastly, the ability of the ITAM peptide to block colony
formation of ITAM-expressing 3T3 fibroblasts was tested (described
further in Example 20 below). In this system, soft agar colony
growth is independent of integrin signaling, which may itself
utilize Syk kinase. As depicted in FIG. 9, chronic treatment of
ITAM.sup.+ fibroblasts with the ITAM peptide was sufficient to
reduce colony formation.
[0161] Thus, synthetic peptides of the present invention were
validated as inhibitors of ITAM signaling in the transformation of
both epithelial and fibroblast cells.
Example 7
Immune Cell Activation is Inhibited by ITAM-Based Peptides of the
Present Invention
[0162] To determine the ability of peptides of the present
invention to block activation of B lymphocytes, these cells were
treated concomitantly with stimulating anti-BCR antibodies and the
ITAM peptide. Peptide treatment resulted in inhibition of
BCR-induced proliferation (FIG. 10A). The ITAM peptide had no
significant effect on stimulation of B lymphocytes through non-ITAM
receptors such as CD40 or LPS (FIG. 10B).
[0163] To verify that the ITAM peptide directly inhibited the
tyrosine kinase activity associated with ITAM-based BCR signaling,
Western blotting for tyrosine phosphorylation was used. When the
relevant peptide concentrations were used, this activity was
visibly reduced (FIG. 10C). Higher concentrations of the peptide,
by contrast, were neither stimulatory nor inhibitory.
[0164] Next, ability of peptides of the present invention to block
degranulation of RBL-2H3 mast cells was tested. The stimulus was,
under these conditions, cross-linking by IgE of the ITAM-containing
high-affinity Fc.epsilon. receptor 1. Indeed, this process was also
significantly inhibited by the ITAM peptide, although in a higher
dose than in other systems (FIG. 11).
[0165] These findings verify that the synthetic peptides of the
present invention have utility in inhibiting ITAM signaling in the
immune system.
Example 8
Modification of Synthetic ITAM Analogues to Improve Potency,
Specificity, and Pharmacological Properties
[0166] The synthetic peptides of the previous Examples are modified
to improve their potency and specificity for interaction between
viral ITAM motifs and cellular proteins that interact therewith,
and/or improved pharmacological properties (e.g. solubility,
bioavailability, biological half-life). The following principles
are applied to the design: [0167] 1. The sequence should contain at
least one YxxL motif of the ITAM. [0168] 2. The sequence and its
ends should provide sufficient hydrophilicity to maintain
significant water solubility and stability in solution. [0169] 3.
The sequence should preferably not contain other known motifs
[0170] The modified peptides are tested as described in the
previous Examples, in order to identify peptides with improved
properties.
Example 9
Treatment of Breast Cancer Using Synthetic ITAM Analogues
[0171] The synthetic peptides of the previous Examples are tested
in animal models of carcinoma (e.g. breast cancer), and found to
reverse the transformation of cells in implanted breast cancer
tumors and to shrink the size of the tumors.
[0172] Alternatively, the synthetic peptides are tested in animal
models of metastatic carcinoma, or ability to prevent metastasis
and are found to reduce the incidence of metastasis.
Example 10
Inhibition of Interactions of Cellular Proteins with an EBV LMP2A
ITAM Using Synthetic ITAM Analogues
[0173] Water-soluble peptides are designed to block the interaction
of cellular proteins with an ITAM motif of Epstein-Barr virus (EBV)
LMP2A protein (e.g. RHSDYQPLGTQDQSLYLGLQHG; SEQ ID No: 50), in
accordance with the principles outlined in Examples 5-9. The
peptides are tested for ability to block transformation of
keratinocytes by LMP2A and treat Burkitt's lymphoma in animal
models, in an analogous manner to the previous Examples.
Example 11
Inhibition of Interactions of Cellular Proteins with an KSHV K1
ITAM Using Synthetic ITAM Analogues
[0174] Water-soluble peptides are designed to block the interaction
of cellular proteins with an ITAM motif of KSHV K1 protein (e.g.
DSNKTVPQQLQDYYSLHDLCTEDYTQP; SEQ ID No: 51), in accordance with the
principles outlined in Examples 5-9. The peptides are tested for
ability to block transformation of fibroblasts by K1 and to treat
animal models of Kaposi's sarcoma, in an analogous manner to the
previous Examples.
Example 12
Inhibition of Interactions of Cellular Proteins with an Hantavirus
G1 ITAM Using Synthetic ITAM Analogues
[0175] Water-soluble peptides are designed to block the interaction
of cellular proteins with an ITAM motif of hantavirus G1 protein
(e.g. KQGCYRTLGVFRYKSRCYVGLVWG, RKGCYRTLGVFRYKSRCYVGLVWG,
KRGCYRTLGVFRYKSRCYVGLVWS, QRGCYRTLGVFRYKSRCYVGLVWN,
KPGCYRTLGVFRYKSRCYVGLVWG, KKGCYRTLGVFRYKSRCYVGLVWC,
KRGCYRTLGVFRYKSRCYVGLVWC, HRGCYRTLGVFRYRSRCYVGLVWG,
RKGCYRTLGVFRYKSRCYVGLVWC, GKGCYRTLGVFRYKSRCYVGLVWC,
KRGCYRTLSVFRYRSRCFVGLVWC, MQGCYRTLSLFRYRSRFFVGLVWC,
KRGLYRTLSMFRYKSKCYVGLVWC, TPGCYRTLNLFRYKSRCYIFTMWI, or
GPGCYRTLNLFRYKSRCYILTMWT, SPGCYRTLNLFRYKSRCYIFTVWV,
GPGCYRTLNLFRYKSRCYILTMWL; SEQ ID No: 52-68, respectively), in
accordance with the principles outlined in Examples 5-9. The
peptides are tested for ability to treat hantavirus pulmonary
syndrome in animal models, in an analogous manner to the previous
Examples.
Example 13
Inhibition of Interactions of Cellular Proteins with an Fc Receptor
Gamma ITAM Using Synthetic ITAM Analogues
[0176] Water-soluble peptides are designed to block the interaction
of cellular proteins with an ITAM motif (e.g.
AATASEKSDGIYTGLSTRTQETYETLKHE, ETADGGYMTLNPRAPTDDDKNIYLTL, or
DYETADGGYMTLNPRAPTDDDKNIYLTL; SEQ ID No: 69-71, respectively) of Fc
receptor gamma-chain protein (e.g. Fc.gamma.RIIa and
Fc.gamma.RIIb), in accordance with the principles outlined in
Examples 5-9. The peptides are tested for ability to treat
pathological thrombosis, autoimmune hemolytic anemia, and
idiopathic thrombocytopenic purpura, in animal models, in an
analogous manner to the previous Examples.
Example 14
Inhibition of Interactions of Cellular Proteins with Immune Protein
ITAMS Using Synthetic ITAM Analogues
[0177] Water-soluble peptides ale designed to block the interaction
of cellular proteins with an ITAM motif of TCR-.zeta./.eta.a,
TCR-.zeta./.eta.b, TCR-.zeta.c, TCR-.zeta./.eta.c, CD3-.gamma.,
CD3-.delta., CD3-.epsilon., Fc.epsilon.RI-.gamma., or
Fc.epsilon.RI-.beta., (e.g. DAGDEYEDENLYEGLNLDDCSMYEDI,
DSKAGMEEDHTYEGLDIDQTATYEDIVTL, DAPAYQQGQNQLYNELNLGRREEYDVL,
DAPAYQHGQNPVYNELNVGRREEYAVL, DAPAYQQGQNQLYNELNLGRREEYDVLDKRR,
ETAANLQDPNQLYNELNLGRREEYDVL, DVPVSPQGHTQLYNELNIGRREEYDVLDKRR,
TAANLQDPNQLYNELNLGRREEYDVLEKK, QQRRRNPQEGVYNALQKDKMAEAYSEIGT,
ERPPPVPNPDYEPIRKGQRDLYSGLNQR, DTQALLRNDQVYQPLRDRDDAQYSHLGGN,
DKQTLLPNDQLYQPLKDREDDQYSHLQGN, DKQTLLNNDQLYQPLKEREDDQYSHLRKK,
EVQALLKNEQLYQPLRDREDTQYSRLGGN, AIASREKADAVYTGLNTRSQETYETLKHE,
AAITSYEKSDGVYTGLSTRNQETYETLKHE, or DIASREKSDAVYTGLNTRNQETYETLKHE;
SEQ ID No: 72-88, respectively), in accordance with the principles
outlined in Examples 5-9. The peptides are tested, in an analogous
manner to the previous Examples, for ability to treat animal models
of B cell activation disorders, mast cell activation disorders,
lymphoma (e.g. Hodgkin's lymphoma or non-Hodgkin's lymphoma),
leukemia (e.g. acute lymphocytic leukemia, acute myelogenous
leukemia, chronic lymphocytic leukemia, chronic myelogenous
leukemia), lymphadenopathy, Kikuchi's disease, Rosai-Dorfman
disease, progressive transformation of germinal centers,
Castleman's disease (e.g. unicentric or multicentric), lymphomatoid
granulomatosis, lymphomatoid papulosis, angioimmunoblastic, or
other lymphoproliferative or immune cell-activation diseases.
Example 15
MAHB: a Nonviral, Membrane-Bound, ITAM-Containing Protein Materials
and Experimental Methods (Examples 15-20)
Cell Lines and Retroviral Infection
[0178] NMuMG and NIH3T3 cell lines were obtained from the American
Type Culture Collection. NMuMG lines were maintained in DMEM
containing 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml
penicillin-streptomycin, and 10 mg/ml insulin at 37.degree. C. and
10% CO.sub.2. All NIH3T3 lines were maintained in DMEM containing
10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml
penicillin-streptomycin, and 50 mM 2-ME at 37.degree. C. and 10%
CO.sub.2.
[0179] Construction of MAHB and its ITAM-mutant variant and cloning
into the MIGR1 retroviral vector is described in Bannish et al
(Ligand-independent signaling functions for the B lymphocyte
antigen receptor and their role in positive selection during B
lymphopoiesis. J Exp Med 2001 Dec. 3; 194(11):1583-96). Fusion
protein constructs were transfected with CaPO.sub.4 into the Bosc23
packaging cell line. To generate stable lines expressing each
construct, 0.25 10.sup.6 NMuMG cells or 0.5 10.sup.6 NIH3T3 cells
were infected by centrifugation at 200 g for 1.5 h at 25.degree. C.
in 4 ml of 1:1 growth medium to retroviral supernatant containing
polybrene (4 mg/ml). After infection, cells were grown in medium
and sorted 48 h post-transduction to obtain pure GFP-expressing
populations.
Flow Cytometry and Immunofluorescence
[0180] Analysis of GFP expression (as an indicator of successful
retroviral infection and protein expression) was performed by flow
cytometry on a FACSCalibur.RTM. (BDBiosciences), using wild-type
NMuMG cells as a negative control. To determine E-cadherin
expression by flow cytometry, 2D cultures of 10.sup.6 NMuMG cells
were used. Rat anti-human E-cadherin (Sigma-Aldrich) was used with
goat anti-rat IgG-AlexaFluor-647 (Molecular Probes), where normal
rat IgG served as the negative control.
[0181] To directly determine expression of MAHB and ITAM-mutant, 2D
cultures of NMuMGs in 16-well slides were used. Cell staining was
performed using the BDCytofix/Cytoperm kit (BDBiosciences) with a
rat monoclonal anti-HA high affinity antibody (clone 3F10, Roche)
followed by goat anti-rat IgG-Alexa Fluor-555 antibody (Molecular
Probes). Images were captured and analyzed using a .times.40
objective on a Zeiss Axiovert 200M inverted epifluorescence
microscope equipped with PCO SensiCam QE high-resolution camera and
Slidebook.RTM. image analysis software (Intelligent Imaging
Innovations).
3D Cultures
[0182] NMuMG cells (1.times.10.sup.4 cells per chamber) were
cultured on recombinant basement membrane (Matrigel.RTM.) cushions,
without exogenous EGF, and structures were analyzed using the
microscope and software. For each independent experiment, 50-200
structures were scored. For inhibitor studies, PP2, Piceatannol,
and Syk Inhibitor 31 (Lai et al, 2003, Bioorg Med Chem Lett 13:
3111-3114) (all from EMD) were added on day 3 of culture and scored
and imaged on day 6. For apoptosis assays, TNF.alpha.
(R&DSystems) or TRAIL (BIOMOL Research Laboratories, Inc.) was
added on day 8 of culture for 20 h, then cells were stained for
cleaved caspase-3. Cell staining was performed with rabbit
anti-cleaved caspase-3 (Cell Signaling), rabbit anti-E-cadherin
(Sigma-Aldrich), rabbit anti-Vimentin (H-84, Santa Cruz
Biotechnology), and anti-rabbit IgG-AlexaFluor-647 (Molecular
Probes).
Colony Formation Assays
[0183] To assay for anchorage-independent growth, NMuMG cells
(2.times.10.sup.4) were suspended in DMEM:F-12 (1:1) medium
containing 20% horse serum and 0.8% methocellulose (Sigma-Aldrich).
Cells were plated in six-well plates pre-coated with 0.6% agar base
in DMEM, were fed with fresh medium every 5 days, and colonies were
counted and measured using a graduated reticule at 10.times.
magnification on day 28.
[0184] NIH3T3 cells (1.times.10.sup.4) were suspended in 0.3% agar
in DMEM containing 10% FBS. Cells from each transduction group were
plated in six-well plates pre-coated with 0.6% agar base in DMEM
containing 10% FBS, were fed with fresh top agar (0.3% agar in DMEM
with 10% FBS) every 5 days, colonies were counted and measured on
day 21.
Focus Formation Assays
[0185] To assay for contact inhibition, NIH3T3 cells
(4.times.10.sup.6) were seeded in a 10 cm dish for 1 week after
reaching confluence. Foci were stained with 0.005% crystal violet,
and foci in a 4 cm.sup.2 area were counted and measured.
Western Blotting and Immunoprecipitation
[0186] 3.times.10.sup.6 NMuMG cells were washed in media without
serum and lysed in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150
mM NaCl, 5 mM EGTA, 0.5% w/v sodium deoxycholate, 1 mM sodium
orthovanadate, protease inhibitor cocktail (Roche), 1 mM
phenylmethylsulfonyl fluoride, 0.5% w/v sodium azide) for 15 min on
ice. Equivalent amounts of protein were separated by SDS-PAGE and
transferred to PVDF. Blots were probed with anti-pTyr (4G10,
Upstate), developed with ECL. Location of MAHB and ITAM-mutant was
determined by stripping the blots in strip buffer (2% SDS, 62.5 mM
Tris, 0.7% .beta.-mercaptoethanol) for 30 min shaking at 62.degree.
C. and probing with anti-HA. 11 antibody (Covance).
[0187] For IP-Western experiments, 2D cultures of 15.times.10.sup.6
NMuMG cells were treated with 50 .mu.M sodium pervanadate,
harvested using cell lifters, and lysed in Phosphosafe.RTM. buffer
(EMD) supplemented with protease inhibitor cocktail (Roche), 1 mM
phenylmethylsulfonyl fluoride, and 0.5% w/v sodium azide for 15 min
at room temperature. Equal amounts of protein were pre=cleared with
Protein G and incubated with mouse IgG or mouse monoclonal HA
antibody (Covance) overnight. Precipitates were collected, washed,
separated by SDS-PAGE, and transferred to PVDF membranes. Blots
were probed with anti-pTyr (4G10, Upstate), anti-HA.11, and
anti-Syk (N-19, Santa Cruz Biotechnology), as described above.
Results
[0188] To determine whether transformation is a general consequence
of expressing ITAM-containing proteins in non-hematopoietic
tissues, a fusion protein termed "MAHB" was created. Instead of
encoding a viral ITAM motif, MAHB encodes the cellular ITAMs of
Ig.alpha. and Ig.beta., which are normally expressed as a
heterodimer in B cells, wherein they are non-transforming, and an
HA tag. A general feature of cellular ITAM-based receptors is their
existence as multi-protein complexes at the cell surface, whose
components cannot be transported individually to the cell surface.
To circumvent the need to express the entire BCR complex, MAHB was
directly targeted to the plasma membrane using the
myristoylation/palmitoylation sequence of Lck (FIG. 12A). In
addition, a signaling-deficient variant of this protein was
generated, ITAM-mutant, in which the ITAM tyrosines were
substituted with phenylalanine (equivalent to Y182F, Y193F in
Ig.alpha. and Y195F, Y206F in Ig.beta.). This variant was used to
confirm that effects observed following MAHB expression was ITAM
dependent. Both the MAHB and ITAM-mutant proteins were cloned into
the MIGR1 retroviral vector that expresses a bicistronic mRNA
encoding either MAHB or ITAM-mutant and GFP (Pui J C et al, Notch1
expression in early lymphopoiesis influences B versus T lineage
determination. Immunity 1999 September; 11 (3):299-308). The
retroviral transduction efficiency for each construct was about
50%, as indicated by GFP expression. Stable cell lines were then
generated by sorting cells expressing high levels of GFP, which
comprised approximately 10-15% of the transduced population. Based
upon GFP or protein expression, MAHB was expressed at lower levels
than ITAM-mutant in both cell types used (FIG. 12B-C). Accordingly,
ITAM-mutant and MIGR express higher protein levels and are thus
valid negative controls for MAHB. Finally, MAHB and the ITAM-mutant
protein expression and membrane localization were confirmed by
immuno-staining for the HA-tagged proteins, as depicted in FIG.
12C. Monitoring growth rates and survival of NMuMG cells in 2D
cultures revealed no differences between cell populations
expressing any of these chimeric proteins.
Example 16
Plasma Membrane Targeted Expression of Ig.alpha./Ig.beta.
Cytoplasmic Domains Triggers ITAM-Dependent Transformation of
Mammary Epithelial Cells
[0189] To determine whether expression of MAHB was able to
transform epithelial cells in vitro, the formation of 3Dacinar
structures by mammary epithelial cells (MEC) was monitored. When
placed on a recombinant basement membrane (Matrigel.RTM.) with the
proteins necessary to provide attachment and survival factors, MECs
develop into growth-arrested, organized acinar structures, as
described in previous Examples. On a scale of zero (normal) to five
(most abnormal), acini expressing MAHB, ITAM-mutant, or the empty
vector (MIGR) were evaluated based on their shape, size, presence
of a lumen, structural integrity, and cellular morphology. For
individual structures, one point was given for each of the
following abnormalities: being nonspherical, enlarged (diameter
over 50 .mu.m), having a malformed (or multiple) lumen(s),
branching from the 3D structure, and having abnormal cellular
morphology. Examples of acini and their respective scores are
depicted in FIG. 13A.
[0190] As depicted in FIG. 13B, MAHB-expressing NMuMG cells
developed depolarized acini, similar to cells expressing MMTV Env
protein (Examples 1-4). By contrast, NMuMG cells transduced with
MIGR formed intact polarized acinar structures with a hollow lumen
similar to wild-type cells and exhibited an average score very
similar to wild-type NMuMG cells. The population of MAHB-expressing
acini as a whole were significantly (P<0.0001) more abnormal
than those expressing GFP alone Additionally, the average score of
the MAHB-expressing NMuMG acini was consistently higher than those
expressing GFP alone or the ITAM-mutant. Moreover, when compared to
MIGR and ITAM-mutant-transduced NMuMG cells, MAHB-expressing cells
formed fewer normal structures, and each of the individual
morphological abnormalities examined occurred with a higher
frequency (FIG. 13C). No correlation was found between low and high
amounts of MAHB within these sorted populations, as determined by
GFP fluorescence, and the degree of acinar depolarization. Acinar
structures formed by ITAM-mutant-expressing NMuMG cells were
normal, indicating that the depolarization induced by MAHB
expression was ITAM dependent (FIG. 13B). This lack of induction of
depolarization of 3Dacini by the ITAM-mutant is not due to low
protein expression, as the variant was expressed at higher levels
than MAHB.
[0191] The ability of anchorage-dependent NMuMG lines to form
colonies in soft agar-methocellulose cultures was also examined.
Inefficient colony formation was observed in MIGR-expressing cells.
By contrast, cells expressing MAHB exhibited more colonies (1586
compared to 140) that were significantly larger (97 .mu.m compared
to 86 .mu.m, P<0.0001) (FIG. 14). Colony formation was reduced
to nearly background (MIGR) levels of both number and size in the
ITAM-mutant-expressing NMuMG cells.
[0192] Thus, expression of cellular ITAM motifs is sufficient,
under the conditions utilized herein, to drive transformation of
epithelial cells.
Example 17
ITAM Expression Induces an EMT Phenotype in Mammary Epithelial
Cells
[0193] The process of epithelial to mesenchymal transition (EMT;
described in Examples 1-4) predicts, in most systems, aggressive
metastatic transformation in vivo. A loss of the epithelial marker
E-cadherin was observed in NMuMG cells expressing MAHB, but not
those expressing ITAM-mutant or transfected with MIGR (FIG. 15A).
In addition, NMuMG cells transduced with MAHB gained expression of
the mesenchymal marker vimentin, whereas MIGR- and
ITAM-mutant-expressing cells did not (FIG. 15B).
[0194] These findings show that these cells are undergoing an EMT
in response to ITAM expression.
Example 18
Sensitivity of Depolarized MAHB-Expressing Acini to Trail and
TNF.alpha.
[0195] Next, the sensitivity to apoptosis induced by TRAIL and
TNF.alpha. was determined for NMuMG cells expressing MAHB in 3D
cultures. In the presence of exogenous pro-apoptotic stimuli,
normal acini are resistant to apoptosis as a consequence of
basement membrane-driven polarization. However, acini that have
become depolarized following oncogene expression become, under the
conditions utilized herein, sensitive to apoptosis induction due to
disrupted integrin signaling. Following treatment with TRAIL and
TNF.alpha., MAHB-expressing acini exhibited caspase-3-dependent
apoptosis, similar to MMTV Env-expressing cells (Example 3),
whereas MIGR- and ITAM-mutant-expressing cells did not (FIG.
16).
[0196] These findings provide further evidence that expression of a
non-viral ITAM-containing protein is capable of transforming
epithelial cells.
Example 19
SRC and SYK Tyrosine Kinases Contribute to ITAM-Induced Acinar
Depolarization
[0197] Under the conditions utilized herein, ITAM signaling
requires the activity of Src and Syk family kinases. The
phosphorylation status of the chimeric proteins was examined in 2D
cultures by anti-phosphotyrosine and anti-HA Western blotting. In
MAHB-expressing NMuMG cells, a constitutively
tyrosine-phosphorylated band with a molecular weight (MW) of 28 kDa
was detected by anti-HA Western blotting, together with two
non-phosphorylated bands with a MW of 25 kDa, present at low levels
(FIG. 17A). In ITAM-mutant-expressing NMuMG cells, only the two
lower MW bands were present.
[0198] In order to determine association of the chimeric proteins
with Syk, cells were treated with pervanadate, immunoprecipitated
with anti-HA antibody, and pellets were probed for Syk. A direct
association was observed between phosphorylated (activated) Syk and
MAHB, but not ITAM-mutant (FIG. 17B).
[0199] To show the dependence on Src and Syk activity of the
MAHB-induced transformation of epithelial cells, cells were treated
with selective inhibitors of each of these kinases. Inhibition of
either Src or Syk prevented the depolarization of acinar
structures; instead, normal polarized structures were observed
(FIG. 17C). In addition, the average score for MAHB-expressing
acini receiving each of the treatments was significantly lower than
those receiving vehicle alone, and was equivalent to the baseline
score of cells expressing MIGR alone (FIG. 13).
[0200] Thus, Src and Syk activity are necessary, under these
conditions, for MAHB-induced depolarization of acinar, structures,
showing that interaction between the cellular ITAM and these
proteins mediates transformation of epithelial cells.
Example 20
Expression of an ITAM-Containing Protein Causes Transformation of
Fibroblasts
[0201] To determine whether MAHB expression could transform
additional cell types, murine 3T3 fibroblasts were stably
transfected with MIGR, MAHB, and ITAM-mutant. To examine the
anchorage dependence of the normally adherent NIH3T3 fibroblasts,
the transfected cell lines were subjected to a soft agar colony
formation assay. NIH3T3 cells transfected with empty vector (MIGR)
formed small colonies after 3 weeks (average size 9077 .mu.m),
similar to wild-type NIH3T3 cells. Expression of MAHB led to the
formation of almost twice as many colonies, that were significantly
(P<0.0001) larger than those the empty vector- or
ITAM-mutant-transfected cells, indicating a loss of anchorage
dependence (13176 .mu.m; FIG. 18A-B), Fibroblasts expressing
ITAM-mutant were nearly indistinguishable from cells transfected
with MIGR, indicating the importance of these motifs in
transformation.
[0202] In addition, the fibroblasts were subjected to focus
formation assays, another assay of transformation. MAHB-expressing
cells formed significantly more foci (approximately threefold;
P<0.05) than those transfected with the empty vector or
ITAM-mutant vector (FIG. 18C). The foci formed by MAHB-expressing
fibroblasts are also larger (average size of 0.715 .mu.m, vs. 0.285
.mu.m for MIGR and 0.290 .mu.m of ITAM-mutant). Thus, the ITAM was
required for loss of contact inhibition.
[0203] Thus, the ability of ITAM-containing proteins to transform
cells is not limited to epithelial cells, but also encompasses
other cell types (e.g. connective tissue cells).
Example 21
Testing of Synthetic ITAM Analogues of the Present Invention in a
3-Dimensional Culture Assay
[0204] To further test the synthetic ITAM analogues of Examples
8-14 and modified variants thereof, epithelial cells transfected
with a viral or cellular ITAM-containing protein are cultured on a
solubilized basement membrane preparation (e.g. Matrigel.RTM.)
comprising laminin, collagen (e.g. collagen IV), and optionally
heparan sulfate proteoglycans and entactin, in the presence of
various peptides in multi-well plates, as described above in
Examples 1, 2, 3, 4, 6, 16, and 19. Exogenous EGF is included, or,
if desired, omitted. After about 6 days, plates are scanned for
wells lacking enlarged structures. The ability to prevent formation
of enlarged structures is indicative of ITAM-inhibitory activity.
Alternatively, mammary epithelial cells are utilized in this assay,
and plates are scanned for wells lacking large spherical
structures, or containing ductal structures. Inhibitory peptides
are titrated to determine the concentration necessary for
inhibition. Untransfected epithelial cells may be used as an
internal standard for background levels of enlarged structures.
[0205] Alternatively, epithelial cells are cultured in multi-well
Invasion Chambers, wherein the bottom chamber contains a
chemo-attractant (e.g, a growth factor in high-calcium medium), and
plates are scanned for wells wherein reduced numbers of cells cross
the membrane, as described above in Example 4.
Example 22
Testing of Synthetic ITAM Analogues of the Present Invention by
Colony Formation in Agar-Methocellulose
[0206] To further test the synthetic ITAM analogues of Examples
8-14 and modified variants thereof, epithelial cells transfected
with a viral or cellular ITAM-containing protein are suspended in
the presence of various peptides in multi-well plates in media
containing agar, growth factors, and methylcellulose or a substance
with similar properties, as described above in Examples 4 and 16.
After about three weeks, plates are scanned for wells exhibiting
reduced colony size or reduced numbers of colonies over a threshold
size. The ability to reduce colony size or number is indicative of
ITAM-inhibitory activity. Inhibitory peptides are titrated to
determine the concentration necessary for inhibition. Untransfected
epithelial cells may be used as an internal standard for background
colony formation levels.
[0207] Alternatively, connective tissue cells (e.g. fibroblasts)
transfected with a viral or cellular ITAM-containing protein are
suspended in the presence of various peptides in multi-well plates
in media containing agar and growth factors, as described above in
Examples 6 and 20. After about three weeks, plates are scanned for
wells exhibiting reduced colony size or reduced numbers of colonies
over a threshold size. The ability to reduce colony size or number
is indicative of ITAM-inhibitory activity. Inhibitory peptides are
titrated to determine the concentration necessary for inhibition.
Untransfected connective tissue cells may be used as an internal
standard for background colony formation levels.
Example 23
Testing of Synthetic ITAM Analogues of the Present Invention by
Co-Ip of ITAM Containing Proteins with Kinases or Substrates
Thereof
[0208] To further test the synthetic ITAM analogues of Examples
8-14 and modified variants thereof, somatic cells (e.g. epithelial
cells or connective tissue cells) transfected with a viral or
cellular ITAM-containing protein are incubated in the presence of
various peptides in multi-well plates, treated with phosphatase
inhibitors, lysed, and immunoprecipitated for the ITAM-containing
protein under relatively low-stringency detergent conditions. In
another embodiment, the phosphate is SHP1. In another embodiment,
the phosphate is SHP2. In another embodiment, the phosphate is any
other phosphatase having an ITAM motif or protein associated
therewith as a substrate. Association of the ITAM-containing
protein with a cellular kinase or substrate thereof (e.g. Syk, Src,
or a family member thereof) is measured by detecting the presence
of the kinase or substrate in the pellet, as described above in
Examples 2 and 19. Alternatively, the lysates are
immunoprecipitated for the cellular kinase, and the ITAM-containing
protein is detected in the pellet. The ability to reduce co-IP is
indicative of ITAM-inhibitory activity. Inhibitory peptides are
titrated to determine the concentration necessary for inhibition.
Untransfected cells may be used as an internal standard for
background Co-IP levels.
Example 24
Testing of Synthetic ITAM Analogues of the Present Invention by
Detecting Markers of EMT
[0209] To further test the synthetic ITAM analogues of Examples
8-14 and modified variants thereof, epithelial cells transfected
with a viral or cellular ITAM-containing, protein are cultured in
collagen-containing media in the presence of various peptides in
multi-well plates, and levels of a marker of epithelial-mesenchymal
transition (e.g. decreased levels of Keratin-18 or E-cadherin
expression, or increased levels of vimentin) are detected, as
described above in Examples 3 and 17. The ability to prevent or
reduce conversion of cells to the transformed phenotype is
indicative of ITAM-inhibitory activity. Inhibitory peptides are
titrated to determine the concentration necessary for inhibition.
Untransfected epithelial cells may be used as an internal standard
for background levels of EMT marker levels.
Example 25
Testing of Synthetic ITAM Analogues of the Present Invention by
Measuring Sensitivity to Apoptosis Induced by Trail or TNF
[0210] To further test the synthetic ITAM analogues of Examples
8-14 and modified variants thereof, epithelial cells transfected
with a viral or cellular ITAM-containing protein are cultured on a
solubilized basement membrane preparation (e.g. Matrigel.RTM.)
comprising laminin, collagen (e.g. collagen IV), and optionally
heparan sulfate proteoglycans and entactin, in the presence of
various peptides in multi-well plates. On about day 5 of culture,
cells are tested for sensitivity to apoptosis induced by TRAIL or
TNF, as described above in Examples 3 and 18. The ability to
abrogate or reduce sensitivity to apoptosis is indicative of
ITAM-inhibitory activity. Inhibitory peptides are titrated to
determine the concentration-necessary for inhibition. Untransfected
epithelial cells may be used as an internal standard for background
levels of apoptosis.
Example 26
Testing of Synthetic ITAM Analogues of the Present Invention by
Inhibition of B Cell Activation and/or Proliferation
[0211] To further test the synthetic ITAM analogues of Examples
8-14 and modified variants thereof, B lymphocytes are incubated in
the presence of anti-BCR antibodies, in the presence of various
peptides in multi-well plates. After about 48 h, activation and/or
proliferation of the cells is measured (e.g. by [.sup.3H] thymidine
incorporation, as described above in Example 7.) The ability to
abrogate or reduce activation and/or proliferation is indicative of
ITAM-inhibitory activity. Inhibitory peptides are titrated to
deter-mine the concentration necessary for inhibition.
Untransfected B lymphocytes may be used as an internal standard for
background levels of activation or proliferation.
Example 27
Testing of Synthetic ITAM Analogues of the Present Invention by
Determining ITAM-Based BCR Signaling
[0212] To further test the synthetic ITAM analogues of Examples
8-14 and modified variants thereof, B lymphocytes are pie-incubated
in multi-well plates in the presence of various peptides, then
stimulated with anti-BCR antibodies. After about 5 min, tyrosine
phosphorylation of downstream targets is measured (e.g. by Western
blotting with anti-phosphotyrosine antibody, as described above in
Example 7). The ability to abrogate or reduce tyrosine
phosphorylation is indicative of ITAM-inhibitory activity.
Inhibitory peptides are titrated to determine the concentration
necessary for inhibition. Untransfected B lymphocytes may be used
as an internal standard for background levels of tyrosine
phosphorylation.
Example 28
Testing of Synthetic ITAM Analogues of the Present Invention by
Inhibition of Mast Cell Degranulation
[0213] To further test the synthetic ITAM analogues of Examples
8-14 and modified variants thereof, mast cells are pre-incubated
incubated overnight with anti-DNP IgE, in multi-well plates, then
degranulation is induced with DNP-HSA in the presence of various
peptides, and degranulation is measured (e.g. by hexosaminidase
activity as described above in Example 7). The ability to abrogate
or reduce degranulation is indicative of ITAM-inhibitory activity.
Inhibitory peptides are titrated to determine the concentration
necessary for inhibition. Untransfected mast cells may be used as
an internal standard for background levels of degranulation.
Example 29
Testing of Synthetic ITAM Analogues of the Present Invention by
Inhibition of Phosphorylation of an ITAM-Containing Protein
[0214] To further test the synthetic ITAM analogues of Examples
8-14 and modified variants thereof somatic cells (e.g. epithelial
cells or connective tissue cells) are cultured in multi-well plates
in the presence of various peptides, then the phosphorylation of a
viral or cellular ITAM-containing protein is measured (e.g. by
Western blotting as described above in Example 19). The ability to
abrogate or reduce phosphorylation is indicative of ITAM-inhibitory
activity, Inhibitory peptides are titrated to determine the
concentration necessary for inhibition. Untransfected cells may be
used as an internal standard for background levels of
phosphorylation.
Example 30
Testing of Synthetic ITAM Analogues of the Present Invention by
Focus Formation Assay
[0215] To further test the synthetic ITAM analogues of Examples
8-14 and modified variants thereof, connective tissue cells (e.g.
fibroblasts) are cultured in multi-well plates in the presence of
various peptides, then focus formation assays as performed (e.g. as
described above in Example 20). The ability to abrogate or reduce
focus formation is indicative of ITAM-inhibitory activity.
Inhibitory peptides are titrated to determine the concentration
necessary for inhibition. Untransfected cells may be used as an
internal standard for background levels of focus formation.
Example 31
Treatment of Sarcoma Using Synthetic ITAM Analogues
[0216] The synthetic peptides of the previous Examples are tested
in animal models of sarcoma (in one embodiment, Kaposi's sarcoma)
and are found to reverse the transformation of the sarcoma cells
and to shrink the size of the tumors.
[0217] Alternatively, the synthetic peptides are tested in animal
models of metastatic sarcoma, or ability to prevent metastasis and
are found to reduce the incidence of metastasis.
Sequence CWU 1
1
98 1 24 PRT Mouse mammary tumor virus 1 Pro Ala Tyr Asp Tyr Ala Ala
Ile Ile Val Lys Arg Pro Pro Tyr Val 1 5 10 15 Leu Leu Pro Val Asp
Ile Gly Asp 20 2 8 PRT Mouse mammary tumor virus ACT_SITE (1)..(1)
2 Lys Arg Pro Pro Tyr Val Leu Leu 1 5 3 26 PRT Artificial
chemically synthesized D/E (1)..(1) "Xaa" can be either aspartate
or glutamate X (2)..(8) "X" can be any naturally occurring amino
acid X (9)..(9) "Xaa" can be any naturally occurring amino acid X
(10)..(11) "X" can be any naturally occurring amino acid X
(13)..(14) "X" can be any naturally occurring amino acid X
(16)..(22) "X" can be any naturally occurring amino acid X
(24)..(25) "X" can be any naturally occurring amino acid D/E
(26)..(26) "Xaa" can be either aspartate or glutamate 3 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Leu Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa 20 25 4 32 PRT Artificial
chemically synthesized D/E (1)..(1) "Xaa" may be aspartate or
glutamate misc_feature (2)..(9) Xaa can be any naturally occurring
amino acid D/E (10)..(10) "Xaa" may be aspartate or glutamate X
(11)..(12) "Xaa" may be any naturally occurring amino acid X
(14)..(15) "Xaa" may be any naturally occurring amino acid X
(17)..(28) "Xaa" may be any naturally occurring amino acid X
(30)..(31) "Xaa" may be any naturally occurring amino acid I/L
(32)..(32) "Xaa" can be leucine or isoleucine 4 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Leu 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa 20 25 30 5 27
PRT Artificial chemically synthesized D/E (1)..(1) Xaa can be
aspartate or glutamate variablelength (2)..(9) 0-1 residues may not
be present X (2)..(9) Xaa may be any naturally occurring amino acid
D/E (10)..(10) Xaa can be aspartate or glutamate misc_feature
(11)..(12) Xaa can be any naturally occurring amino acid
misc_feature (14)..(15) Xaa can be any naturally occurring amino
acid misc_feature (17)..(23) Xaa can be any naturally occurring
amino acid misc_feature (25)..(26) Xaa can be any naturally
occurring amino acid I/L (27)..(27) "Xaa" can be leucine or
isoleucine 5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Xaa Xaa Leu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa
20 25 6 32 PRT Artificial chemically synthesized D/E (1)..(1) "Xaa"
can be aspartate or glutamate Variablelength (2)..(9) 0-1 residues
may not be present X (2)..(9) Xaa may be any naturally occurring
amino acid D/E (10)..(10) "Xaa" can be aspartate or glutamate
misc_feature (11)..(12) Xaa can be any naturally occurring amino
acid misc_feature (14)..(15) Xaa can be any naturally occurring
amino acid misc_feature (17)..(28) Xaa can be any naturally
occurring amino acid misc_feature (30)..(31) Xaa can be any
naturally occurring amino acid I/L (32)..(32) "Xaa" can be leucine
or isoleucine 6 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Xaa Xaa Leu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Tyr Xaa Xaa Xaa 20 25 30 7 31 PRT Artificial chemically
synthesized D/E (1)..(1) "Xaa" can be aspartate or glutamate
misc_feature (2)..(8) Xaa can be any naturally occurring amino acid
D/E (9)..(9) "Xaa" can be aspartate or glutamate misc_feature
(10)..(11) Xaa can be any naturally occurring amino acid
misc_feature (13)..(14) Xaa can be any naturally occurring amino
acid Variablelength (16)..(27) 0-5 residues may not be present X
(16)..(27) Xaa may be any naturally occurring amino acid
misc_feature (29)..(30) Xaa can be any naturally occurring amino
acid I/L (31)..(31) "Xaa" can be leucine or isoleucine 7 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Leu Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa 20 25
30 8 32 PRT Artificial chemically synthesized D/E (1)..(1) "Xaa"
can be aspartate or glutamate misc_feature (2)..(9) Xaa can be any
naturally occurring amino acid D/E (10)..(10) "Xaa" can be
aspartate or glutamate misc_feature (11)..(12) Xaa can be any
naturally occurring amino acid misc_feature (14)..(15) Xaa can be
any naturally occurring amino acid Variablelength (17)..(28) 0-5
residues may not be present X (17)..(28) Xaa may be any naturally
occurring amino acid misc_feature (30)..(31) Xaa can be any
naturally occurring amino acid I/L (32)..(32) "Xaa" can be leucine
or isoleucine 8 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Xaa Xaa Leu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Tyr Xaa Xaa Xaa 20 25 30 9 16 PRT Artificial chemically
synthesized misc_feature (2)..(3) Xaa can be any naturally
occurring amino acid I/L (4)..(4) "Xaa" can be leucine or
isoleucine Variablelength (5)..(12) 0-2 residues may not be present
X (5)..(12) Xaa may be any naturally occurring amino acid
misc_feature (14)..(15) Xaa can be any naturally occurring amino
acid I/L (16)..(16) "Xaa" can be leucine or isoleucine 9 Tyr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa 1 5 10 15
10 19 PRT Artificial synthetic construct D/E (1)..(1) "Xaa" can be
aspartate or glutamate Variablelength (2)..(3) 0-2 residues may not
be present X (2)..(3) Xaa may be any naturally occurring amino acid
misc_feature (5)..(6) Xaa can be any naturally occurring amino acid
I/L (7)..(7) "Xaa" can be leucine or isoleucine Variablelength
(8)..(15) 0-2 residues may not be present X (8)..(15) Xaa may be
any naturally occurring amino acid misc_feature (17)..(18) Xaa can
be any naturally occurring amino acid I/L (19)..(19) "Xaa" can be
leucine or isoleucine 10 Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Tyr 1 5 10 15 Xaa Xaa Xaa 11 4 PRT Artificial
synthetic construct misc_feature (2)..(3) Xaa can be any naturally
occurring amino acid I/L (4)..(4) "Xaa" can be leucine or
isoleucine 11 Tyr Xaa Xaa Xaa 1 12 19 PRT Artificial synthetic
construct D/E (1)..(1) "Xaa" can be aspartate or glutamate
Variablelength (2)..(3) 0-2 residues may not be present X (2)..(3)
Xaa may be any naturally occurring amino acid Variablelength
(8)..(15) 0-2 residues may not be present X (8)..(15) Xaa may be
any naturally occurring amino acid 12 Xaa Xaa Xaa Tyr Ala Ala Ile
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr 1 5 10 15 Val Leu Leu 13 17 PRT
Artificial synthetic construct D/E (1)..(1) "Xaa" can be aspartate
or glutamate Variablelength (6)..(13) 0-2 residues may not be
present X (6)..(13) Xaa may be any naturally occurring amino acid
13 Xaa Tyr Ala Ala Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Val Leu
1 5 10 15 Leu 14 17 PRT Artificial synthetic construct D/E (1)..(1)
"Xaa" can be aspartate or glutamate Variablelength (2)..(3) 0-2
residues may not be present X (2)..(3) Xaa may be any naturally
occurring amino acid misc_feature (8)..(13) Xaa can be any
naturally occurring amino acid 14 Xaa Xaa Xaa Tyr Ala Ala Ile Xaa
Xaa Xaa Xaa Xaa Xaa Tyr Val Leu 1 5 10 15 Leu 15 15 PRT Artificial
synthetic construct D/E (1)..(1) "Xaa" can be aspartate or
glutamate misc_feature (6)..(11) Xaa can be any naturally occurring
amino acid 15 Xaa Tyr Ala Ala Ile Xaa Xaa Xaa Xaa Xaa Xaa Tyr Val
Leu Leu 1 5 10 15 16 28 PRT Artificial synthetic construct D/E
(1)..(1) "Xaa" can be aspartate or glutamate Variablelength
(2)..(9) 0-1 residues may not be present X (2)..(9) Xaa may be any
naturally occurring amino acid D/E (10)..(10) "Xaa" can be
aspartate or glutamate Variablelength (11)..(12) 0-2 residues may
not be present X (11)..(12) Xaa may be any naturally occurring
amino acid misc_feature (14)..(15) Xaa can be any naturally
occurring amino acid I/L (16)..(16) "Xaa" can be leucine or
isoleucine Variablelength (17)..(24) 0-2 residues may not be
present X (17)..(24) Xaa may be any naturally occurring amino acid
misc_feature (26)..(27) Xaa can be any naturally occurring amino
acid I/L (28)..(28) "Xaa" can be leucine or isoleucine 16 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa 20 25 17 28 PRT
Artificial synthetic construct D/E (1)..(1) "Xaa" can be aspartate
or glutamate Variablelength (2)..(9) 0-1 residues may not be
present X (2)..(9) Xaa may be any naturally occurring amino acid
D/E (10)..(10) "Xaa" can be aspartate or glutamate Variablelength
(11)..(12) 0-2 residues may not be present X (11)..(12) Xaa may be
any naturally occurring amino acid misc_feature (14)..(15) Xaa can
be any naturally occurring amino acid Variablelength (17)..(24) 0-2
residues may not be present X (17)..(24) Xaa may be any naturally
occurring amino acid misc_feature (26)..(27) Xaa can be any
naturally occurring amino acid I/L (28)..(28) "Xaa" can be leucine
or isoleucine 17 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Tyr Xaa Xaa Leu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa
Xaa Xaa 20 25 18 26 PRT Mus musculus 18 Asp Met Pro Asp Asp Tyr Glu
Asp Glu Asn Leu Tyr Glu Gly Leu Asn 1 5 10 15 Leu Asp Asp Cys Ser
Met Tyr Glu Asp Ile 20 25 19 28 PRT Artificial synthetic construct
D/E (1)..(1) "Xaa" can be aspartate or glutamate Variablelength
(2)..(9) 0-1 residues may not be present X (2)..(9) Xaa may be any
naturally occurring amino acid D/E (10)..(10) "Xaa" can be
aspartate or glutamate Variablelength (11)..(12) 0-2 residues may
not be present X (11)..(12) Xaa may be any naturally occurring
amino acid Variablelength (17)..(24) 0-2 residues may not be
present X (17)..(24) Xaa may be any naturally occurring amino acid
19 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Gly Leu
1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Asp Ile 20 25 20
27 PRT Artificial synthetic construct D/E (1)..(1) "Xaa" can be
aspartate or glutamate misc_feature (2)..(8) Xaa can be any
naturally occurring amino acid D/E (9)..(9) "Xaa" can be aspartate
or glutamate misc_feature (10)..(11) Xaa can be any naturally
occurring amino acid Variablelength (16)..(23) 0-2 residues may not
be present X (16)..(23) Xaa may be any naturally occurring amino
acid 20 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Gly Leu
Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Asp Ile 20 25 21
27 PRT Artificial synthetic construct D/E (1)..(1) "Xaa" can be
aspartate or glutamate Variablelength (2)..(9) 0-1 residues may not
be present X (2)..(9) Xaa may be any naturally occurring amino acid
D/E (10)..(10) "Xaa" can be aspartate or glutamate Variablelength
(11)..(12) 0-2 residues may not be present X (11)..(12) Xaa may be
any naturally occurring amino acid misc_feature (17)..(23) Xaa can
be any naturally occurring amino acid 21 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Gly Leu 1 5 10 15 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Tyr Glu Asp Ile 20 25 22 26 PRT Artificial synthetic
construct D/E (1)..(1) "Xaa" can be aspartate or glutamate
misc_feature (2)..(8) Xaa can be any naturally occurring amino acid
D/E (9)..(9) "Xaa" can be aspartate or glutamate misc_feature
(10)..(11) Xaa can be any naturally occurring amino acid
misc_feature (16)..(22) Xaa can be any naturally occurring amino
acid 22 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Gly Leu
Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Asp Ile 20 25 23 31
PRT Mus musculus 23 Glu Lys Phe Gly Val Asp Met Pro Asp Asp Tyr Glu
Asp Glu Asn Leu 1 5 10 15 Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser
Met Tyr Glu Asp Ile 20 25 30 24 36 PRT Artificial synthetic
construct D/E (1)..(1) "Xaa" can be aspartate or glutamate
Variablelength (2)..(9) 0-1 residues may not be present X (2)..(9)
Xaa may be any naturally occurring amino acid D/E (10)..(10) "Xaa"
can be aspartate or glutamate Variablelength (11)..(12) 0-2
residues may not be present X (11)..(12) Xaa may be any naturally
occurring amino acid Variablelength (17)..(24) 0-9 residues may not
be present X (17)..(24) Xaa may be any naturally occurring amino
acid misc_feature (25)..(32) Xaa can be any naturally occurring
amino acid 24 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Glu Asp Glu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 Tyr Glu Asp Ile 35 25 34 PRT
Artificial synthetic construct D/E (1)..(1) "Xaa" can be aspartate
or glutamate Variablelength (2)..(11) 0-1 residues may not be
present X (2)..(11) Xaa may be any naturally occurring amino acid
D/E (12)..(12) "Xaa" can be aspartate or glutamate Variablelength
(13)..(13) residue may not be present X (13)..(13) Xaa may be any
naturally occurring amino acid misc_feature (18)..(30) Xaa can be
any naturally occurring amino acid 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Asp 1 5 10 15 Glu Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu 20 25 30 Asp Ile 26 33
PRT Artificial synthetic construct D/E (1)..(1) "Xaa" can be
aspartate or glutamate Variablelength (2)..(9) 0-1 residues may not
be present X (2)..(9) Xaa may be any naturally occurring amino acid
D/E (10)..(10) "Xaa" can be aspartate or glutamate Variablelength
(11)..(12) 0-2 residues may not be present X (11)..(12) Xaa may be
any naturally occurring amino acid misc_feature (17)..(18) Xaa can
be any naturally occurring amino acid misc_feature (23)..(29) Xaa
can be any naturally occurring amino acid 26 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Asp Glu 1 5 10 15 Xaa Xaa Tyr
Glu Gly Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Asp 20 25 30 Ile 27
34 PRT Artificial synthetic construct D/E (1)..(1) "Xaa" can be
aspartate or glutamate Variablelength (2)..(11) 0-1 residues may
not be present X (2)..(11) Xaa may be any naturally occurring amino
acid D/E (12)..(12) "Xaa" can be aspartate or glutamate
Variablelength (13)..(13) residue may not be present X (13)..(13)
Xaa may be any naturally occurring amino acid misc_feature
(18)..(19) Xaa can be any naturally occurring amino acid
misc_feature (24)..(30) Xaa can be any naturally occurring amino
acid 27 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu
Asp 1 5 10 15 Glu Xaa Xaa Tyr Glu Gly Leu Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Tyr Glu 20 25 30 Asp Ile 28 29 PRT Mus musculus 28 Asp Lys Asp
Asp Gly Lys Ala Gly Met Glu Glu Asp His Thr Tyr Glu 1 5 10 15 Gly
Leu Asn Ile Asp Gln Thr Ala Thr Tyr Glu Asp Ile 20 25 29 28 PRT
Artificial synthetic construct D/E (1)..(1) "Xaa" can be aspartate
or glutamate Variablelength (2)..(9) 0-1 residues may not be
present X (2)..(9) Xaa may be any naturally occurring amino acid
D/E (10)..(10) "Xaa" can be aspartate or glutamate Variablelength
(11)..(12) 0-2 residues may not be present X (11)..(12) Xaa may be
any naturally occurring amino acid Variablelength (17)..(24) 0-2
residues may not be present X (17)..(24) Xaa may be any naturally
occurring amino acid 29 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Tyr Glu Gly Leu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Glu Asp Ile 20 25 30 28 PRT Artificial synthetic construct D/E
(1)..(1) "Xaa" can be aspartate or glutamate Variablelength
(2)..(9) 0-1 residues may not be present X (2)..(9) Xaa may be any
naturally occurring amino acid D/E (10)..(10) "Xaa" can be
aspartate or glutamate misc_feature (11)..(12) Xaa can be any
naturally occurring amino acid Variablelength (17)..(24) 0-2
residues may not be present X (17)..(24) Xaa may be any naturally
occurring amino acid 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Tyr Glu Gly Leu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Glu Asp Ile 20 25 31 27 PRT Artificial synthetic construct D/E
(1)..(1) "Xaa" can be aspartate or glutamate Variablelength
(2)..(9) 0-1 residues may not be present X (2)..(9) Xaa may be any
naturally occurring amino acid D/E (10)..(10) "Xaa" can be
aspartate or glutamate Variablelength (11)..(12) 0-2 residues may
not be present X (11)..(12) Xaa may be any naturally occurring
amino acid misc_feature (17)..(23) Xaa can be any naturally
occurring amino acid 31 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Tyr Glu Gly Leu 1
5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Asp Ile 20 25 32 27 PRT
Artificial synthetic construct D/E (1)..(1) "Xaa" can be aspartate
or glutamate Variablelength (2)..(9) 0-1 residues may not be
present X (2)..(9) Xaa may be any naturally occurring amino acid
D/E (10)..(10) "Xaa" can be aspartate or glutamate misc_feature
(11)..(12) Xaa can be any naturally occurring amino acid
misc_feature (17)..(23) Xaa can be any naturally occurring amino
acid 32 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Gly
Leu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Asp Ile 20 25 33
14 PRT Mouse mammary tumor virus 33 Lys Arg Pro Pro Tyr Val Leu Leu
Pro Val Asp Ile Gly Asp 1 5 10 34 8 PRT Mus musculus 34 Asp Cys Ser
Met Tyr Glu Asp Ile 1 5 35 8 PRT Mus musculus 35 Gln Thr Ala Thr
Tyr Glu Asp Ile 1 5 36 29 PRT Homo sapiens 36 Ser Ser Cys Arg Leu
Thr Asn Cys Leu Asp Ser Ser Ala Tyr Val Tyr 1 5 10 15 Ala Ala Ile
Ile Val Leu Met Pro Pro Tyr Val Leu Leu 20 25 37 20 PRT Artificial
synthetic construct D/E (1)..(1) "Xaa" can be aspartate or
glutamate X (2)..(6) Xaa may be any naturally occurring amino acid
X (11)..(16) Xaa may be any naturally occurring amino acid 37 Xaa
Xaa Xaa Xaa Xaa Xaa Tyr Ala Ala Ile Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Tyr Val Leu Leu 20 38 26 PRT Homo sapiens 38 Arg Leu Thr Asn Cys
Leu Asp Ser Ser Ala Tyr Asp Tyr Ala Ala Ile 1 5 10 15 Ile Val Lys
Arg Pro Pro Tyr Val Leu Leu 20 25 39 20 PRT Artificial synthetic
construct D/E (1)..(1) "Xaa" can be aspartate or glutamate X
(2)..(5) Xaa may be any naturally occurring amino acid X (11)..(16)
Xaa may be any naturally occurring amino acid 39 Xaa Xaa Xaa Xaa
Xaa Asp Tyr Ala Ala Ile Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Tyr Val
Leu Leu 20 40 20 PRT Homo sapiens 40 Asp Ser Ser Ala Tyr Asp Tyr
Ala Ala Ile Ile Val Lys Arg Pro Pro 1 5 10 15 Tyr Val Leu Leu 20 41
20 PRT Artificial synthetic construct X (2)..(5) Xaa may be any
naturally occurring amino acid X (11)..(16) Xaa may be any
naturally occurring amino acid 41 Asp Xaa Xaa Xaa Xaa Asp Tyr Ala
Ala Ile Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Tyr Val Leu Leu 20 42 41
DNA Artificial primer 42 ccacctgttc ctgtatgtgc tgctatgaag
atctcgagct c 41 43 41 DNA Artificial primer 43 ggtggacaag
gacatacacg acgatacttc tagagctcga g 41 44 44 DNA Artificial primer
44 cctgttcctg aagaggccat atgtgctgct atagagatct cgag 44 45 47 DNA
Artificial primer 45 cgacaaggac ttctccggcg gtatacacga cgatatctct
agagctc 47 46 8 PRT Artificial synthetic construct biotin (1)..(1)
46 Lys Arg Pro Pro Ala Val Leu Leu 1 5 47 8 PRT Mouse mammary tumor
virus biotin (1)..(1) 47 Lys Arg Pro Pro Tyr Val Leu Leu 1 5 48 8
PRT Mouse mammary tumor virus 48 Lys Arg Pro Pro Tyr Val Leu Leu 1
5 49 4 PRT Mouse mammary tumor virus dsRed (1)..(1) 49 Tyr Val Leu
Leu 1 50 22 PRT Epstein-Barr virus 50 Arg His Ser Asp Tyr Gln Pro
Leu Gly Thr Gln Asp Gln Ser Leu Tyr 1 5 10 15 Leu Gly Leu Gln His
Gly 20 51 27 PRT Kaposi's sarcoma-associated herpesvirus 51 Asp Ser
Asn Lys Thr Val Pro Gln Gln Leu Gln Asp Tyr Tyr Ser Leu 1 5 10 15
His Asp Leu Cys Thr Glu Asp Tyr Thr Gln Pro 20 25 52 24 PRT
Hantavirus sp. 52 Lys Gln Gly Cys Tyr Arg Thr Leu Gly Val Phe Arg
Tyr Lys Ser Arg 1 5 10 15 Cys Tyr Val Gly Leu Val Trp Gly 20 53 24
PRT Hantavirus sp. 53 Arg Lys Gly Cys Tyr Arg Thr Leu Gly Val Phe
Arg Tyr Lys Ser Arg 1 5 10 15 Cys Tyr Val Gly Leu Val Trp Gly 20 54
24 PRT Hantavirus sp. 54 Lys Arg Gly Cys Tyr Arg Thr Leu Gly Val
Phe Arg Tyr Lys Ser Arg 1 5 10 15 Cys Tyr Val Gly Leu Val Trp Ser
20 55 24 PRT Hantavirus sp. 55 Gln Arg Gly Cys Tyr Arg Thr Leu Gly
Val Phe Arg Tyr Lys Ser Arg 1 5 10 15 Cys Tyr Val Gly Leu Val Trp
Asn 20 56 24 PRT Hantavirus sp. 56 Lys Pro Gly Cys Tyr Arg Thr Leu
Gly Val Phe Arg Tyr Lys Ser Arg 1 5 10 15 Cys Tyr Val Gly Leu Val
Trp Gly 20 57 24 PRT Hantavirus sp. 57 Lys Lys Gly Cys Tyr Arg Thr
Leu Gly Val Phe Arg Tyr Lys Ser Arg 1 5 10 15 Cys Tyr Val Gly Leu
Val Trp Cys 20 58 24 PRT Hantavirus sp. 58 Lys Arg Gly Cys Tyr Arg
Thr Leu Gly Val Phe Arg Tyr Lys Ser Arg 1 5 10 15 Cys Tyr Val Gly
Leu Val Trp Cys 20 59 24 PRT Hantavirus sp. 59 His Arg Gly Cys Tyr
Arg Thr Leu Gly Val Phe Arg Tyr Arg Ser Arg 1 5 10 15 Cys Tyr Val
Gly Leu Val Trp Gly 20 60 24 PRT Hantavirus sp. 60 Arg Lys Gly Cys
Tyr Arg Thr Leu Gly Val Phe Arg Tyr Lys Ser Arg 1 5 10 15 Cys Tyr
Val Gly Leu Val Trp Cys 20 61 24 PRT Hantavirus sp. 61 Gly Lys Gly
Cys Tyr Arg Thr Leu Gly Val Phe Arg Tyr Lys Ser Arg 1 5 10 15 Cys
Tyr Val Gly Leu Val Trp Cys 20 62 24 PRT Hantavirus sp. 62 Lys Arg
Gly Cys Tyr Arg Thr Leu Ser Val Phe Arg Tyr Arg Ser Arg 1 5 10 15
Cys Phe Val Gly Leu Val Trp Cys 20 63 24 PRT Hantavirus sp. 63 Met
Gln Gly Cys Tyr Arg Thr Leu Ser Leu Phe Arg Tyr Arg Ser Arg 1 5 10
15 Phe Phe Val Gly Leu Val Trp Cys 20 64 24 PRT Hantavirus sp. 64
Lys Arg Gly Leu Tyr Arg Thr Leu Ser Met Phe Arg Tyr Lys Ser Lys 1 5
10 15 Cys Tyr Val Gly Leu Val Trp Cys 20 65 24 PRT Hantavirus sp.
65 Thr Pro Gly Cys Tyr Arg Thr Leu Asn Leu Phe Arg Tyr Lys Ser Arg
1 5 10 15 Cys Tyr Ile Phe Thr Met Trp Ile 20 66 24 PRT Hantavirus
sp. 66 Gly Pro Gly Cys Tyr Arg Thr Leu Asn Leu Phe Arg Tyr Lys Ser
Arg 1 5 10 15 Cys Tyr Ile Leu Thr Met Trp Thr 20 67 24 PRT
Hantavirus sp. 67 Ser Pro Gly Cys Tyr Arg Thr Leu Asn Leu Phe Arg
Tyr Lys Ser Arg 1 5 10 15 Cys Tyr Ile Phe Thr Val Trp Val 20 68 24
PRT Hantavirus sp. 68 Gly Pro Gly Cys Tyr Arg Thr Leu Asn Leu Phe
Arg Tyr Lys Ser Arg 1 5 10 15 Cys Tyr Ile Leu Thr Met Trp Leu 20 69
29 PRT Homo sapiens 69 Ala Ala Thr Ala Ser Glu Lys Ser Asp Gly Ile
Tyr Thr Gly Leu Ser 1 5 10 15 Thr Arg Thr Gln Glu Thr Tyr Glu Thr
Leu Lys His Glu 20 25 70 26 PRT Homo sapiens 70 Glu Thr Ala Asp Gly
Gly Tyr Met Thr Leu Asn Pro Arg Ala Pro Thr 1 5 10 15 Asp Asp Asp
Lys Asn Ile Tyr Leu Thr Leu 20 25 71 28 PRT Homo sapiens 71 Asp Tyr
Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu Asn Pro Arg Ala 1 5 10 15
Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu Thr Leu 20 25 72 26 PRT
Homo sapiens 72 Asp Ala Gly Asp Glu Tyr Glu Asp Glu Asn Leu Tyr Glu
Gly Leu Asn 1 5 10 15 Leu Asp Asp Cys Ser Met Tyr Glu Asp Ile 20 25
73 29 PRT Homo sapiens 73 Asp Ser Lys Ala Gly Met Glu Glu Asp His
Thr Tyr Glu Gly Leu Asp 1 5 10 15 Ile Asp Gln Thr Ala Thr Tyr Glu
Asp Ile Val Thr Leu 20 25 74 27 PRT Homo sapiens 74 Asp Ala Pro Ala
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 1 5 10 15 Asn Leu
Gly Arg Arg Glu Glu Tyr Asp Val Leu 20 25 75 27 PRT Homo sapiens 75
Asp Ala Pro Ala Tyr Gln His Gly Gln Asn Pro Val Tyr Asn Glu Leu 1 5
10 15 Asn Val Gly Arg Arg Glu Glu Tyr Ala Val Leu 20 25 76 31 PRT
Homo sapiens 76 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
Asn Glu Leu 1 5 10 15 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
Asp Lys Arg Arg 20 25 30 77 27 PRT Homo sapiens 77 Glu Thr Ala Ala
Asn Leu Gln Asp Pro Asn Gln Leu Tyr Asn Glu Leu 1 5 10 15 Asn Leu
Gly Arg Arg Glu Glu Tyr Asp Val Leu 20 25 78 31 PRT Homo sapiens 78
Asp Val Pro Val Ser Pro Gln Gly His Thr Gln Leu Tyr Asn Glu Leu 1 5
10 15 Asn Ile Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
20 25 30 79 29 PRT Homo sapiens 79 Thr Ala Ala Asn Leu Gln Asp Pro
Asn Gln Leu Tyr Asn Glu Leu Asn 1 5 10 15 Leu Gly Arg Arg Glu Glu
Tyr Asp Val Leu Glu Lys Lys 20 25 80 29 PRT Homo sapiens 80 Gln Gln
Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln 1 5 10 15
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr 20 25 81 28 PRT
Homo sapiens 81 Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro
Ile Arg Lys 1 5 10 15 Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln
Arg 20 25 82 29 PRT Homo sapiens 82 Asp Thr Gln Ala Leu Leu Arg Asn
Asp Gln Val Tyr Gln Pro Leu Arg 1 5 10 15 Asp Arg Asp Asp Ala Gln
Tyr Ser His Leu Gly Gly Asn 20 25 83 29 PRT Homo sapiens 83 Asp Lys
Gln Thr Leu Leu Pro Asn Asp Gln Leu Tyr Gln Pro Leu Lys 1 5 10 15
Asp Arg Glu Asp Asp Gln Tyr Ser His Leu Gln Gly Asn 20 25 84 29 PRT
Homo sapiens 84 Asp Lys Gln Thr Leu Leu Asn Asn Asp Gln Leu Tyr Gln
Pro Leu Lys 1 5 10 15 Glu Arg Glu Asp Asp Gln Tyr Ser His Leu Arg
Lys Lys 20 25 85 29 PRT Homo sapiens 85 Glu Val Gln Ala Leu Leu Lys
Asn Glu Gln Leu Tyr Gln Pro Leu Arg 1 5 10 15 Asp Arg Glu Asp Thr
Gln Tyr Ser Arg Leu Gly Gly Asn 20 25 86 29 PRT Homo sapiens 86 Ala
Ile Ala Ser Arg Glu Lys Ala Asp Ala Val Tyr Thr Gly Leu Asn 1 5 10
15 Thr Arg Ser Gln Glu Thr Tyr Glu Thr Leu Lys His Glu 20 25 87 30
PRT Homo sapiens 87 Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val
Tyr Thr Gly Leu 1 5 10 15 Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr
Leu Lys His Glu 20 25 30 88 29 PRT Homo sapiens 88 Asp Ile Ala Ser
Arg Glu Lys Ser Asp Ala Val Tyr Thr Gly Leu Asn 1 5 10 15 Thr Arg
Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu 20 25 89 8 PRT
Artificial synthetic construct 89 Lys Arg Pro Pro Tyr Leu Val Val 1
5 90 28 PRT Cercopithecine herpesvirus 15 90 Pro Tyr Asp Ala Glu
Asp Gly Gly Asp Gly Gly Pro Tyr Gln Pro Leu 1 5 10 15 Arg Gly Gln
Asp Pro Asn Gln Leu Tyr Ala Arg Leu 20 25 91 46 PRT Cercopithecine
herpesvirus 15 91 Gly Pro Tyr Gln Pro Leu Arg Gly Gln Asp Pro Asn
Gln Leu Tyr Ala 1 5 10 15 Arg Leu Gly Gly Gly Gly Gly Asn Gly Thr
Leu Pro Pro Pro Pro Tyr 20 25 30 Ser Pro Gln Arg Glu Thr Ser Leu
His Leu Tyr Glu Glu Ile 35 40 45 92 28 PRT Cercopithecine
herpesvirus 15 92 Glu Asp Pro Tyr Trp Gly Asn Gly Asp Arg His Ser
Asp Tyr Gln Pro 1 5 10 15 Leu Gly Thr Gln Asp Gln Ser Leu Tyr Leu
Gly Leu 20 25 93 31 PRT Human herpesvirus 4 93 Pro Pro Tyr Glu Asp
Leu Asp Trp Gly Asn Gly Asp Arg His Ser Asp 1 5 10 15 Tyr Gln Pro
Leu Gly Asn Gln Asp Pro Ser Leu Tyr Leu Gly Leu 20 25 30 94 31 PRT
Human herpesvirus 4 94 Tyr Asp Ala Pro Ser His Arg Pro Pro Ser Tyr
Gly Gly Ser Gly Gly 1 5 10 15 Tyr Ala Thr Leu Gly Gln Gln Glu Pro
Ser Leu Tyr Ala Gly Leu 20 25 30 95 41 PRT Human herpesvirus 4 95
Asp Arg Asp Gly Asp Pro Val Pro Pro Asp Tyr Asp Ala Pro Ser His 1 5
10 15 Arg Pro Pro Ser Tyr Gly Gly Ser Gly Gly Tyr Ala Thr Leu Gly
Gln 20 25 30 Gln Glu Pro Ser Leu Tyr Ala Gly Leu 35 40 96 48 PRT
Cercopithecine herpesvirus 12 96 Leu Ser Lys Leu Thr Ala Leu Val
Ala Val Ala Thr Trp Phe Ala Ile 1 5 10 15 Leu Met Thr Tyr Leu Val
Leu Pro Ser Ala Asn Asn Ile Ile Val Leu 20 25 30 Ser Leu Leu Val
Ala Ala Glu Gly Ile Gln Ser Ile Tyr Leu Leu Val 35 40 45 97 38 PRT
Human herpesvirus 4 97 Glu Ser Asn Glu Glu Pro Pro Pro Pro Tyr Glu
Asp Pro Tyr Trp Gly 1 5 10 15 Asn Gly Asp Arg His Ser Asp Tyr Gln
Pro Leu Gly Thr Gln Asp Gln 20 25 30 Ser Leu Tyr Leu Gly Leu 35 98
28 PRT Human herpesvirus 4 98 Glu Asp Ser Asp Trp Gly Asn Gly Asp
Arg His Ser Asp Tyr Gln Pro 1 5 10 15 Leu Gly Asn Gln Asp Pro Ser
Leu Tyr Leu Gly Leu 20 25
* * * * *