U.S. patent application number 14/819909 was filed with the patent office on 2016-01-28 for antigen presenting cell targeted cancer vaccines.
The applicant listed for this patent is Baylor Research Institute. Invention is credited to Keiko Akagawa, Jacques F. Banchereau, Anne-Laure Flamar, Peter Klucar, SangKon Oh, Gerard Zurawski, Sandra Zurawski.
Application Number | 20160022792 14/819909 |
Document ID | / |
Family ID | 42729026 |
Filed Date | 2016-01-28 |
United States Patent
Application |
20160022792 |
Kind Code |
A1 |
Zurawski; Gerard ; et
al. |
January 28, 2016 |
ANTIGEN PRESENTING CELL TARGETED CANCER VACCINES
Abstract
The present invention includes compositions and methods for the
expression, secretion and use of novel compositions for use as,
e.g., vaccines and antigen delivery vectors, to delivery antigens
to antigen presenting cells. In one embodiment, the vector is an
anti-CD40 antibody, or fragments thereof, and one or more antigenic
peptides linked to the anti-CD40 antibody or fragments thereof,
including humanized antibodies.
Inventors: |
Zurawski; Gerard;
(Midlothian, TX) ; Banchereau; Jacques F.;
(Montclair, NJ) ; Flamar; Anne-Laure; (New York,
NY) ; Klucar; Peter; (Dallas, TX) ; Akagawa;
Keiko; (Tokyo, JP) ; Zurawski; Sandra;
(Midlothian, TX) ; Oh; SangKon; (Baltimore,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baylor Research Institute |
Dallas |
TX |
US |
|
|
Family ID: |
42729026 |
Appl. No.: |
14/819909 |
Filed: |
August 6, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12717778 |
Mar 4, 2010 |
9102734 |
|
|
14819909 |
|
|
|
|
61159055 |
Mar 10, 2009 |
|
|
|
61159062 |
Mar 10, 2009 |
|
|
|
61159059 |
Mar 10, 2009 |
|
|
|
Current U.S.
Class: |
424/134.1 ;
530/387.3 |
Current CPC
Class: |
A61K 39/0011 20130101;
A61P 35/00 20180101; A61P 31/04 20180101; A61P 31/18 20180101; C07K
16/2878 20130101; C07K 2319/91 20130101; C07K 2319/00 20130101;
A61P 37/02 20180101; A61K 39/001129 20180801; Y02A 50/30 20180101;
A61P 31/14 20180101; A61K 2039/6056 20130101; A61P 35/02 20180101;
C07K 14/435 20130101; C07K 2319/33 20130101; A61P 31/12
20180101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/435 20060101 C07K014/435; C07K 16/28 20060101
C07K016/28 |
Goverment Interests
STATEMENT OF FEDERALLY FUNDED RESEARCH
[0002] This invention was made with U.S. Government support under
Contract No. 1U19AI057234-0100003 awarded by the NIH. The
government has certain rights in this invention.
Claims
1. A fusion protein comprising the formula: Ab-(PL-Ag)x;
Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x; Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or
Ab-(Ag-PL)x-Ag; wherein Ab is an antibody or fragment thereof; PL
is at least one peptide linker comprising at least one
glycosylation site; Ag is at least one cancer antigen; and x is an
integer from 1 to 20.
2. The fusion protein of claim 1, wherein the fusion protein has
more stability in solution than the same fusion protein without the
glycosylation site.
3. The fusion protein of claim 1, wherein the Ag is selected from
tumor associated antigens selected from CEA, prostate specific
antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12,
MUC-related protein (Mucin) (MUC-1, MUC-2, etc.), GM2 and GD2
gangliosides, ras, myc, tyrosinase, MART (melanoma antigen),
MARCO-MART, cyclin B1, cyclin D, Pmel 17(gp100), GnT-V intron V
sequence (N-acetylglucoaminyltransferase V intron V sequence),
Prostate Ca psm, prostate serum antigen (PSA), PRAME (melanoma
antigen), .beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene
product), GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10,
c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6,
gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance
protein (LRP), Bcl-2, and Ki-67.
4. The fusion protein of claim 1, wherein the Ag is selected from
tumor associated antigens comprising antigens from leukemias and
lymphomas, neurological tumors such as astrocytomas or
glioblastomas, melanoma, breast cancer, lung cancer, head and neck
cancer, gastrointestinal tumors, gastric cancer, colon cancer,
liver cancer, pancreatic cancer, genitourinary tumors such cervix,
uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate
cancer or penile cancer, bone tumors, vascular tumors, or cancers
of the lip, nasopharynx, pharynx and oral cavity, esophagus,
rectum, gall bladder, biliary tree, larynx, lung and bronchus,
bladder, kidney, brain and other parts of the nervous system,
thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple
myeloma and leukemia.
5. The fusion protein of claim 1, wherein the Ag is selected from
at least one of: TABLE-US-00113 (SEQ ID NO.: 74)
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAV CGGVLVHPQWV; (SEQ
ID NO.: 75) LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNR
FLRPGDDSSHD; (SEQ ID NO.: 76)
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPK KLQCVDLHVIS; (SEQ
ID NO.: 77) NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITS
WGSEPCALPERP; or (SEQ ID NO.: 78) SLYTKVVHYRKWIKDTIVANP,
and fragments thereof.
6. The fusion protein of claim 1, wherein the Ag is selected from
at least one of: TABLE-US-00114 (SEQ ID NO.: 113) IMDQVPFSV; (SEQ
ID NO.: 114) ITDQVPFSV; (SEQ ID NO.: 115) YLEPGPVTV; (SEQ ID NO.:
116) YLEPGPVTA; (SEQ ID NO.: 117) KTWGQYWQV; (SEQ ID NO.: 122)
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEA
QRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIW
VNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVW
KTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPLAH
SSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQ; (SEQ ID NO.: 124)
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGP
VTAQVVLQAAIPLTSCGSSPVPAS; (SEQ ID NO.: 126)
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTT
EVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEV SIVVLSGTTAA; (SEQ
ID NO.: 128) QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLR
LVKRQVPLDCVLYRYGSFSVTLDIVQ; and (SEQ ID NO.: 130)
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRL
CQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIVPGI LLTGQEAGLGQ,
and fragments thereof.
7. The fusion protein of claim 1, wherein the Ag is selected from
at least one of: TABLE-US-00115 (SEQ ID NO.: 132)
MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDY; and (SEQ ID
NO.: 133) DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK.
8. The fusion protein of claim 1, wherein the Ag is selected from
at least one of: TABLE-US-00116 (SEQ ID NO.: 141)
MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV; (SEQ ID NO.: 142)
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKS
RLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELL; (SEQ ID NO.: 143)
LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATD VKFISNPPSMV; and
(SEQ ID NO.: 144) AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIE
ALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI,
and fragments thereof.
9. The fusion protein of claim 1, wherein the Ag is 19 to 32 amino
acids long.
10. The fusion protein of claim 1, wherein the Ag is 17 to 60 amino
acids long and is selected from a cytotoxic T lymphocyte (CTL)
epitope identified in PSA or cyclin 1.
11. The fusion protein of claim 1, wherein x comprises 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.
12. The fusion protein of claim 1, wherein the Ag comprises two or
more cancer peptides from different cancer antigens separated by
the PL.
13. The fusion protein of claim 1, wherein the Ag is separated by
at least one PL comprising an alanine and a serine.
14. The fusion protein of claim 1, wherein the Ag is selected from
SEQ ID NOS.: 74-78, 79-86, 87-92, 93-95, 113-117, 122-130, 132-133,
and 141-144.
15. The fusion protein of claim 1, wherein the Ab comprises at
least the variable region of the antibody anti-CD40.sub.--12E12.3F3
(ATCC Accession No. PTA-9854), anti-CD40.sub.--12B4.2C10 (ATCC
Submission No. HS446, Accession No. ______), and
anti-CD40.sub.--11B6.1C3 (ATCC Submission No. HS440, Accession No.
______).
16. The fusion protein of claim 1, wherein the Ab is expressed by a
nucleic acid expression vector comprising SEQ ID NOS.: 40 and
41.
17. The fusion protein of claim 1, wherein the Ab comprises at
least one variable domain having 90, 95, 99 or 100% sequence
identity with a heavy chain variable domain of SEQ ID NOS.: 148,
150 and 153 or a light chain variable domains of SEQ ID NOS.: 149,
151, 152 or 154, or both.
18. The fusion protein of claim 1, wherein the PL is selected from:
TABLE-US-00117 (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP; (SEQ ID
NO.: 12) PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
19. The fusion protein of claim 1, wherein the PL comprises an
alanine and a serine.
20. An antigen delivery vector that expresses an anti-CD40 antibody
or fragment thereof and two or more cancer peptides at the
carboxy-terminus of the light chain, the heavy chain or both the
light and heavy chains of the anti-CD40 antibody, wherein when two
or more cancer peptides are present, the cancer peptides are
separated by the one or more peptide linkers that comprise at least
one glycosylation site.
21. The vector of claim 1, wherein the one or more peptide linkers
are selected from: TABLE-US-00118 (SEQ ID NO.: 11)
SSVSPTTSVHPTPTSVPPTPTKSSP; (SEQ ID NO.: 12)
PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
22. An anti-CD40 fusion protein comprising an anti-CD40 antibody or
fragment thereof and one or more cancer peptides at the
carboxy-terminus of the anti-CD40 antibody, wherein when two or
more cancer peptides are present the cancer peptides are separated
by the one or more linker peptides that comprise at least one
glycosylation site.
23. The fusion protein of claim 22, wherein the antibody comprises
at least the variable region of the antibody
anti-CD40.sub.--12E12.3F3 (ATCC Accession No. PTA-9854),
anti-CD40.sub.--12B4.2C10 (ATCC Submission No. HS446, Accession No.
______), and anti-CD40.sub.--11B6.1C3 (ATCC Submission No. HS440,
Accession No. ______).
24. The fusion protein of claim 22, wherein the Ab comprises at
least one variable domain having 90, 95, 99 or 100% sequence
identity with a heavy chain variable domain of SEQ ID NOS.: 148,
150 and 153 or a light chain variable domains of SEQ ID NOS.: 149,
151, 152 or 154, or both.
25. The fusion protein of claim 22, wherein the cancer antigen is
selected from SEQ ID NOS.: 74-78, 79-86, 87-92, 93-95, 113-117,
122-130, 132-133, and 141-144.
26. A method of stabilizing cancer peptides comprising:
incorporating one or more cancer peptides that are unstable or
insoluble into a fusion protein with an antibody, wherein the
antibody and the cancer peptides are separated by one or more
peptide linkers that comprise one or more glycosylation sites.
27. The method of claim 26, wherein the fusion protein comprises
two or more cancer peptides and the cancer peptides are separated
by the one or more peptide linkers.
28. The method of claim 26, wherein the fusion protein comprises
two or more cancer peptides and the peptides are separated by the
one or more peptide linkers.
29. The method of claim 26, wherein the fusion protein comprises
two or more cancer peptides and the peptides are separated by one
or more linkers comprising an alanine and a serine.
30. The method of claim 26, wherein Ag is selected from tumor
associated antigens selected from CEA, prostate specific antigen
(PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12, MUC-related
protein (Mucin) (MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides,
ras, myc, tyrosinase, MART (melanoma antigen), MARCO-MART, cyclin
B1, cyclin D, Pmel 17(gp100), GnT-V intron V sequence
(N-acetylglucoaminyltransferase V intron V sequence), Prostate Ca
psm, prostate serum antigen (PSA), PRAME (melanoma antigen),
.beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene product),
GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10, c-ERB2
(Her2/neu), EBNA (Epstein-Ban Virus nuclear antigen) 1-6, gp75,
human papilloma virus (HPV) E6 and E7, p53, lung resistance protein
(LRP), Bcl-2, and Ki-67.
31. The method of claim 26, wherein the Ag is selected from tumor
associated antigens comprising antigens from leukemias and
lymphomas, neurological tumors such as astrocytomas or
glioblastomas, melanoma, breast cancer, lung cancer, head and neck
cancer, gastrointestinal tumors, gastric cancer, colon cancer,
liver cancer, pancreatic cancer, genitourinary tumors such cervix,
uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate
cancer or penile cancer, bone tumors, vascular tumors, or cancers
of the lip, nasopharynx, pharynx and oral cavity, esophagus,
rectum, gall bladder, biliary tree, larynx, lung and bronchus,
bladder, kidney, brain and other parts of the nervous system,
thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple
myeloma and leukemia.
32. The method of claim 26, wherein the Ag is selected from at
least one of: TABLE-US-00119 (SEQ ID NO.: 74)
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC GGVLVHPQWV; (SEQ
ID NO.: 75) LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRF
LRPGDDSSHD; (SEQ ID NO.: 76)
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKK LQCVDLHVIS; (SEQ
ID NO.: 77) NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWG
SEPCALPERP; or (SEQ ID NO.: 78) SLYTKVVHYRKWIKDTIVANP.
33. The method of claim 26, wherein the Ag is selected from at
least one of: TABLE-US-00120 (SEQ ID NO.: 113) IMDQVPFSV; (SEQ ID
NO.: 114) ITDQVPFSV; (SEQ ID NO.: 115) YLEPGPVTV; (SEQ ID NO.: 116)
YLEPGPVTA; (SEQ ID NO.: 117) KTWGQYWQV; (SEQ ID NO.: 122)
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEA
QRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIW
VNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVW
KTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPLAH
SSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQ; (SEQ ID NO.: 124)
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGP
VTAQVVLQAAIPLTSCGSSPVPAS; (SEQ ID NO.: 126)
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTT
EVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEV SIVVLSGTTAA; (SEQ
ID NO.: 128) QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLR
LVKRQVPLDCVLYRYGSFSVTLDIVQ; and (SEQ ID NO.: 130)
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRL
CQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIVPGI LLTGQEAGLGQ,
and fragments thereof.
34. The method of claim 26, wherein the Ag is selected from at
least one of: TABLE-US-00121 (SEQ ID NO.: 132)
MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDY; and (SEQ ID
NO.: 133) DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK.
35. The method of claim 26, wherein the Ag is selected from at
least one of: TABLE-US-00122 (SEQ ID NO.: 141)
MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV; (SEQ ID NO.: 142)
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKS
RLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELL; (SEQ ID NO.: 143)
LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATD VKFISNPPSMV; and
(SEQ ID NO.: 144) AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIE
ALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI,
and fragments thereof.
36. The method of claim 26, wherein the Ag is 19 to 32 amino acids
long.
37. The method of claim 26, wherein the Ag is 17 to 60 amino acids
long and is selected from a cytotoxic T lymphocyte (CTL) epitope
identified in PSA or cyclin 1.
38. The method of claim 26, wherein x comprises 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.
39. The method of claim 26, wherein the fusion protein comprises
cancer peptides from different antigens separated by different
peptide linkers.
40. The method of claim 26, wherein the fusion protein comprises
two or more cancer peptides separated by one or more peptide
linkers and the peptide linkers comprise an alanine and a
serine.
41. The method of claim 26, wherein the antibody comprises SEQ ID
NOS.: 38 and 39.
42. The method of claim 26, wherein the fusion protein is expressed
by a nucleic acid expression vector comprising SEQ ID NOS.: 40 and
41.
43. The method of claim 26, wherein the peptide linker is selected
from: TABLE-US-00123 (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP;
(SEQ ID NO.: 12) PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
44. A method of enhancing T cell responses comprising: immunizing a
subject in need of vaccination with an effective amount of a
vaccine comprising a fusion protein comprising an anti-CD40
antibody or portion thereof and one or more cancer peptides linked
to the carboxy-terminus of the anti-CD40 antibody.
45. The method of claim 44, wherein the cancer peptides are
selected from tumor associated antigens selected from CEA, prostate
specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12,
MUC (Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides,
ras, myc, tyrosinase, MART (melanoma antigen), MARCO-MART, cyclin
B1, cyclin D, Pmel 17(gp100), GnT-V intron V sequence
(N-acetylglucoaminyltransferase V intron V sequence), Prostate Ca
psm, prostate serum antigen (PSA), PRAME (melanoma antigen),
.beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene product),
GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10, c-ERB2
(Her2/neu), EBNA (Epstein-Ban Virus nuclear antigen) 1-6, gp75,
human papilloma virus (HPV) E6 and E7, p53, lung resistance protein
(LRP), Bcl-2, and Ki-67.
46. The method of claim 44, wherein the cancer peptides are
selected from tumor associated antigens comprising antigens from
leukemias and lymphomas, neurological tumors such as astrocytomas
or glioblastomas, melanoma, breast cancer, lung cancer, head and
neck cancer, gastrointestinal tumors, gastric cancer, colon cancer,
liver cancer, pancreatic cancer, genitourinary tumors such cervix,
uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate
cancer or penile cancer, bone tumors, vascular tumors, or cancers
of the lip, nasopharynx, pharynx and oral cavity, esophagus,
rectum, gall bladder, biliary tree, larynx, lung and bronchus,
bladder, kidney, brain and other parts of the nervous system,
thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple
myeloma and leukemia.
47. A method of making an anti-CD40-antigen fusion protein
comprising: expressing a fusion protein comprising an anti-CD40
antibody or fragment thereof in a host cell, the fusion protein
comprising one or more cancer peptides at the carboxy-terminus of
the anti-CD40 antibody or fragment thereof, wherein when two or
more cancer peptides are separated by one or more linkers, at least
one linker comprising a glycosylation site; and isolating the
fusion protein.
48. The method of claim 47, wherein the antibody comprises at least
the variable region of the antibody anti-CD40.sub.--12E12.3F3 (ATCC
Accession No. PTA-9854), anti-CD40.sub.--12B4.2C10 (ATCC Submission
No. HS446, Accession No. ______), and anti-CD40.sub.--11B6.1C3
(ATCC Submission No. HS440, Accession No. ______).
49. The method of claim 47, wherein the host is a eukaryotic
cell.
50. The method of claim 47, wherein the cancer peptides are
selected from tumor associated antigens selected from CEA, prostate
specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12,
MUC-related protein (Mucin) (MUC-1, MUC-2, etc.), GM2 and GD2
gangliosides, ras, myc, tyrosinase, MART (melanoma antigen),
MARCO-MART, cyclin B1, cyclin D, Pmel 17(gp100), GnT-V intron V
sequence (N-acetylglucoaminyltransferase V intron V sequence),
Prostate Ca psm, prostate serum antigen (PSA), PRAME (melanoma
antigen), .beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene
product), GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10,
c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6,
gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance
protein (LRP), Bcl-2, and Ki-67.
51. The method of claim 47, wherein the cancer peptides are
selected from tumor associated antigens comprising antigens from
leukemias and lymphomas, neurological tumors such as astrocytomas
or glioblastomas, melanoma, breast cancer, lung cancer, head and
neck cancer, gastrointestinal tumors, gastric cancer, colon cancer,
liver cancer, pancreatic cancer, genitourinary tumors such cervix,
uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate
cancer or penile cancer, bone tumors, vascular tumors, or cancers
of the lip, nasopharynx, pharynx and oral cavity, esophagus,
rectum, gall bladder, biliary tree, larynx, lung and bronchus,
bladder, kidney, brain and other parts of the nervous system,
thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple
myeloma and leukemia.
52. The method of claim 47, wherein the cancer peptides are
selected from at least one of: TABLE-US-00124 (SEQ ID NO.: 74)
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC GGVLVHPQWV; (SEQ
ID NO.: 75) LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRF
LRPGDDSSHD; (SEQ ID NO.: 76)
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKK LQCVDLHVIS; (SEQ
ID NO.: 77) NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWG
SEPCALPERP; or (SEQ ID NO.: 78) SLYTKVVHYRKWIKDTIVANP.
53. The method of claim 47, wherein the cancer peptides are
selected from at least one of: TABLE-US-00125 (SEQ ID NO.: 113)
IMDQVPFSV; (SEQ ID NO.: 114) ITDQVPFSV; (SEQ ID NO.: 115)
YLEPGPVTV; (SEQ ID NO.: 116) YLEPGPVTA; (SEQ ID NO.: 117)
KTWGQYWQV; (SEQ ID NO.: 122)
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEA
QRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIW
VNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVW
KTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPLAH
SSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQ; (SEQ ID NO.: 124)
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGP
VTAQVVLQAAIPLTSCGSSPVPAS; (SEQ ID NO.: 126)
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTT
EVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEV SIVVLSGTTAA; (SEQ
ID NO.: 128) QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLR
LVKRQVPLDCVLYRYGSFSVTLDIVQ; and (SEQ ID NO.: 130)
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRL
CQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIVPGI LLTGQEAGLGQ,
and fragments thereof.
54. The method of claim 47, wherein the cancer peptides are
selected from at least one of: TABLE-US-00126 (SEQ ID NO.: 132)
MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDY; and (SEQ ID
NO.: 133) DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK.
55. The method of claim 47, wherein the cancer peptides are
selected from at least one of: TABLE-US-00127 (SEQ ID NO.: 141)
MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV; (SEQ ID NO.: 142)
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKS
RLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELL; (SEQ ID NO.: 143)
LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATD VKFISNPPSMV; and
(SEQ ID NO.: 144) AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIE
ALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI,
and fragments thereof.
56. A method of expanding antigen-specific T cells in vitro
comprising: isolating peripheral blood mononuclear cells (PBMCs)
from a cancer patient; incubating the isolated PBMCs with an
immunogenic amount of an .alpha.CD40-(PL-Ag)x or
.alpha.CD40-(Ag-PL)x vaccine, wherein Ag is a tumor associated
antigen and x is an integer 1 to 20; expanding the PBMCs in the
presence of an effective amount of IL-2; harvesting the cells; and
assessing the cytokine production by the cells to determine the
presence of anti-cancer specific T cells.
57. A tumor associated antigen-specific T cells made by the method
comprising: isolating peripheral blood mononuclear cells (PBMCs)
from a cancer patient; incubating the isolated PBMCs with an
immunogenic amount of an .alpha.CD40-(PL-Ag)x or
.alpha.CD40-(Ag-PL)x vaccine, wherein Ag is a tumor associated
antigen and x is an integer 1 to 20; expanding the PBMCs in the
presence of an effective amount of IL-2; harvesting the cells; and
assessing the cytokine production by the cells to determine the
presence of tumor associated antigen-specific T cells.
58. A therapeutic vaccine comprising a fusion protein comprising
the formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x;
Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an
antibody or fragment thereof; PL is at least one peptide linker
comprising at least one glycosylation site; Ag is at least one
cancer antigen; and x is an integer from 1 to 20.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/717,778, filed Mar. 4, 2010, which claims priority to
U.S. Provisional Application Ser. No. 61/159,055, filed Mar. 10,
2009, U.S. Provisional Application Ser. No. 61/159,062, filed Mar.
10, 2009, and U.S. Provisional Application Ser. No. 61/159,059,
filed Mar. 10, 2009, the entire contents of each are incorporated
herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0003] The present invention relates in general to the field of
immunization, and more particularly, to novel anti-CD40 based
vaccines against cancer.
BACKGROUND OF THE INVENTION
[0004] Without limiting the scope of the invention, its background
is described in connection with antigen presentation.
[0005] One example of vaccines and methods for antigen presentation
is taught in U.S. Pat. No. 7,118,751, issued to Ledbetter, et al.,
for DNA vaccines encoding an amino-terminus antigen linked to a
carboxy-terminus domain that binds CD40. Briefly, vaccines are
taught that target one or more antigens to a cell surface receptor
to improve the antigen-specific humoral and cellular immune
response. Antigen(s) linked to a domain that binds to a cell
surface receptor are internalized, carrying antigen(s) into an
intracellular compartment where the antigen(s) are digested into
peptides and loaded onto MHC molecules. T cells specific for the
peptide antigens are activated, leading to an enhanced immune
response. The vaccine may comprise antigen(s) linked to a domain
that binds at least one receptor or a DNA plasmid encoding
antigen(s) linked to a domain that binds at least one receptor. A
preferred embodiment of the invention targets HIV-1 env antigen to
the CD40 receptor, resulting in delivery of antigen to CD40
positive cells, and selective activation of the CD40 receptor on
cells presenting HIV-1 env antigens to T cells.
[0006] Another example is found in United States Patent Application
No. 20080254026, filed by Li, et al., for antagonist anti-CD40
monoclonal antibodies and methods for their use. Briefly,
compositions and methods are disclosed for use in therapy for
treating diseases mediated by stimulation of CD40 signaling on
CD40-expressing cells are provided. The methods comprise
administering a therapeutically effective amount of an antagonist
anti-CD40 antibody or antigen-binding fragment thereof to a patient
in need thereof. The antagonist anti-CD40 antibody or
antigen-binding fragment thereof is free of significant agonist
activity, but exhibits antagonist activity when the antibody binds
a CD40 antigen on a human CD40-expressing cell. Antagonist activity
of the anti-CD40 antibody or antigen-binding fragment thereof
beneficially inhibits proliferation and/or differentiation of human
CD40-expressing cells, such as B cells.
[0007] Yet another example is taught in United States Patent
Application No. 20080241139, filed by Delucia for an adjuvant
combination comprising a microbial TLR agonist, a CD40 or 4-1BB
agonist, and optionally an antigen and the use thereof for inducing
a synergistic enhancement in cellular immunity. Briefly, this
application is said to teach adjuvant combinations comprising at
least one microbial TLR agonist such as a whole virus, bacterium or
yeast or portion thereof such a membrane, spheroplast, cytoplast,
or ghost, a CD40 or 4-1BB agonist and optionally an antigen wherein
all 3 moieties may be separate or comprise the same recombinant
microorganism or virus are disclosed. The use of these immune
adjuvants for treatment of various chronic diseases such as cancers
and HIV infection is also provided.
[0008] United States Patent Application No. 20080199471, filed by
Bernett, et al., is directed to optimized CD40 antibodies and
methods of using the same. Briefly, this application is said to
teach antibodies that target CD40, wherein the antibodies comprise
at least one modification relative to a parent antibody, wherein
the modification alters affinity to an Fc.gamma.R or alters
effector function as compared to the parent antibody. Also
disclosed are methods of using the antibodies of the invention.
[0009] Finally, United States Patent Application No. 20080181915,
file by Tripp, et al., is directed to a CD40 ligand adjuvant for
respiratory syncytial virus. Briefly, this application is said to
teach methods and adjuvants for enhancing an immune response to RSV
in a host, wherein the methods and adjuvants comprise a source of a
CD40 binding protein. Preferably, the CD40 binding protein is CD40L
and the source is a vector comprising a promoter operatively linked
to a CD40L coding region. The enhanced immune response produced by
the adjuvants and methods of the current invention includes both
increased expression of Th1 cytokines and increased production of
antibody.
SUMMARY OF THE INVENTION
[0010] In one embodiment, the present invention is a fusion protein
comprising the formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x;
Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an
antibody or fragment thereof; wherein PL is at least one peptide
linker comprising at least one glycosylation site; wherein Ag is at
least one antigen; and wherein x is an integer from 1 to 20, the
fusion protein having more stability in solution than the same
fusion protein without the glycosylation site. In one aspect, Ag is
selected from a viral antigen, a tumor antigen, an infectious
disease antigen, an autoimmune antigen, a toxin or combinations
thereof. In another aspect, the Ag is a peptide concatamer. In
another aspect, the PL is a peptide concatamer. In another aspect,
the -(PL-Ag)x, -(Ag-PL)x, -(PL-Ag-PL)x, or -(Ag-PL-Ag)x are located
at the carboxy terminus of the Ab heavy chain or fragment thereof.
In another aspect, the Ag elicits a humoral immune response and/or
cellular immune response in a host. In one aspect, the Ab comprises
at least the variable region of anti-CD40.sub.--12E12.3F3 (American
Type Culture Collection (ATTC) Accession No. PTA-9854),
anti-CD40.sub.--12B4.2C10 (ATTC Submission No. HS446, Accession No.
PTA-10653), and anti-CD40.sub.--11B6.1C3 (ATCC Submission No.
HS440, Accession No. PTA-10652).
[0011] In one aspect, the Ag is selected from autoimmune diseases
or disorders associated with antigens involved in autoimmune
disease selected from glutamic acid decarboxylase 65 (GAD 65),
native DNA, myelin basic protein, myelin proteolipid protein,
acetylcholine receptor components, thyroglobulin, and the thyroid
stimulating hormone (TSH) receptor. In another aspect, the Ag is
selected from infectious disease antigens selected from bacterial,
viral, parasitic, and fungal antigens. In another aspect, x
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, or 19. In another aspect, the fusion protein comprises two
or more Ags from different antigens separated by at least one PL.
In another aspect, the fusion protein comprises two or more Ags
separated by at least one PL comprising an alanine and a serine. In
another aspect, the Ab is an antibody fragment selected from Fv,
Fab, Fab', F(ab')2, Fc, or a ScFv.
[0012] In one aspect, the Ab binds specifically to an MHC class I,
MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16,
CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57,
CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40,
BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1,
B7-2, IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fc.gamma.
receptor, T cell receptor, or lectin. In another aspect, the Ab is
an IgA, IgD, IgE, IgG or IgM or isotype thereof. In another aspect,
the Ab is a human antibody or a humanized antibody. In another
aspect, the PL comprises an alanine and a serine. In another
aspect, the PL is selected from:
TABLE-US-00001 (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP; (SEQ ID
NO.: 12) PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
[0013] Yet another embodiment of the present invention is a nucleic
acid expression vector encoding a fusion protein comprising: a
first polynucleotide encoding an antibody light chain or fragment
thereof; and a second polynucleotide encoding an antibody heavy
chain or fragment thereof; wherein the fusion protein comprises the
following formula: Ab-(PL-Ag)x or Ab-(Ag-PL)x; wherein Ab is an
antibody or fragment thereof; wherein PL is at least one peptide
linker comprising at least one glycosylation site; wherein Ag is at
least one antigen; and wherein x is an integer from 1 to 20, the
fusion protein having more stability in solution than the same
fusion protein without the glycosylation site. In one aspect, the
(PL-Ag)x or (Ag-PL)x are located at the carboxy terminus of the Ab
heavy chain or fragment thereof. In another aspect, the first and
second polynucleotide are on a single expression vector. In another
aspect, the Ag is selected from infectious disease antigens
selected from bacterial, viral, parasitic, and fungal antigens. In
another aspect, x comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, or 19. In another aspect, the fusion
protein comprises two or more Ags from different antigens separated
by at least one PL. In another aspect, the fusion protein comprises
two or more Ags separated by at least on PL comprising an alanine
and a serine. In another aspect, the Ab is an antibody fragment
selected from Fv, Fab, Fab', F(ab')2, Fc, or a ScFv. In another
aspect, the Ab binds specifically to an MHC class I, MHC class II,
CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD20, CD29,
CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86,
CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO,
DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2,
IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fc.gamma. receptor,
T cell receptor, or lectin. In another aspect, the Ab is an IgA,
IgD, IgE, IgG or IgM or isotype thereof. In another aspect, the Ab
is a human antibody or a humanized antibody. In another aspect, the
PL is comprises an alanine and a serine and/or the PL is selected
from:
TABLE-US-00002 (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP; (SEQ ID
NO.: 12) PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
In another aspect, the first and second polynucleotides are
downstream from a constitutive promoter.
[0014] Yet another embodiment of the present invention is a stable,
secretable fusion protein comprising the formula:
NH.sub.2-Ab-(PL-Ag)x-COOH or NH2-Ab-(Ag-PL)x-COOH; wherein Ab is an
antibody or fragment thereof; wherein PL is at least one peptide
linker comprising at least one glycosylation site; wherein Ag is at
least one immunogenic antigen; and wherein x is an integer from 1
to 20, the fusion protein being stable and soluble in solution as
compared to an Ab-Ag protein alone that is not soluble or
stable.
[0015] Another embodiment is a method of stabilizing antigenic
peptides comprising: incorporating one or more antigenic peptides
that are unstable or insoluble into a fusion protein, wherein the
fusion protein has the following structure: Ab-(PL-Ag)x or
Ab-(Ag-PL)x; wherein Ab is an antibody or fragment thereof; wherein
PL is at least one peptide linker comprising at least one
glycosylation site; wherein Ag is at least one antigen; and wherein
x is an integer from 1 to 20, the fusion protein being stable and
soluble in solution wherein the Ab-Ag is not soluble or stable.
[0016] Yet another embodiment of the present invention is a host
cell comprising a nucleic acid expression vector comprising: a
first polynucleotide encoding an antibody light chain; and a second
polynucleotide encoding an antibody heavy chain fusion protein, the
fusion protein comprising the following formula: Ab-(PL-Ag)x or
Ab-(Ag-PL)x; wherein Ab is an antibody or fragment thereof; wherein
PL is at least one peptide linker comprising at least one
glycosylation site; wherein Ag is at least one antigen; and wherein
x is an integer from 1 to 20, the fusion protein having more
stability is solution than the fusion protein without the
glycosylation site. In another embodiment, the host cell comprises
an expression vector that produces a fusion protein comprising the
formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x; Ab-(Ag-PL-Ag)x;
Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an antibody or
fragment thereof; wherein PL is at least one peptide linker
comprising at least one glycosylation site; wherein Ag is at least
one antigen; and wherein x is an integer from 1 to 20, the fusion
protein having more stability in solution than the same fusion
protein without the glycosylation site.
[0017] The present invention also includes a pharmaceutical
composition comprising the antibody having the formula comprising
the formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x;
Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an
antibody or fragment thereof; wherein PL is at least one peptide
linker comprising at least one glycosylation site; wherein Ag is at
least one antigen; and wherein x is an integer from 1 to 20, the
fusion protein having more stability in solution than the same
fusion protein without the glycosylation site.
[0018] Yet another embodiment of the present invention is a fusion
protein comprising the formula: Ab-(PL-Ag)x-(PLy-Agz)n; or
Ab-(Ag-PL)x-(PLy-Agz)n; wherein Ab is an antibody or fragment
thereof; wherein PL is at least one peptide linker comprising at
least one glycosylation site; wherein Ag is at least one antigen;
and wherein x is an integer from 1 to 20; wherein n is 0 to 19; and
wherein y or z is 0 to 10, wherein the fusion protein has more
stability in solution than the same fusion protein without the
glycosylation site.
[0019] Another embodiment is an isolated and purified vaccine
comprising: a heavy chain selected from at least one of SEQ ID
NOS.: 6, 7, 8, 9, 10, 16, 17, 18, 19, 20, 36, 37, 96, 97, 98, 99,
110, 111, 112, 118, 119, 134, 136, 138, 146, and 147 that binds
specifically to CD40; and a light chain that binds specifically to
CD40. In one aspect, the antibody is defined further as a humanized
antibody.
[0020] Yet another embodiment of the present invention is a fusion
protein comprising the formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x;
Ab-(PL-Ag-PL)x; Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag;
wherein Ab is an antibody or fragment thereof; PL is at least one
peptide linker comprising at least one glycosylation site; Ag is at
least one viral antigen; and x is an integer from 1 to 20. In one
aspect, the fusion protein has more stability is solution than the
PL without the glycosylation site. In another aspect, the Ag
comprises a peptide from an adenovirus, retrovirus, picornavirus,
herpesvirus, rotaviruses, hantaviruses, coronavirus, togavirus,
flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus,
bunyavirus, arenavirus, reovirus, papilomavirus, parvovirus,
poxvirus, hepadnavirus, or spongiform virus. In another aspect, the
Ag comprises a peptide from at least one of HIV, CMV, hepatitis A,
B, and C, influenza; measles, polio, smallpox, rubella, respiratory
syncytial, herpes simplex, varicella zoster, Epstein-Barr, Japanese
encephalitis, rabies, flu, or cold viruses.
[0021] In another aspect, the Ag is selected from:
TABLE-US-00003 Nef (66-97): (SEQ ID NO.: 1)
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL; Nef (116-145): (SEQ ID NO.: 2)
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL; Gag p17 (17-35): (SEQ ID NO.: 3)
EKIRLRPGGKKKYKLKHIV; Gag p17-p24 (253-284): (SEQ ID NO.: 4)
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD; or Pol 325-355 (RT 158-188) is:
(SEQ ID NO.: 5) AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY.
In another aspect, the Ag is 19 to 32 residues. In another aspect,
the Ag is selected from a cytotoxic T lymphocyte (CTL) epitope
identified in the HIV-1 Nef, Gag and Env proteins presented in the
context of MHC-class I molecules. In another aspect, the Ag is
selected from HIV gp120, gp41, Gag, p17, p24, p2, p7, p1, p6, Tat,
Rev, PR, RT, IN, Vif, Vpr, Vpx, Vpu and Nef. In another aspect, x
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18 or 19. In another aspect, the Ag comprises virus peptides
from different antigens separated by different peptide linkers. In
another aspect, the Ag is separated by at least one PL comprising
an alanine and a serine. In another aspect, the fusion protein is
selected from SEQ ID NOS.: 21, 22, 23, 24, 25, 26 or 36. In another
aspect, the fusion protein is isolated from a cell that comprises a
polynucleotide vector that encodes the fusion protein, the
polynucleotide vector comprising SEQ ID NOS.: 21, 22, 23, 24, 25,
26 or 36. In another aspect, the Ab comprises SEQ ID NOS.: 37 and
38.
[0022] In another aspect, the fusion protein is isolated from a
cell that comprises a polynucleotide vector that expresses the
fusion protein and the Ab portion comprises SEQ ID NOS.: 39 and 40.
In another aspect, Ag is selected from at least one of SEQ ID NOS.:
52-56, 58-60, 61-69, 70-72, or 73-77. In another aspect, the Ag is
17 to 60 residues. In another aspect, the Ag is 8, 10, 12, 14, 15,
16, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 to 60 residues long. In
another aspect, the Ag comprises at least one lipopeptide. In
another aspect, the Ag is at the carboxy-terminus and further
comprises a carboxy-terminus (Palm)-NH.sub.2 group. In another
aspect, the PL is selected from:
TABLE-US-00004 (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP; (SEQ ID
NO.: 12) PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
In another aspect, the PL comprises an alanine and a serine.
[0023] Another embodiment is the present invention is a viral
antigen delivery vector comprising: a fusion protein comprising an
anti-CD40 antibody or fragment thereof and one or more viral
peptides at the carboxy-terminus of the anti-CD40 antibody, wherein
when two or more viral peptides are present the viral peptides are
separated by the one or more peptide linkers comprising at least
one potential glycosylation site. In another aspect, an antigen
delivery vector is an anti-CD40 antibody or fragment thereof and
two or more viral peptides at the carboxy-terminus of the light
chain, the heavy chain or both the light and heavy chains of the
anti-CD40 antibody, wherein when two or more viral peptides are
separated by the one or more peptide linkers that comprise at least
one potential glycosylation site.
[0024] Yet another embodiment of the present invention is a method
of stabilizing viral peptides comprising: incorporating one or more
viral peptides that are unstable or insoluble into a fusion protein
with an antibody, wherein the antibody and the viral peptides are
separated by one or more peptide linkers that comprise one or more
glycosylation sites. Yet another embodiment is a method of
enhancing T cell responses comprising: immunizing a subject in need
of vaccination with an effective amount of a vaccine comprising the
formula: Ab-(PL-Ag)x or Ab-(Ag-PL)x; wherein Ab is an antibody or
fragment thereof; PL is at least one peptide linker comprising at
least one glycosylation site; Ag is at least one viral antigen; and
x is an integer from 1 to 20. In one aspect, the fusion protein has
more stability in solution than an identical fusion protein without
the glycosylation site. In another aspect, the at least one viral
antigen comprise peptides from adenovirus, retrovirus,
picornavirus, herpesvirus, rotaviruses, hantaviruses, coronavirus,
togavirus, flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus,
bunyavirus, arenavirus, reovirus, papilomavirus, parvovirus,
poxvirus, hepadnavirus, or spongiform virus. In another aspect, the
at least one viral antigen comprise peptides from at least one of
HIV, CMV, hepatitis A, B, and C, influenza; measles, polio,
smallpox, rubella; respiratory syncytial, herpes simplex, varicella
zoster, Epstein-Barr, Japanese encephalitis, rabies, flu, or cold
viruses.
[0025] In one aspect, the Ag is selected from:
TABLE-US-00005 Nef (66-97): (SEQ ID NO.: 1)
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL; Nef (116-145): (SEQ ID NO.: 2)
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL; Gag p17 (17-35): (SEQ ID NO.: 3)
EKIRLRPGGKKKYKLKHIV; Gag p17-p24 (253-284): (SEQ ID NO.: 4)
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD; and/or Pol 325-355 (RT 158-188)
is: (SEQ ID NO.: 5) AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY.
In another aspect, the Ag is 19 to 32 residues and is selected from
a cytotoxic T lymphocyte (CTL) epitope identified in the HIV-1 Nef,
Gag and Env proteins presented in the context of MHC-class I
molecules. In another aspect, the Ag is selected from HIV gp120,
gp41, Gag, p17, p24, p2, p7, p1, p6, Tat, Rev, PR, RT, IN, Vif,
Vpr, Vpx, Vpu and Nef. In another aspect, x comprises 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. In another
aspect, the Ag comprises two or more viral antigens from different
viruses. In another aspect, PL comprises an alanine and a serine.
In another aspect, the vaccine is selected from SEQ ID NOS.: 21,
22, 23, 24, 25, 26 or 36. In another aspect, the Ab comprises SEQ
ID NOS.: 37 and 38. In another aspect, the Ag is selected from at
least one of SEQ ID NOS.: 52-56, 58-60, 61-69, 70-72, or 73-77. In
another aspect, the Ag is 17 to 60 residues. In another aspect, the
Ag is 8, 10, 12, 14, 15, 16, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55
to 60 residues long. In another aspect, the Ag is 8, 10, 12, 14,
15, 16, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 to 60 residues long.
In another aspect, the Ag comprise a lipopeptide. In another
aspect, the Ag is at the carboxy-terminus and comprises a
carboxy-terminus (Palm)-NH.sub.2 group. In another aspect, the PL
is selected from:
TABLE-US-00006 (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP; (SEQ ID
NO.: 12) PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
[0026] Yet another embodiment of the present invention is a method
of making HIV peptide-specific IFN' producing T cells comprising:
immunizing a subject with a fusion protein comprising an anti-CD40
antibody, or fragment thereof, with one or more HIV peptides at the
carboxy-terminus of the antibody; and isolating peripheral blood
mononuclear cells from the subject, wherein the isolated peripheral
mononuclear cells are enriched for anti-HIV IFN' producing T cells,
wherein the anti-CD40 antibody comprises SEQ ID NOS.: 37 and 38 or
fragments thereof. In one aspect, the subject is a patient
suspected of having an HIV infection. In another aspect, the fusion
protein comprises two or more HIV peptides and the peptides are
separated by one or more peptide linkers. In another aspect, the
fusion protein comprises two or more HIV peptides and the peptides
are separated by the one or more peptide linkers comprise
glycosylation sequences. In another aspect, the fusion protein
comprises two or more HIV peptides and the peptides are separated
by one or more peptide linkers comprising an alanine and a serine.
In another aspect, the one or more HIV peptides comprise at least
one lipopeptide. In another aspect, the one or more HIV peptides
comprise a carboxy-terminus (Palm)-NH.sub.2 group. In another
aspect, the one or more HIV peptides are 19- to 32-amino-acid long
and are selected from a cytotoxic T lymphocyte (CTL) epitopes
identified in the HIV-1 Nef, Gag and Env proteins in the context of
different MHC-class I molecules. In another aspect, the one or more
HIV peptides are selected from HIV gp120, gp41, Gag, p17, p24, p2,
p'7, p1, p6, Tat, Rev, PR, RT, IN, Vif, Vpr, Vpx, Vpu and Nef. In
another aspect, the one or more viral peptides are selected from at
least one of:
TABLE-US-00007 Nef (66-97): (SEQ ID NO.: 1)
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL; Nef (116-145): (SEQ ID NO.: 2)
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL; Gag p17 (17-35): (SEQ ID NO.: 3)
EKIRLRPGGKKKYKLKHIV; Gag p17-p24 (253-284): (SEQ ID NO.: 4)
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD; and/or Pol 325-355 (RT 158-188)
is: (SEQ ID NO.: 5) AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY.
[0027] Yet another embodiment of the present invention is a fusion
protein comprising an anti-CD40 antibody, or fragment thereof, with
one or more viral peptides at the carboxy-terminus of the antibody
separated by a PL comprising at least one alanine and one serine.
In one aspect, the one or more viral peptides are HIV peptides. In
another aspect, the one or more viral peptides are selected from at
least one of:
TABLE-US-00008 Nef (66-97): (SEQ ID NO.: 1)
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL; Nef (116-145): (SEQ ID NO.: 2)
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL; Gag p17 (17-35): (SEQ ID NO.: 3)
EKIRLRPGGKKKYKLKHIV; Gag p17-p24 (253-284): (SEQ ID NO.: 4)
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD; and/or Pol 325-355 (RT 158-188)
is: (SEQ ID NO.: 5) AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY.
[0028] The present invention also includes a method of making a
fusion protein comprising: inserting into an expression vector a
nucleic acid construct comprising polynucleotides that encode a
protein having the formula: Ab-(PL-Ag)x or Ab-(Ag-PL)x; wherein Ab
is an antibody or fragment thereof; PL is at least one peptide
linker comprising at least one glycosylation site; Ag is at least
one viral antigen; and x is an integer from 1 to 20; and culturing
the vector under conditions sufficient to permit expression of the
fusion protein. In one aspect, the fusion protein has more
stability in solution than an identical fusion protein without the
glycosylation site. In another aspect, the at least one viral
antigen comprise peptides from an adenovirus, retrovirus,
picornavirus, herpesvirus, rotaviruses, hantaviruses, coronavirus,
togavirus, flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus,
bunyavirus, arenavirus, reovirus, papilomavirus, parvovirus,
poxvirus, hepadnavirus, or spongiform virus. In another aspect, the
at least one viral antigen comprise peptides from at least one of
HIV, CMV, hepatitis A, B, and C, influenza; measles, polio,
smallpox, rubella, respiratory syncytial, herpes simplex, varicella
zoster, Epstein-Barr, Japanese encephalitis, rabies, flu, or cold
viruses. In another aspect, the fusion protein is the Ab's light
chain, the Ab's heavy chain or both the Ab's light and heavy
chains. In another aspect, the Ag is selected from:
TABLE-US-00009 Nef (66-97): (SEQ ID NO.: 1)
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL; Nef (116-145): (SEQ ID NO.: 2)
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL; Gag p17 (17-35): (SEQ ID NO.: 3)
EKIRLRPGGKKKYKLKHIV; Gag p17-p24 (253-284): (SEQ ID NO.: 4)
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD; and/or Pol 325-355 (RT 158-188)
is: (SEQ ID NO.: 5) AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY.
[0029] Yet another embodiment of the present invention includes a
method of expanding antigen-specific T cells in vitro comprising:
isolating PBMCs from an HIV patient; incubating the isolated PBMCs
with an effective amount of a .alpha.CD40.LIPO5 HIV peptide
vaccine; expanding the PBMCs in the presence of an effective amount
of IL-2; harvesting the cells; and assessing the cytokine
production by the cells to determine the presence of anti-HIV
specific T cells. Another embodiment is an HIV antigen-specific T
cells made by the method comprising: isolating PBMCs from an HIV
patient; incubating the isolated PBMCs with an effective amount of
a .alpha.CD40.LIPO5 HIV peptide vaccine; expanding the PBMCs in the
presence of an effective amount of IL-2; harvesting the cells; and
assessing the cytokine production by the cells to determine the
presence of anti-HIV specific T cells. Another embodiment is a
method of making a therapeutic vaccine comprising: loading a
dendritic cell with .alpha.CD40.LIPO5 HIV peptide vaccine
comprising: isolating HIV patient monocytes; differentiating the
monocytes into dendritic cells with IFN.alpha. and GM-CSF; and
exposing the differentiated dendritic cells to an .alpha.CD40.LIPO5
HIV peptide, wherein the loaded dendritic cells are capable of
stimulating autologous HIV-peptide specific T cells in vitro.
[0030] The present invention also includes a therapeutic vaccine
made by the method comprising: loading a dendritic cell with
.alpha.CD40.LIPO5 HIV peptide vaccine comprising: isolating HIV
patient monocytes; differentiating the monocytes into dendritic
cells with IFN.alpha. and GM-CSF; and exposing the differentiated
dendritic cells to an .alpha.CD40.LIPO5 HIV peptide, wherein the
loaded dendritic cells are capable of stimulating autologous
HIV-peptide specific T cells in vitro. Another embodiment is a
therapeutic vaccine comprising a polypeptide comprising at least
one of SEQ ID NOS.: 21, 22, 23, 24, 25, 26 or 36. Yet another
embodiment is a therapeutic vaccine comprising a fusion protein
comprising the formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x;
Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an
antibody or fragment thereof; PL is at least one peptide linker
comprising at least one glycosylation site; Ag is at least one
viral antigen; and x is an integer from 1 to 20.
[0031] Yet another embodiment of the present invention includes a
fusion protein comprising the formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x;
Ab-(PL-Ag-PL)x; Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag;
wherein Ab is an antibody or fragment thereof; PL is at least one
peptide linker comprising at least one glycosylation site; Ag is at
least one cancer antigen; and x is an integer from 1 to 20. In one
aspect, the fusion protein has more stability in solution than the
same fusion protein without the glycosylation site. In another
aspect, the Ag is selected from tumor associated antigens selected
from CEA, prostate specific antigen (PSA), HER-2/neu, BAGE, GAGE,
MAGE 1-4, 6 and 12, MUC-related protein (Mucin) (MUC-1, MUC-2,
etc.), GM2 and GD2 gangliosides, ras, myc, tyrosinase, MART
(melanoma antigen), MARCO-MART, cyclin B1, cyclin D, Pmel
17(gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase
V intron V sequence), Prostate Ca psm, prostate serum antigen
(PSA), PRAME (melanoma antigen), .beta.-catenin, MUM-1-B (melanoma
ubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE
(melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Ban Virus
nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7,
p53, lung resistance protein (LRP), Bcl-2, and Ki-67. In another
aspect, the Ag is selected from tumor associated antigens
comprising antigens from leukemias and lymphomas, neurological
tumors such as astrocytomas or glioblastomas, melanoma, breast
cancer, lung cancer, head and neck cancer, gastrointestinal tumors,
gastric cancer, colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such cervix, uterus, ovarian cancer, vaginal
cancer, testicular cancer, prostate cancer or penile cancer, bone
tumors, vascular tumors, or cancers of the lip, nasopharynx,
pharynx and oral cavity, esophagus, rectum, gall bladder, biliary
tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease,
non-Hodgkin's lymphoma, multiple myeloma and leukemia.
[0032] In another aspect, the Ag is selected from at least one
of:
TABLE-US-00010 (SEQ ID NO.: 74)
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC GGVLVHPQWV; (SEQ
ID NO.: 75) LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRF
LRPGDDSSHD; (SEQ ID NO.: 76)
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKK LQCVDLHVIS; (SEQ
ID NO.: 77) NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWG
SEPCALPERP; or (SEQ ID NO.: 78) SLYTKVVHYRKWIKDTIVANP,
and fragments thereof. In another aspect, the Ag is selected from
at least one of
TABLE-US-00011 (SEQ ID NO.: 113) IMDQVPFSV; (SEQ ID NO.: 114)
ITDQVPFSV; (SEQ ID NO.: 115) YLEPGPVTV; (SEQ ID NO.: 116)
YLEPGPVTA; (SEQ ID NO.: 117) KTWGQYWQV; (SEQ ID NO.: 122)
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQ
RLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVN
NTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTW
GQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPLAHSSSA
FTITDQVPFSVSVSQLRALDGGNKHFLRNQ; (SEQ ID NO.: 124)
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGPV
TAQVVLQAAIPLTSCGSSPVPAS; (SEQ ID NO.: 126)
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTE
VISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSI VVLSGTTAA; (SEQ
ID NO.: 128) QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRL
VKRQVPLDCVLYRYGSFSVTLDIVQ; and (SEQ ID NO.: 130)
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLC
QPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIVPGILL TGQEAGLGQ,
and fragments thereof.
[0033] In another aspect, the Ag is selected from at least one
of:
TABLE-US-00012 (SEQ ID NO.: 132)
MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLD Y; and (SEQ ID
NO.: 133) DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK.
In another aspect, the Ag is selected from at least one of:
TABLE-US-00013 (SEQ ID NO.: 141)
MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV; (SEQ ID NO.: 142)
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSR
LQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELL; (SEQ ID NO.: 143)
LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDV KFISNPPSMV; and
(SEQ ID NO.: 144)
AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEA
LLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI,
and fragments thereof. In another aspect, the Ag is 19 to 32 amino
acids long. In another aspect, the Ag is 17 to 60 amino acids long
and is selected from a cytotoxic T lymphocyte (CTL) epitope
identified in PSA or cyclin 1. In another aspect, x comprises 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In
another aspect, the Ag comprises two or more cancer peptides from
different cancer antigens separated by the PL. In another aspect,
the Ag is separated by at least one PL comprising an alanine and a
serine. In another aspect, the Ag is selected from SEQ ID NOS.:
74-78, 79-86, 87-92, 93-95, 113-117, 122-130, 132-133, and 141-144.
In another aspect, the Ab comprises SEQ ID NOS.: 38 and 39. In
another aspect, the Ab is expressed by a nucleic acid expression
vector comprising SEQ ID NOS.: 40 and 41. In another aspect, the PL
is selected from:
TABLE-US-00014 (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP; (SEQ ID
NO.: 12) PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
In another aspect, the PL comprises an alanine and a serine.
[0034] Yet another embodiment of the present invention includes a
antigen delivery vector that expresses an anti-CD40 antibody or
fragment thereof and two or more cancer peptides at the
carboxy-terminus of the light chain, the heavy chain or both the
light and heavy chains of the anti-CD40 antibody, wherein when two
or more cancer peptides are present, the cancer peptides are
separated by the one or more peptide linkers that comprise at least
one glycosylation site. In one aspect, the one or more peptide
linkers are selected from:
TABLE-US-00015 (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP; (SEQ ID
NO.: 12) PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
[0035] Yet another embodiment of the present invention includes an
anti-CD40 fusion protein comprising an anti-CD40 antibody or
fragment thereof and one or more cancer peptides at the
carboxy-terminus of the anti-CD40 antibody, wherein when two or
more cancer peptides are present the cancer peptides are separated
by the one or more linker peptides that comprise at least one
glycosylation site. In one aspect, the antibody fragment is
selected from an Fv, Fab, Fab', F(ab').sub.2, Fc, or a ScFv
fragment. In another aspect, the Ag is selected from SEQ ID NOS.:
74-78, 79-86, 87-92, 93-95, 113-117, 122-130, 132-133, and
141-144.
[0036] Yet another embodiment of the present invention includes a
method of stabilizing cancer peptides comprising: incorporating one
or more cancer peptides that are unstable or insoluble into a
fusion protein with an antibody, wherein the antibody and the
cancer peptides are separated by one or more peptide linkers that
comprise one or more glycosylation sites. In another aspect, the
fusion protein comprises two or more cancer peptides and the cancer
peptides are separated by the one or more peptide linkers. In
another aspect, the fusion protein comprises two or more cancer
peptides and the peptides are separated by the one or more peptide
linkers. In another aspect, the fusion protein comprises two or
more cancer peptides and the peptides are separated by one or more
linkers comprising an alanine and a serine. In another aspect, the
cancer peptide is selected from tumor associated antigens selected
from CEA, prostate specific antigen (PSA), HER-2/neu, BAGS, GAGE,
MAGE 1-4, 6 and 12, MUC-related protein (Mucin) (MUC-1, MUC-2,
etc.), GM2 and GD2 gangliosides, ras, myc, tyrosinase, MART
(melanoma antigen), MARCO-MART, cyclin B1, cyclin D, Pmel
17(gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase
V intron V sequence), Prostate Ca psm, prostate serum antigen
(PSA), PRAME (melanoma antigen), .beta.-catenin, MUM-1-B (melanoma
ubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE
(melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr
Virus nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6
and E7, p53, lung resistance protein (LRP), Bcl-2, and Ki-67. In
another aspect, the Ag is selected from tumor associated antigens
comprising antigens from leukemias and lymphomas, neurological
tumors such as astrocytomas or glioblastomas, melanoma, breast
cancer, lung cancer, head and neck cancer, gastrointestinal tumors,
gastric cancer, colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such cervix, uterus, ovarian cancer, vaginal
cancer, testicular cancer, prostate cancer or penile cancer, bone
tumors, vascular tumors, or cancers of the lip, nasopharynx,
pharynx and oral cavity, esophagus, rectum, gall bladder, biliary
tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease,
non-Hodgkin's lymphoma, multiple myeloma and leukemia.
[0037] In another aspect, the Ag is selected from at least one
of:
TABLE-US-00016 (SEQ ID NO.: 74)
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC GGVLVHPQWV; (SEQ
ID NO.: 75) LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRF
LRPGDDSSHD; (SEQ ID NO.: 76)
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKK LQCVDLHVIS; (SEQ
ID NO.: 77) NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWG
SEPCALPERP; or (SEQ ID NO.: 78) SLYTKVVHYRKWIKDTIVANP.
[0038] In another aspect, the Ag is selected from at least one
of:
TABLE-US-00017 (SEQ ID NO.: 113) IMDQVPFSV; (SEQ ID NO.: 114)
ITDQVPFSV; (SEQ ID NO.: 115) YLEPGPVTV; (SEQ ID NO.: 116)
YLEPGPVTA; (SEQ ID NO.: 117) KTWGQYWQV; (SEQ ID NO.: 122)
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQ
RLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVN
NTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTW
GQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPLAHSSSA
FTITDQVPFSVSVSQLRALDGGNKHFLRNQ; (SEQ ID NO.: 124)
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGPV
TAQVVLQAAIPLTSCGSSPVPAS; (SEQ ID NO.: 126)
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTE
VISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSI VVLSGTTAA; (SEQ
ID NO.: 128) QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRL
VKRQVPLDCVLYRYGSFSVTLDIVQ; and (SEQ ID NO.: 130)
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLC
QPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIVPGILL TGQEAGLGQ,
and fragments thereof.
[0039] In another aspect, the Ag is selected from at least one
of:
TABLE-US-00018 (SEQ ID NO.: 132)
MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDY; and (SEQ ID
NO.: 133) DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK.
[0040] In another aspect, the Ag is selected from at least one
of:
TABLE-US-00019 (SEQ ID NO.: 141)
MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV; (SEQ ID NO.: 142)
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKS
RLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELL; (SEQ ID NO.: 143)
LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDV KFISNPPSMV; and
(SEQ ID NO.: 144)
AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEA
LLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI,
and fragments thereof. In another aspect, the Ag is 19 to 32 amino
acids long. In another aspect, the Ag is 17 to 60 amino acids long
and is selected from a cytotoxic T lymphocyte (CTL) epitope
identified in PSA or cyclin 1. In another aspect, x comprises 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In
another aspect, the fusion protein comprises cancer peptides from
different antigens separated by different peptide linkers. In
another aspect, the fusion protein comprises two or more cancer
peptides separated by one or more peptide linkers comprising an
alanine and a serine. In another aspect, the antibody comprises SEQ
ID NOS.: 38 and 39. In another aspect, the fusion protein is
expressed by a nucleic acid expression vector comprising SEQ ID
NOS.: 40 and 41. In another aspect, the peptide linker is selected
from:
TABLE-US-00020 (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP; (SEQ ID
NO.: 12) PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
[0041] Yet another embodiment of the present invention includes a
method of enhancing T cell responses comprising: immunizing a
subject in need of vaccination with an effective amount of a
vaccine comprising a fusion protein comprising an anti-CD40
antibody or portion thereof and one or more cancer peptides linked
to the carboxy-terminus of the anti-CD40 antibody. In another
aspect, the cancer peptides are selected from tumor associated
antigens selected from CEA, prostate specific antigen (PSA),
HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12, MUC (Mucin) (e.g.,
MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc,
tyrosinase, MART (melanoma antigen), MARCO-MART, cyclin B1, cyclin
D, Pmel 17(gp100), GnT-V intron V sequence
(N-acetylglucoaminyltransferase V intron V sequence), Prostate Ca
psm, prostate serum antigen (PSA), PRAME (melanoma antigen),
.beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene product),
GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10, c-ERB2
(Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6, gp75,
human papilloma virus (HPV) E6 and E7, p53, lung resistance protein
(LRP), Bcl-2, and Ki-67. In another aspect, the cancer peptides is
selected from tumor associated antigens comprising antigens from
leukemias and lymphomas, neurological tumors such as astrocytomas
or glioblastomas, melanoma, breast cancer, lung cancer, head and
neck cancer, gastrointestinal tumors, gastric cancer, colon cancer,
liver cancer, pancreatic cancer, genitourinary tumors such cervix,
uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate
cancer or penile cancer, bone tumors, vascular tumors, or cancers
of the lip, nasopharynx, pharynx and oral cavity, esophagus,
rectum, gall bladder, biliary tree, larynx, lung and bronchus,
bladder, kidney, brain and other parts of the nervous system,
thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple
myeloma and leukemia.
[0042] Yet another embodiment of the present invention includes a
method of making an anti-CD40-antigen fusion protein comprising:
expressing a fusion protein comprising an anti-CD40 antibody or
fragment thereof in a host cell, the fusion protein comprising one
or more cancer peptides at the carboxy-terminus of the anti-CD40
antibody or fragment thereof, wherein when two or more cancer
peptides are separated by one or more linkers, at least one linker
comprising a glycosylation site; and isolating the fusion protein.
In another aspect, the fusion protein expressed in the host is
further isolated and purified. In another aspect, the host is a
eukaryotic cell. In another aspect, the cancer peptides are
selected from tumor associated antigens selected from CEA, prostate
specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12,
MUC-related protein (Mucin) (MUC-1, MUC-2, etc.), GM2 and GD2
gangliosides, ras, myc, tyrosinase, MART (melanoma antigen),
MARCO-MART, cyclin B1, cyclin D, Pmel 17(gp100), GnT-V intron V
sequence (N-acetylglucoaminyltransferase V intron V sequence),
Prostate Ca psm, prostate serum antigen (PSA), PRAME (melanoma
antigen), .beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene
product), GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10,
c-ERB2 (Her2/neu), EBNA (Epstein-Ban Virus nuclear antigen) 1-6,
gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance
protein (LRP), Bcl-2, and Ki-67. In another aspect, the cancer
peptides are selected from tumor associated antigens comprising
antigens from leukemias and lymphomas, neurological tumors such as
astrocytomas or glioblastomas, melanoma, breast cancer, lung
cancer, head and neck cancer, gastrointestinal tumors, gastric
cancer, colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such cervix, uterus, ovarian cancer, vaginal
cancer, testicular cancer, prostate cancer or penile cancer, bone
tumors, vascular tumors, or cancers of the lip, nasopharynx,
pharynx and oral cavity, esophagus, rectum, gall bladder, biliary
tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease,
non-Hodgkin's lymphoma, multiple myeloma and leukemia. In another
aspect, the cancer peptides are selected from at least one of:
TABLE-US-00021 (SEQ ID NO.: 74)
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC GGVLVHPQWV; (SEQ
ID NO.: 75) LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRF
LRPGDDSSHD; (SEQ ID NO.: 76)
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKK LQCVDLHVIS; (SEQ
ID NO.: 77) NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWG
SEPCALPERP; or (SEQ ID NO.: 78) SLYTKVVHYRKWIKDTIVANP.
[0043] In another aspect, the cancer peptides are selected from at
least one of:
TABLE-US-00022 (SEQ ID NO.: 113) IMDQVPFSV; (SEQ ID NO.: 114)
ITDQVPFSV; (SEQ ID NO.: 115) YLEPGPVTV; (SEQ ID NO.: 116)
YLEPGPVTA; (SEQ ID NO.: 117) KTWGQYWQV; (SEQ ID NO.: 122)
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQ
RLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVN
NTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTW
GQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPLAHSSSA
FTITDQVPFSVSVSQLRALDGGNKHFLRNQ; (SEQ ID NO.: 124)
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGPV
TAQVVLQAAIPLTSCGSSPVPAS; (SEQ ID NO.: 126)
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTE
VISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSI VVLSGTTAA; (SEQ
ID NO.: 128) QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRL
VKRQVPLDCVLYRYGSFSVTLDIVQ; and (SEQ ID NO.: 130)
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLC
QPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIVPGILL TGQEAGLGQ,
and fragments thereof.
[0044] In another aspect, the cancer peptides are selected from at
least one of:
TABLE-US-00023 (SEQ ID NO.: 132)
MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDY; and (SEQ ID
NO.: 133) DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK.
[0045] In another aspect, the cancer peptides are selected from at
least one of:
TABLE-US-00024 (SEQ ID NO.: 141)
MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV; (SEQ ID NO.: 142)
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKS
RLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELL; (SEQ ID NO.: 143)
LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATD VKFISNPPSMV; and
(SEQ ID NO.: 144) AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIE
ALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI,
and fragments thereof.
[0046] Yet another embodiment of the present invention includes a
method of expanding antigen-specific T cells in vitro comprising:
isolating peripheral blood mononuclear cells (PBMCs) from a cancer
patient; incubating the isolated PBMCs with an immunogenic amount
of an .alpha.CD40-(PL-Ag)x or .alpha.CD40-(Ag-PL)x vaccine, wherein
Ag is a tumor associated antigen and x is an integer 1 to 20;
expanding the PBMCs in the presence of an effective amount of IL-2;
harvesting the cells; and assessing the cytokine production by the
cells to determine the presence of anti-cancer specific T
cells.
[0047] Yet another embodiment of the present invention includes a
tumor associated antigen-specific T cells made by the method
comprising: isolating peripheral blood mononuclear cells (PBMCs)
from a cancer patient; incubating the isolated PBMCs with an
immunogenic amount of an .alpha.CD40-(PL-Ag)x or
.alpha.CD40-(Ag-PL)x vaccine, wherein Ag is a tumor associated
antigen and x is an integer 1 to 20; expanding the PBMCs in the
presence of an effective amount of IL-2; harvesting the cells; and
assessing the cytokine production by the cells to determine the
presence of tumor associated antigen-specific T cells.
[0048] Yet another embodiment of the present invention includes a
therapeutic vaccine comprising a fusion protein comprising the
formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x; Ab-(Ag-PL-Ag)x;
Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an antibody or
fragment thereof; PL is at least one peptide linker comprising at
least one glycosylation site; Ag is at least one cancer antigen;
and x is an integer from 1 to 20.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] For a more complete understanding of the features and
advantages of the present invention, reference is now made to the
detailed description of the invention along with the accompanying
figures and in which:
[0050] FIG. 1 shows protein A affinity recombinant antibodies fused
to various HIV peptides (lanes 1 to 5) secreted from transfected
293F cells, analyzed by reducing SDS.PAGE and Coomassie Brilliant
Blue staining.
[0051] FIG. 2 shows protein A affinity purified recombinant
antibodies fused to various HIV peptides (Lanes 1 and 2) secreted
from transfected 293F cells, then analyzed by reducing SDS.PAGE and
Coomassie Brilliant Blue staining.
[0052] FIG. 3 shows protein A affinity purified recombinant
antibodies fused to various HIV peptide strings (Lanes 1 to 5)
secreted from transfected 293F cells, then analyzed by reducing
SDS.PAGE and Coomassie Brilliant Blue staining.
[0053] FIG. 4 shows protein A affinity purified recombinant
antibodies fused to various HIV peptide strings (Lanes 1 to 6)
secreted from transfected 293F cells, then analyzed by reducing
SDS.PAGE and Coomassie Brilliant Blue staining.
[0054] FIG. 5 describes the protocol used in vitro to assay the
potency of .alpha.CD40.LIPO5 HIV peptide fusion recombinant
antibody (.alpha.CD40.LIPO5 rAb) to elicit the expansion of
antigen-specific T cells in the context of a PBMC culture.
[0055] FIG. 6A-C shows HIV peptide-specific IFN' production in
PBMCs from HIV patients incubated with various concentrations of
anti-CD40.LIPO5 peptide string vaccine. C is the control group,
which received no vaccine, and defines the baseline response of the
culture to each peptide.
[0056] FIG. 7 is a summary of .alpha.CD40.LIPO5 peptide vaccine
responses against the 5 peptide regions from 8 HIV patients.
[0057] FIG. 8A-B shows that the .alpha.CD40.LIPO5 HIV peptide
vaccine elicits expansion of HIV peptide-specific T cells capable
of secreting multiple cytokines--a desirable feature in a vaccine.
FIG. 8A-B also shows that the .alpha.CD40.LIPO5 HIV peptide vaccine
elicits gag253, nef66, nef116 and pol325 peptide-specific responses
characterized by production of multiple cytokines (patient A5).
[0058] FIG. 9 shows the protocol for testing .alpha.CD40.LIPO5 HIV
peptide vaccine for its ability to direct the expansion of
antigen-specific T cells resulting from targeted uptake by DCs and
presentation of peptide epitopes on their surface MHC complex.
[0059] FIG. 10A-B shows the cytokine secretion in response to HIV
peptides from DC-T cell co-cultures treated with various doses of
.alpha.CD40.LIPO5 HIV peptide vaccine (patient A10).
[0060] FIG. 11A-B shows PBMCs from patient A4 treated with the
.alpha.CD40.LIPO5 HIV peptide vaccine elicit expansion of
antigen-specific T cells with specificity to the gag253 region, but
not to the flexible linker sequences.
[0061] FIG. 12A is the .alpha.CD40.LIPO5 HIV peptide vaccine heavy
chain sequence showing flexible linker regions in bold, joining
sequences underlined and HIV peptide regions shaded in grey. FIG.
12A shows PBMCs from patient A3 treated with the .alpha.CD40.LIPO5
HIV peptide vaccine elicit expansion of antigen-specific T cells
with specificities to the gag253, nef66, and nef116 regions, but
not to the flexible linker sequences. FIGS. 12B-1 and 12-B2 shows
HIV antigen-specific T cell responses evoked from HIV patient A17
PBMCs incubated with 30 nM of three different HIV5 peptide DC
targeting vaccines. FIGS. 12C-1 and 12-C2 is a similar study to
that show in FIGS. 12B-1 and 12-B2, except that the PBMCs are from
a different HIV patient (A2). FIG. 12D shows 15 different HIV
peptide responses [5 peptide regions sampled in 3 patients], it was
found that the anti-CD40.HIV5pep vaccine was superior to
anti-DCIR.HIV5pep, anti-LOX-1.HIV5pep and non-LIPO5 mix for
eliciting a broad range of HIV peptide-specific CD8+ and CD4+ T
responses.
[0062] FIG. 13 shows the internalization of anti-CD40 mAb:IL-4DC.
IL-4DCs were treated with 500 ng/ml of anti-CD40-Alexa 568.
[0063] FIG. 14 shows CD4 and CD8 T cell proliferation by DCs
targeted with anti-CD40-HA1. 5.times.10e3 IFNDCs loaded with 2
ug/ml of anti-CD40-HA or control Ig-HA1 were co-cultured with
CFSE-labeled autologous CD4+ or CD8+ T cells (2.times.10e5) for 7
days. Cells were then stained with anti-CD4 or anti-CD8 antibodies.
Cell proliferation was tested by measuring CFSE-dilution.
[0064] FIG. 15 shows a titration of HA1 fusion protein on CD4+ T
proliferation. IFNDCs (5K) loaded with fusion proteins were
co-cultured with CFSE-labeled CD4+ T cells (200K) for 7 days.
[0065] FIG. 16 shows IFNDCs targeted with anti-CD40-HA1 activate
HA1-specific CD4+ T cells. CD4+ T cells were re-stimulated with DCs
loaded with 5 uM of indicated peptides, and then intracellular
IFN.gamma. was stained.
[0066] FIG. 17 shows IFNDCs targeted with anti-CD40-HA1 activate
HA1-specific CD4+ T cells. CD4+ T cells were re-stimulated with DCs
loaded with indicated peptides for 36 h, and then culture
supernatant was analyzed for measuring IFN.gamma..
[0067] FIG. 18 shows that targeting CD40 results in enhanced
cross-priming of MART-1 specific CD8+ T cells. IFNDCs (5K/well)
loaded with fusion proteins were co-cultured with purified CD8+ T
cells for 10 days. Cells were stained with anti-CD8 and tetramer.
Cells are from healthy donors (HLA-A*0201+).
[0068] FIG. 19 shows targeting CD40 results in enhanced
cross-priming of MART-1 specific CD8+ T cells (Summary of
8-repeated experiments using cells from different healthy
donors).
[0069] FIG. 20 shows CD8+ CTL induced with IFNDCs targeted with
anti-CD40-MART-1 are functional. CD8+ T cells co-cultured with
IFNDCs targeted with fusion proteins were mixed with T2 cells
loaded with 10 uM peptide epitope.
[0070] FIG. 21 shows CD8+ CTL induced with IFNDCs targeted with
anti-CD40-Flu M1 are functional. CD8+ T cells co-cultured with
IFNDCs targeted with fusion proteins were mixed with T2 cells
loaded with 1.0 nM peptide epitope.
[0071] FIG. 22 shows an outline of protocol to test the ability a
vaccine composed of anti-CD4012E12 linked to PSA (prostate specific
antigen) to elicit the expansion from a naive T cell population.
PSA-specific CD4+ T cells corresponding to a broad array of PSA
epitopes. Briefly, DCs derived by culture with IFN.alpha. and
GM-CSF of monocytes from a healthy donor are incubated with the
vaccine. The next day, cells are placed in fresh medium and pure
CD4+ T cells from the same donor are added. Several days later, PSA
peptides are added and, after four hours, secreted gamma-IFN levels
in the culture supernatants are determined.
[0072] FIG. 23 shows that many PSA peptides elicit potent
gamma-IFN-production responses indicating that anti-CD4012E12 and
similar anti-CD40 agents can efficiently deliver antigen to DCs,
resulting in the priming of immune responses against multiple
epitopes of the antigen.
[0073] FIG. 24 shows DCs targeted with anti-CD40-PSA induce
PSA-specific CD8+ T cell responses. IFNDCs were targeted with 1 ug
mAb fusion protein with PSA. Purified autologous CD8+ T cells were
co-cultured for 10 days. Cells were stained with anti-CD8 and PSA
(KLQCVDLHV)-tetramer. Cells are from a HLA-A*0201 positive healthy
donor. The results demonstrate that anti-CD40 effectively deliver
PSA to the DCs, which in turn elicit the expansion of PSA-specific
CD8+ T cells.
[0074] FIG. 25 a scheme (left) and the IFN' production by T cells
of the pools of peptides and control for Donor 2. 5.times.10e3
IFNDCs loaded with 2 ug/ml of anti-CD40-Cyclin D1 were co-cultured
with purified autologous CD4+ T cells (2.times.10e5) for 8 days.
Cells were then re-stimulated with 5 uM of individual peptides
derived from CyclinD1 for 5 h in the presence of Brefeldin A. Cells
were stained for measuring intracellular IFN.gamma. expression.
[0075] FIG. 26 shows a peptide scan and IFN.gamma. production by T
cells obtained from the pools of peptides shown in FIG. 25 and
control for Donor 2. 5.times.10e3 IFNDCs loaded with 2 ug/ml of
anti-CD40-Cyclin D1 were co-cultured with purified autologous CD4+
T cells (2.times.10e5) for 8 days. Cells were then re-stimulated
with 5 uM of individual peptides derived from CyclinD1 for 5 h in
the presence of Brefeldin A. Cells were stained for measuring
intracellular IFN.gamma. expression.
[0076] FIG. 27 shows the expression and construct design for
anti-CD40-MART-1 peptide antibodies.
[0077] FIG. 28 is a summary of the CD4.sup.+ and CD8.sup.+
immunodominant epitopes for MART-1.
[0078] FIG. 29 shows the expression and construct design for
anti-CD40-gp100 peptide antibodies.
[0079] FIG. 30 shows the design for additional anti-CD40-gp100
peptide antibodies.
[0080] FIG. 31 shows the expression and construct design for
additional anti-CD40-gp100 peptide antibodies.
[0081] FIG. 32 is a summary of the CD4.sup.+ and CD8.sup.+
immunodominant epitopes for gp100.
[0082] FIG. 33 shows the expression and construct design for
additional anti-CD40-gp100 peptide antibodies.
[0083] FIG. 34A shows that full-length Cyclin B1 fused to the
C-terminus of either antibody H chain or cohesion fail to be
secreted from mammalian 293F cells. FIG. 34B shows that full-length
Cyclin B1 fused to the C-terminus of either antibody H chain or
cohesion fail to be secreted from mammalian 293F cells.
[0084] FIG. 35 shows Cyclin B1 segmentation strategy based on known
or predicted structural domain regions.
[0085] FIG. 36 shows that Cyclin D1 segments p1, p3, and p4, but
not p2 express well as direct fusions to the H chain
C-terminus.
[0086] FIG. 37 shows the relative expression levels of various
Cyclin D1 segments as direct fusions to the H chain C-terminus in
various combinations with flexible linker sequences.
[0087] FIG. 38 show a summary of various H chain-Cyclin D1 segment
constructs and their relative expressibility as vaccines.
[0088] FIG. 39 shows that full-length Cyclin D1 fused to the
C-terminus of a DC targeting antibody H chain is very poorly
expressed as a secreted recombinant antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0089] While the making and using of various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts that can be embodied in a wide variety of
specific contexts. The specific embodiments discussed herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention.
[0090] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity, but
include the general class of which a specific example may be used
for illustration. The terminology herein is used to describe
specific embodiments of the invention, but their usage does not
delimit the invention, except as outlined in the claims.
[0091] The invention includes also variants and other modification
of an antibody (or "Ab") of fragments thereof, e.g., anti-CD40
fusion protein (antibody is used interchangeably with the term
"immunoglobulin"). As used herein, the term "antibodies or
fragments thereof," includes whole antibodies or fragments of an
antibody, e.g., Fv, Fab, Fab', F(ab').sub.2, Fc, and single chain
Fv fragments (ScFv) or any biologically effective fragments of an
immunoglobulins that binds specifically to, e.g., CD40. Antibodies
from human origin or humanized antibodies have lowered or no
immunogenicity in humans and have a lower number or no immunogenic
epitopes compared to non-human antibodies. Antibodies and their
fragments will generally be selected to have a reduced level or no
antigenicity in humans.
[0092] As used herein, the terms "Ag" or "antigen" refer to a
substance capable of either binding to an antigen binding region of
an immunoglobulin molecule or of eliciting an immune response,
e.g., a T cell-mediated immune response by the presentation of the
antigen on Major Histocompatibility Antigen (MHC) cellular
proteins. As used herein, "antigen" includes, but is not limited
to, antigenic determinants, haptens, and immunogens which may be
peptides, small molecules, carbohydrates, lipids, nucleic acids or
combinations thereof. The skilled immunologist will recognize that
when discussing antigens that are processed for presentation to T
cells, the term "antigen" refers to those portions of the antigen
(e.g., a peptide fragment) that is a T cell epitope presented by
MHC to the T cell receptor. When used in the context of a B cell
mediated immune response in the form of an antibody that is
specific for an "antigen", the portion of the antigen that binds to
the complementarity determining regions of the variable domains of
the antibody (light and heavy) the bound portion may be a linear or
three-dimensional epitope. In the context of the present invention,
the term antigen is used on both contexts, that is, the antibody is
specific for a protein antigen (CD40), but also carries one or more
peptide epitopes for presentation by MHC to T cells. In certain
cases, the antigens delivered by the vaccine or fusion protein of
the present invention are internalized and processed by antigen
presenting cells prior to presentation, e.g., by cleavage of one or
more portions of the antibody or fusion protein.
[0093] As used herein, the term "antigenic peptide" refers to that
portion of a polypeptide antigen that is specifically recognized by
either B-cells or T-cells. B-cells respond to foreign antigenic
determinants via antibody production, whereas T-lymphocytes are the
mediate cellular immunity. Thus, antigenic peptides are those parts
of an antigen that are recognized by antibodies, or in the context
of an MHC, by T-cell receptors.
[0094] As used herein, the term "epitope" refers to any protein
determinant capable of specific binding to an immunoglobulin or of
being presented by a Major Histocompatibility Complex (MHC) protein
(e.g., Class I or Class II) to a T-cell receptor. Epitopic
determinants are generally short peptides 5-30 amino acids long
that fit within the groove of the MHC molecule that presents
certain amino acid side groups toward the T cell receptor and has
certain other residues in the groove, e.g., due to specific charge
characteristics of the groove, the peptide side groups and the T
cell receptor. Generally, an antibody specifically binds to an
antigen when the dissociation constant is 1 mM, 100 nM or even 10
nM.
[0095] As used herein, the term "vector" is used in two different
contexts. When using the term "vector" with reference to a vaccine,
a vector is used to describe a non-antigenic portion that is used
to direct or deliver the antigenic portion of the vaccine. For
example, an antibody or fragments thereof may be bound to or form a
fusion protein with the antigen that elicits the immune response.
For cellular vaccines, the vector for delivery and/or presentation
of the antigen is the antigen presenting cell, which is delivered
by the cell that is loaded with antigen. In certain cases, the
cellular vector itself may also process and present the antigen(s)
to T cells and activate an antigen-specific immune response. When
used in the context of nucleic acids, a "vector" refers a construct
which is capable of delivering, and preferably expressing, one or
more genes or polynucleotide sequences of interest in a host cell.
Examples of vectors include, but are not limited to, viral vectors,
naked DNA or RNA expression vectors, DNA or RNA expression vectors
associated with cationic condensing agents, DNA or RNA expression
vectors encapsulated in liposomes, and certain eukaryotic cells,
such as producer cells.
[0096] The compositions and methods of the present invention can be
used with a wide variety of peptides and/or protein in which the
antibody or fragment thereof and the peptide linker or "PL" create
a protein that is stable and/or soluble.
[0097] As used herein, the compositions and methods use an antigen
delivery vector comprising the formula: Ab-(PL-Ag)x or Ab-(Ag-PL)x;
wherein Ab is an antibody or fragment thereof; PL is at least one
peptide linker comprising at least one glycosylation site; Ag is at
least one viral antigen; and x is an integer from 1 to 20. One
example of an antibody for use with the present invention comprises
at least the variable region of anti-CD40.sub.--12E12.3F3 (American
Type Culture Collection (ATCC) 10801 University Boulevard Manassas,
Va. 20110-2209 USA Accession No. PTA-9854, Deposited Feb. 26,
2009), anti-CD40.sub.--12B4.2C10 (ATCC Submission Deposit No.
HS446, ATCC Accession No. PTA-10653, Deposited Feb. 17, 2010), and
anti-CD40.sub.--11B6.1C3 (ATCC Submission Deposit No. HS440, ATCC
Accession No. PTA-10652, Deposited Feb. 17, 2010).
[0098] As used herein, the terms "stable" and "unstable" when
referring to proteins is used to describe a peptide or protein that
maintains its three-dimensional structure and/or activity (stable)
or that loses immediately or over time its three-dimensional
structure and/or activity (unstable). As used herein, the term
"insoluble" refers to those proteins that when produced in a cell
(e.g., a recombinant protein expressed in a eukaryotic or
prokaryotic cell or in vitro) are not soluble in solution absent
the use of denaturing conditions or agents (e.g., heat or chemical
denaturants, respectively). The antibody or fragment thereof and
the linkers taught herein have been found to convert antibody
fusion proteins with the peptides from insoluble and/or unstable
into proteins that are stable and/or soluble. Another example of
stability versus instability is when the domain of the protein with
a stable conformation has a higher melting temperature (T.sub.m)
than the unstable domain of the protein when measured in the same
solution. A domain is stable compared to another domain when the
difference in the T.sub.m is at least about 2.degree. C., more
preferably about 4.degree. C., still more preferably about
7.degree. C., yet more preferably about 10.degree. C., even more
preferably about 15.degree. C., still more preferably about
20.degree. C., even still more preferably about 25.degree. C., and
most preferably about 30.degree. C., when measured in the same
solution.
[0099] As used herein, "polynucleotide" or "nucleic acid" refers to
a strand of deoxyribonucleotides or ribonucleotides in either a
single- or a double-stranded form (including known analogs of
natural nucleotides). A double-stranded nucleic acid sequence will
include the complementary sequence. The polynucleotide sequence may
encode variable and/or constant region domains of immunoglobulin
that are formed into a fusion protein with one or more linkers. For
use with the present invention, multiple cloning sites (MCS) may be
engineered into the locations at the carboxy-terminal end of the
heavy and/or light chains of the antibodies to allow for in-frame
insertion of peptide for expression between the linkers. As used
herein, the term "isolated polynucleotide" refers to a
polynucleotide of genomic, cDNA, or synthetic origin or some
combination thereof. By virtue of its origin the "isolated
polynucleotide" (1) is not associated with all or a portion of a
polynucleotide in which the "isolated polynucleotides" are found in
nature, (2) is operably linked to a polynucleotide which it is not
linked to in nature, or (3) does not occur in nature as part of a
larger sequence. The skilled artisan will recognize that to design
and implement a vector having the formula Ab-(PL-Ag)x or
Ab-(Ag-PL)x, can be manipulated at the nucleic acid level by using
techniques known in the art, such as those taught in Current
Protocols in Molecular Biology, 2007 by John Wiley and Sons,
relevant portions incorporated herein by reference. Briefly, the
Ab, Ag and PL encoding nucleic acid sequences can be inserted using
polymerase chain reaction, enzymatic insertion of oligonucleotides
or polymerase chain reaction fragments in a vector, which may be an
expression vector. To facilitate the insertion of (PL-Ag)x or
(Ag-PL)x at the carboxy terminus of the antibody light chain, the
heavy chain, or both, a multiple cloning site (MCS) may be
engineered in sequence with the antibody sequences.
[0100] As used herein, the term "polypeptide" refers to a polymer
of amino acids and does not refer to a specific length of the
product; thus, peptides, oligopeptides, and proteins are included
within the definition of polypeptide. This term also does not refer
to or exclude post expression modifications of the polypeptide, for
example, glycosylations, acetylations, phosphorylations and the
like. Included within the definition are, for example, polypeptides
containing one or more analogs of an amino acid (including, for
example, unnatural amino acids, etc.), polypeptides with
substituted linkages, as well as other modifications known in the
art, both naturally occurring and non-naturally occurring. The term
"domain," or "polypeptide domain" refers to that sequence of a
polypeptide that folds into a single globular region in its native
conformation, and that may exhibit discrete binding or functional
properties.
[0101] A polypeptide or amino acid sequence "derived from" a
designated nucleic acid sequence refers to a polypeptide having an
amino acid sequence identical to that of a polypeptide encoded in
the sequence, or a portion thereof wherein the portion consists of
at least 3-5 amino acids, preferably at least 4-7 amino acids, more
preferably at least 8-10 amino acids, and even more preferably at
least 11-15 amino acids, or which is immunologically identifiable
with a polypeptide encoded in the sequence. This terminology also
includes a polypeptide expressed from a designated nucleic acid
sequence.
[0102] As used herein, "pharmaceutically acceptable carrier" refers
to any material that when combined with an immunoglobulin (Ig)
fusion protein of the present invention allows the Ig to retain
biological activity and is generally non-reactive with the
subject's immune system. Examples include, but are not limited to,
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as an oil/water emulsion,
and various types of wetting agents. Certain diluents may be used
with the present invention, e.g., for aerosol or parenteral
administration, that may be phosphate buffered saline or normal
(0.85%) saline.
[0103] The invention provides an CD40 binding molecule comprising
at least one immunoglobulin light chain variable domain (V.sub.L)
which comprises in sequence hypervariable regions CDR1.sub.L,
CDR2.sub.L and CDR3.sub.L, the CDR1.sub.L having the amino acid
sequence SASQGISNYLN (SEQ ID NO.:41) the CDR2.sub.L having the
amino acid sequence YTSILHS (SEQ ID NO.:42) and the CDR3.sub.L
having the amino acid sequence QQFNKLPPT (SEQ ID NO.:43) the amino
acid sequences of which are shown in SEQ ID NO. 37; and direct
equivalent thereof.
[0104] Accordingly the invention provides an CD40 binding molecule
which comprises an antigen binding site comprising at least one
immunoglobulin heavy chain variable domain (V.sub.H) which
comprises in sequence hypervariable regions CDR1.sub.H, CDR2.sub.H
and CDR3.sub.H, the CDR1.sub.H having the amino acid sequence
GFTFSDYYMY (SEQ ID NO.:44), the CDR2.sub.H having the amino acid
sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:45), and the CDR3.sub.H
having the amino acid sequence RGLPFHAMDY (SEQ ID NO.:46), the
amino acid sequences of which are shown in SEQ ID NO. 38; and
direct equivalents thereof.
[0105] In one aspect the invention provides a single domain CD40
binding molecule comprising an isolated immunoglobulin light chain
comprising a heavy chain variable domain (V.sub.L) as defined
above. In another aspect the invention provides a single domain
CD40 binding molecule comprising an isolated immunoglobulin heavy
chain comprising a heavy chain variable domain (V.sub.H) as defined
above.
[0106] In another aspect the invention also provides an CD40
binding molecule comprising both heavy (V.sub.H) and light chain
(V.sub.L) variable domains in which the CD40 binding molecule
comprises at least one antigen binding site comprising: a) an
immunoglobulin heavy chain variable domain (V.sub.L) which
comprises in sequence hypervariable regions CDR1.sub.L, CDR2.sub.L
and CDR3.sub.L, the CDR1.sub.L having the amino acid sequence
SASQGISNYLN (SEQ ID NO.:41), the CDR2.sub.L having the amino acid
sequence YTSILHS (SEQ ID NO.:42), and the CDR3.sub.L having the
amino acid sequence QQFNKLPPT (SEQ ID NO.:43), the amino acid
sequences of which are shown in SEQ ID. NO. 1, and b) an
immunoglobulin light chain variable domain (V.sub.H) which
comprises in sequence hypervariable regions CDR1.sub.H, CDR2.sub.H
and CDR3.sub.H, the CDR1.sub.H having the amino acid sequence
GFTFSDYYMY (SEQ ID NO.:44), the CDR2' having the amino acid
sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:45), and the CDR3.sub.H
having the amino acid sequence RGLPFHAMDY (SEQ ID NO.:46), the
amino acid sequences of which are shown in SEQ ID NO. 38; and
direct equivalents thereof.
[0107] Unless otherwise indicated, any polypeptide chain is herein
described as having an amino acid sequence starting at the
N-terminal end and ending at the C-terminal end. When the antigen
binding site comprises both the V.sub.H and V.sub.L domains, these
may be located on the same polypeptide molecule or, preferably,
each domain may be on a different chain, the V.sub.H domain being
part of an immunoglobulin heavy chain or fragment thereof and the
V.sub.L being part of an immunoglobulin light chain or fragment
thereof.
[0108] Non-limiting examples for antigens targeted by the
antibodies of the present invention include, but are not limited
to: cell surface marker selected from MHC class I, MHC class II,
CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD20, CD29,
CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86,
CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO,
DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2,
IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fc.gamma. receptor,
T cell receptors, lectins, or other immune cell receptors. In one
specific example, the antigens that are targeted by the antibody
portion of the present invention are specifically expressed by
antigen presenting cells, e.g., dendritic cells, Langerhans cells,
macrophages, and B cells.
[0109] As used herein, the term "CD40 binding molecule" refers to
any molecule capable of binding to the CD40 antigen either alone or
associated with other molecules having one or more the V.sub.L and
V.sub.H CDRs taught herein, in some cases 2, 3, 4, 5, or all 6
CDRs. The binding reaction may be shown by standard methods
(qualitative assays) including, for example, a bioassay for
determining by blocking the binding of other molecules to CD40 or
any kind of binding or activity assays (e.g., activation, reduction
or modulation of an immune response), with reference to a negative
control test in which an antibody of unrelated specificity but of
the same isotype, e.g., an anti-CD25 or anti-CD80 antibody, is
used.
[0110] The present invention may also be made into a single chain
antibody having the variable domains of the heavy and light chains
of an antibody covalently bound by a peptide linker usually
including from 10 to 30 amino acids, preferably from 15 to 25 amino
acids. Therefore, such a structure does not include the constant
part of the heavy and light chains and it is believed that the
small peptide spacer should be less antigenic than a whole constant
part.
[0111] As used herein, the term "chimeric antibody" refers to an
antibody in which the constant regions of heavy or light chains or
both are of human origin while the variable domains of both heavy
and light chains are of non-human (e.g., mouse, hamster or rat)
origin or of human origin but derived from a different human
antibody.
[0112] As used herein, the term "CDR-grafted antibody" refers to an
antibody in which the hypervariable complementarity determining
regions (CDRs) are derived from a donor antibody, such as a
non-human (e.g., mouse) antibody or a different human antibody,
while all or substantially all the other parts of the
immunoglobulin (e.g., the conserved regions of the variable
domains, i.e., framework regions), are derived from an acceptor
antibody (in the case of a humanized antibody--an antibody of human
origin). A CDR-grafted antibody may include a few amino acids of
the donor sequence in the framework regions, for instance in the
parts of the framework regions adjacent to the hypervariable
regions.
[0113] As used herein, the term "human antibody" refers to an
antibody in which the constant and variable regions of both the
heavy and light chains are all of human origin, or substantially
identical to sequences of human origin, not necessarily from the
same antibody and includes antibodies produced by mice in which the
mouse, hamster or rat immunoglobulin variable and constant part
genes have been replaced by their human counterparts, e.g. as
described in general terms in EP 0546073 B1, U.S. Pat. No.
5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S.
Pat. No. 5,633,425, U.S. Pat. No. 5,661,016, U.S. Pat. No.
5,770,429, EP 0 438474 B1 and EP 0 463151 B1, relevant portions
incorporated herein by reference.
[0114] The CD40 binding molecule of the invention can be a
humanized antibody that comprises the CDRs obtained from the
anti-CD40.sub.--12E12.3F3, the anti-CD40.sub.--12E12.3F3, or the
anti-CD40.sub.--12B4.2C10 antibodies. One example of a chimeric
antibody includes the variable domains of both heavy and light
chains are of human origin, for instance those variable domains of
the anti-CD40.sub.--12E12.3F3 antibody that are part of SEQ ID NO.:
148 and SEQ ID NO.: 149, anti-CD40.sub.--12B4.2C10 in SEQ ID NO.:
150 and SEQ ID NO.: 151 or SEQ ID NO.: 152, and/or
anti-CD40.sub.--11B6.1C3, SEQ ID NO.: 153 and SEQ ID NO.: 154, or
combination thereof. The constant region domains preferably also
comprise suitable human constant region domains, for instance as
described in "Sequences of Proteins of Immunological Interest",
Kabat E. A. et al, US Department of Health and Human Services,
Public Health Service, National Institute of Health. The nucleic
acid sequences can be found in, e.g., SEQ ID NOS.: 8 and 9.
[0115] Hypervariable regions may be associated with any kind of
framework regions, e.g., of human origin. Suitable framework
regions were described Kabat E. A. One heavy chain framework is a
heavy chain framework, for instance that of
anti-CD40.sub.--12E12.3F3 antibody that are part of SEQ ID NO.:
149; anti-CD40.sub.--12B4.2C10--SEQ ID NO.: 151 or SEQ ID NO.: 152,
and/or anti-CD40.sub.--11B6.1C3--SEQ ID NO.: 154, or combination
thereof, e.g., FR1.sub.L, FR2.sub.L, FR3.sub.L and FR4.sub.L
regions. In a similar manner, SEQ ID NO. 148 shows the
anti-CD40.sub.--12E12.3F3 (or the equivalents for
anti-CD40.sub.--12B4.2C10 and anti-CD40.sub.--11B6.1C3, SEQ ID
NOS.: 150 and 153, respectively) heavy chain framework that
includes the sequence of FR1.sub.H, FR2.sub.H, FR3.sub.H and
FR4.sub.H regions. The CDRs may be added to a human antibody
framework, such as those described in U.S. Pat. No. 7,456,260,
issued to Rybak, et al., which teach new human variable chain
framework regions and humanized antibodies comprising the framework
regions, relevant portions and framework sequences incorporated
herein by reference. To accomplish the engraftment at a genetic
level, the present invention also includes the underlying nucleic
acid sequences for the V.sub.L AND V.sub.H regions as well as the
complete antibodies and the humanized versions thereof. The nucleic
acid sequences of the present invention include SEQ ID NOS.: 155
and 156, which are the anti-CD40 antibody light and the heavy
chains, respectively, as well as those nucleic acid sequences that
include variable codon usage for the same amino acid sequences and
conservative variations thereof having 85, 90, 95 or 100% sequence
identity at the nucleic or amino acid level. Likewise, the CDRs may
have 85, 90, 95 or 100% sequence identity at the nucleic or amino
acid level, individually, in groups or 2, 3, 4 or 5 or all
together.
[0116] Monoclonal antibodies raised against a protein naturally
found in all humans are typically developed in a non-human system
e.g. in mice, and as such are typically non-human proteins. As a
direct consequence of this, a xenogenic antibody as produced by a
hybridoma, when administered to humans, elicits an undesirable
immune response that is predominantly mediated by the constant part
of the xenogenic immunoglobulin. Xenogeneic antibodies tend to
elicit a host immune response, thereby limiting the use of such
antibodies as they cannot be administered over a prolonged period
of time. Therefore, it is particularly useful to use single chain,
single domain, chimeric, CDR-grafted, or especially human
antibodies that are not likely to elicit a substantial allogenic
response when administered to humans. The present invention
includes antibodies with minor changes in an amino acid sequence
such as deletion, addition or substitution of one, a few or even
several amino acids which are merely allelic forms of the original
protein having substantially identical properties.
[0117] The inhibition of the binding of CD40 to its receptor may be
conveniently tested in various assays including such assays are
described hereinafter in the text. By the term "to the same extent"
is meant that the reference and the equivalent molecules exhibit,
on a statistical basis, essentially identical CD40 binding
inhibition curves in one of the assays referred to above. For
example, the assay used may be an assay of competitive inhibition
of binding of CD40 by the binding molecules of the invention.
[0118] Among various uses of the immunoconjugates of the invention
are included a variety of disease conditions caused by specific
human cells that may be eliminated by the toxic action of the
fusion protein. For example, for the humanized
anti-CD40.sub.--12E12.3F3 (ATCC Accession No. PTA-9854),
anti-CD40.sub.--12B4.2C10 (ATCC SubmissionAccession No. HS446,
AccessionSubmission No. PTA-10653AB13-22.12B4.2C10 (HS446)), and
anti-CD40.sub.--11B6.1C3 (ATCC SubmissionAccession No. HS440,
AccessionSubmission No. PTA-10652AB13-22.11B6.1C3 (HS440)),
antibodies disclosed herein, one preferred application for
immunoconjugates is the treatment of malignant cells expressing
CD40.
[0119] A CD40 binding molecule of the invention may be produced by
recombinant DNA techniques. In view of this, one or more DNA
molecules encoding the binding molecule must be constructed, placed
under appropriate control sequences and transferred into a suitable
host organism for expression.
[0120] In a very general manner, there are accordingly provided:
(i) DNA molecules encoding a single domain CD40 binding molecule of
the invention, a single chain CD40 binding molecule of the
invention, a heavy or light chain or fragments thereof of a CD40
binding molecule of the invention; and (ii) the use of the DNA
molecules of the invention for the production of a CD40 binding
molecule of the invention by recombinant methods.
[0121] The present state of the art is such that the skilled worker
in the art can synthesize the DNA molecules of the invention given
the information provided herein, i.e., the amino acid sequences of
the hypervariable regions and the DNA sequences coding for them. A
method for constructing a variable domain gene is for example
described in EPA 239 400, relevant portions incorporated herein by
reference. Briefly, a gene encoding a variable domain of a MAb is
cloned. The DNA segments encoding the framework and hypervariable
regions are determined and the DNA segments encoding the
hypervariable regions are removed so that the DNA segments encoding
the framework regions are fused together with suitable restriction
sites at the junctions. The restriction sites may be generated at
the appropriate positions by mutagenesis of the DNA molecule by
standard procedures. Double stranded synthetic CDR cassettes are
prepared by DNA synthesis according to the sequences given in SEQ
ID NO. 1 and 3 or 2 and 4 (amino acid and nucleic acid sequences,
respectively). These cassettes are often provided with sticky ends
so that they can be ligated at the junctions of the framework.
[0122] It is not necessary to have access to the mRNA from a
producing hybridoma cell line in order to obtain a DNA construct
coding for the CD40 binding molecules of the invention. For
example, PCT application WO 90/07861 gives full instructions for
the production of an antibody by recombinant DNA techniques given
only written information as to the nucleotide sequence of the gene,
relevant portions incorporated herein by reference. Briefly, the
method comprises the synthesis of a number of oligonucleotides,
their amplification by the PCR method, and their splicing to give
the desired DNA sequence.
[0123] Expression vectors comprising a suitable promoter or genes
encoding heavy and light chain constant parts are publicly
available. Thus, once a DNA molecule of the invention is prepared
it may be conveniently transferred in an appropriate expression
vector. DNA molecules encoding single chain antibodies may also be
prepared by standard methods, for example, as described in WO
88/1649. In view of the foregoing, no hybridoma or cell line
deposit is necessary to comply with the criteria of sufficiency of
description.
[0124] For example, first and second DNA constructs are made that
bind specifically to CD40. Briefly, a first DNA construct encodes a
light chain or fragment thereof and comprises a) a first part which
encodes a variable domain comprising alternatively framework and
hypervariable regions, the hypervariable regions being in sequence
CDR1.sub.L, CDR2.sub.L and CDR3.sub.L the amino acid sequences of
which are shown in SEQ ID NO. 1; this first part starting with a
codon encoding the first amino acid of the variable domain and
ending with a codon encoding the last amino acid of the variable
domain, and b) a second part encoding a light chain constant part
or fragment thereof which starts with a codon encoding the first
amino acid of the constant part of the heavy chain and ends with a
codon encoding the last amino acid of the constant part or fragment
thereof, followed by a stop codon.
[0125] The first part encodes a variable domain having an amino
acid sequence substantially identical to the amino acid sequence as
shown in SEQ ID NO. 1. A second part encodes the constant part of a
human heavy chain, more preferably the constant part of the human
.gamma.1 chain. This second part may be a DNA fragment of genomic
origin (comprising introns) or a cDNA fragment (without
introns).
[0126] The second DNA construct encodes a heavy chain or fragment
thereof and comprises a) a first part which encodes a variable
domain comprising alternatively framework and hypervariable
regions; the hypervariable regions being CDR1.sub.H and optionally
CDR2.sub.H and CDR3.sub.H, the amino acid sequences of which are
shown in SEQ ID NO. 2; this first part starting with a codon
encoding the first amino acid of the variable domain and ending
with a codon encoding the last amino acid of the variable domain,
and b) a second part encoding a heavy chain constant part or
fragment thereof which starts with a codon encoding the first amino
acid of the constant part of the light chain and ends with a codon
encoding the last amino acid of the constant part or fragment
thereof followed by a stop codon.
[0127] The first part encodes a variable domain having an amino
acid sequence substantially identical to the amino acid sequence as
shown in SEQ ID NO. 2. The first part has the nucleotide sequence
as shown in SEQ ID NO. 2 starting with the nucleotide at position 1
and ending with the nucleotide at position 321. Also preferably the
second part encodes the constant part of a human light chain, more
preferably the constant part of the human .kappa. chain.
[0128] The invention also includes CD40 binding molecules in which
one or more of the residues of CDR1.sub.L, CDR2.sub.L, CDR3.sub.L,
CDR1.sub.H, CDR2.sub.H or CDR3.sub.H or the frameworks, typically
only a few (e.g. FR1-4.sub.L or .sub.H), are changed from the
residues shown in SEQ ID NO. 37 and SEQ ID NO. 38; by, e.g., site
directed mutagenesis of the corresponding DNA sequences. The
invention includes the DNA sequences coding for such changed CD40
binding molecules. In particular the invention includes a CD40
binding molecules in which one or more residues of CDR1.sub.L,
CDR2.sub.L and/or CDR3.sub.L have been changed from the residues
shown in SEQ ID NO. 37 and one or more residues of CDR1.sub.H,
CDR2.sub.H and/or CDR3.sub.H have been changed from the residues
shown in SEQ ID NO. 38.
[0129] Each of the DNA constructs are placed under the control of
suitable control sequences, in particular under the control of a
suitable promoter. Any kind of promoter may be used, provided that
it is adapted to the host organism in which the DNA constructs will
be transferred for expression. However, if expression is to take
place in a mammalian cell, an immunoglobulin gene promoter may be
used in B cells. The first and second parts may be separated by an
intron, and, an enhancer may be conveniently located in the intron
between the first and second parts. The presence of such an
enhancer that is transcribed but not translated, may assist in
efficient transcription. In particular embodiments the first and
second DNA constructs comprise the enhancer of, e.g., a heavy chain
human gene.
[0130] The desired antibody may be produced in a cell culture or in
a transgenic animal. A suitable transgenic animal may be obtained
according to standard methods that include micro injecting into
eggs the first and second DNA constructs placed under suitable
control sequences transferring the so prepared eggs into
appropriate pseudo-pregnant females and selecting a descendant
expressing the desired antibody.
[0131] The invention also provides an expression vector able to
replicate in a prokaryotic or eukaryotic cell line, which comprises
at least one of the DNA constructs above described. Each expression
vector containing a DNA construct is then transferred into a
suitable host organism. When the DNA constructs are separately
inserted on two expression vectors, they may be transferred
separately, i.e. one type of vector per cell, or co-transferred,
this latter possibility being preferred. A suitable host organism
may be a bacterium, a yeast or a mammalian cell line, this latter
being preferred. More preferably, the mammalian cell line is of
lymphoid origin, e.g., a myeloma, hybridoma or a normal
immortalized B-cell, which conveniently does not express any
endogenous antibody heavy or light chain.
[0132] When the antibody chains are produced in a cell culture, the
DNA constructs must first be inserted into either a single
expression vector or into two separate but compatible expression
vectors, the latter possibility being preferred. For expression in
mammalian cells it is preferred that the coding sequence of the
CD40 binding molecule is integrated into the host cell DNA within a
locus which permits or favors high level expression of the CD40
binding molecule.
[0133] In a further aspect of the invention there is provided a
process for the product of a CD40 binding molecule that comprises:
(i) culturing an organism which is transformed with an expression
vector as defined above; and (ii) recovering the CD40 binding
molecule from the culture.
[0134] In accordance with the present invention it has been found
that the anti-CD40.sub.--12E12.3F3, anti-CD40.sub.--12B4.2C10
and/or anti-CD40.sub.--11B6.1C3 antibody appears to have binding
specificity for the antigenic epitope of human CD40. It is
therefore most surprising that antibodies to this epitope, e.g. the
anti-CD40.sub.--12E12.3F3 antibody, are capable of delivering
antigen efficiently into dendritic cells (DCs). Antibodies, in
particular chimeric and CDR-grafted antibodies and especially human
antibodies, which have binding specificity for the antigenic
epitope of mature human CD40; and use of such antibodies for DC
antigen loading are novel and are included within the scope of the
present invention.
[0135] To use the anti-CD40 antibody of the present invention for
treatment indications, the appropriate dosage will, of course, vary
depending upon, for example, the antibody disclosed herein to be
employed, the host, the mode of administration and the nature and
severity of the condition being treated. However, in prophylactic
use, satisfactory results are generally found at dosages from about
0.05 mg to about 10 mg per kilogram body weight more usually from
about 0.1 mg to about 5 mg per kilogram body weight. The frequency
of dosing for prophylactic uses will normally be in the range from
about once per week up to about once every 3 months, more usually
in the range from about once every 2 weeks up to about once every
10 weeks, e.g., once every 4 to 8 weeks. The anti-CD40 antibody of
the present can be administered parenterally, intravenously, e.g.,
into the antecubital or other peripheral vein, intramuscularly, or
subcutaneously.
[0136] Pharmaceutical compositions of the invention may be
manufactured in conventional manner, e.g., in a lyophilized form.
For immediate administration it is dissolved in a suitable aqueous
carrier, for example sterile water for injection or sterile
buffered physiological saline. If it is considered desirable to
make up a solution of larger volume for administration by infusion
rather as a bolus injection, it is advantageous to incorporate
human serum albumin or the patient's own heparinized blood into the
saline at the time of formulation. The presence of an excess of
such physiologically inert protein prevents loss of antibody by
adsorption onto the walls of the container and tubing used with the
infusion solution. If albumin is used, a suitable concentration is
from 0.5 to 4.5% by weight of the saline solution.
[0137] One embodiment of the present invention provides an
immunoconjugate comprising a humanized antibody of the invention,
e.g., a humanized anti-CD40 antibody, linked to one or more
effector molecules, antigen(s) and/or a detectable label(s).
Preferably, the effector molecule is a therapeutic molecule such
as, for example, one or more peptides that comprise one or more T
cell epitopes, a toxin, a small molecule, a cytokine or a
chemokine, an enzyme, or a radiolabel.
[0138] Exemplary toxins include, but are not limited to,
Pseudomonas exotoxin or diphtheria toxin. Examples of small
molecules include, but are not limited to, chemotherapeutic
compounds such as taxol, doxorubicin, etoposide, and bleiomycin.
Exemplary cytokines include, but are not limited to, IL-1, IL-2,
IL-4, IL-5, IL-6, and IL-12, IL-17, and IL-25. Exemplary enzymes
include, but are not limited to, RNAses, DNAses, proteases,
kinases, and caspases. Exemplary radioisotopes include, but are not
limited to, .sup.32P and .sup.125I.
[0139] As used herein, the term "epitope" refers to a molecule or
substance capable of stimulating an immune response. In one
example, epitopes include but are not limited to a polypeptide and
a nucleic acid encoding a polypeptide, wherein expression of the
nucleic acid into a polypeptide is capable of stimulating an immune
response when the polypeptide is processed and presented on a Major
Histocompatibility Complex (MHC) molecule. Generally, epitopes
include peptides presented on the surface of cells non-covalently
bound to the binding groove of Class I or Class II MHC, such that
they can interact with T cell receptors and the respective T cell
accessory molecules.
[0140] Proteolytic Processing of Antigens. Epitopes that are
displayed by MHC on antigen presenting cells are cleavage peptides
or products of larger peptide or protein antigen precursors. For
MHC I epitopes, protein antigens are often digested by proteasomes
resident in the cell. Intracellular proteasomal digestion produces
peptide fragments of about 3 to 23 amino acids in length that are
then loaded onto the MHC protein. Additional proteolytic activities
within the cell, or in the extracellular milieu, can trim and
process these fragments further. Processing of MHC Class II
epitopes generally occurs via intracellular proteases from the
lysosomal/endosomal compartment. The present invention includes, in
one embodiment, pre-processed peptides that are attached to the
anti-CD40 antibody (or fragment thereof) that directs the peptides
against which an enhanced immune response is sought directly to
antigen presenting cells.
[0141] To identify epitopes potentially effective as immunogenic
compounds, predictions of MHC binding alone are useful but often
insufficient. The present invention includes methods for
specifically identifying the epitopes within antigens most likely
to lead to the immune response sought for the specific sources of
antigen presenting cells and responder T cells.
[0142] The present invention allows for a rapid and easy assay for
the identification of those epitopes that are most likely to
produce the desired immune response using the patient's own antigen
presenting cells and T cell repertoire. The compositions and
methods of the present invention are applicable to any protein
sequence, allowing the user to identify the epitopes that are
capable of binding to MHC and are properly presented to T cells
that will respond to the antigen. Accordingly, the invention is not
limited to any particular target or medical condition, but instead
encompasses and MHC epitope(s) from any useful source.
[0143] As used herein, the term "veneered" refers to a humanized
antibody framework onto which antigen-binding sites or CDRs
obtained from non-human antibodies (e.g., mouse, rat or hamster),
are placed into human heavy and light chain conserved structural
framework regions (FRs), for example, in a light chain or heavy
chain polynucleotide to "graft" the specificity of the non-human
antibody into a human framework. The polynucleotide expression
vector or vectors that express the veneered antibodies can be
transfected mammalian cells for the expression of recombinant human
antibodies which exhibit the antigen specificity of the non-human
antibody and will undergo posttranslational modifications that will
enhance their expression, stability, solubility, or combinations
thereof.
[0144] Antigens.
[0145] Examples of viral antigens for use with the present
invention include, but are not limited to, e.g., HIV, HCV, CMV,
adenoviruses, retroviruses, picornaviruses, etc. Non-limiting
example of retroviral antigens such as retroviral antigens from the
human immunodeficiency virus (HIV) antigens such as gene products
of the gag, pol, and env genes, the Nef protein, reverse
transcriptase, and other HIV components; hepatitis viral antigens
such as the S, M, and L proteins of hepatitis B virus, the pre-S
antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis
A, B, and C, viral components such as hepatitis C viral RNA;
influenza viral antigens such as hemagglutinin and neuraminidase
and other influenza viral components; measles viral antigens such
as the measles virus fusion protein and other measles virus
components; rubella viral antigens such as proteins E1 and E2 and
other rubella virus components; rotaviral antigens such as VP7sc
and other rotaviral components; cytomegaloviral antigens such as
envelope glycoprotein B and other cytomegaloviral antigen
components; respiratory syncytial viral antigens such as the RSV
fusion protein, the M2 protein and other respiratory syncytial
viral antigen components; herpes simplex viral antigens such as
immediate early proteins, glycoprotein D, and other herpes simplex
viral antigen components; varicella zoster viral antigens such as
gpI, gpII, and other varicella zoster viral antigen components;
Japanese encephalitis viral antigens such as proteins E, M-E,
M-E-NS1, NS1, NS1-NS2A, 80% E, and other Japanese encephalitis
viral antigen components; rabies viral antigens such as rabies
glycoprotein, rabies nucleoprotein and other rabies viral antigen
components. See Fundamental Virology, Second Edition, eds. Fields,
B. N. and Knipe, D. M. (Raven Press, New York, 1991) for additional
examples of viral antigens. The at least one viral antigen may be
peptides from an adenovirus, retrovirus, picornavirus, herpesvirus,
rotaviruses, hantaviruses, coronavirus, togavirus, flavirvirus,
rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus, arenavirus,
reovirus, papilomavirus, parvovirus, poxvirus, hepadnavirus, or
spongiform virus. In certain specific, non-limiting examples, the
at least one viral antigen are peptides obtained from at least one
of HIV, CMV, hepatitis A, B, and C, influenza, measles, polio,
smallpox, rubella; respiratory syncytial, herpes simplex, varicella
zoster, Epstein-Barr, Japanese encephalitis, rabies, flu, and/or
cold viruses.
[0146] In one aspect, the one or more of the antigenic peptides are
selected from at least one of:
TABLE-US-00025 Nef (66-97): (SEQ ID NO.: 1)
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL; Nef (116-145): (SEQ ID NO.: 2)
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL; Gag p17 (17-35): (SEQ ID NO.: 3)
EKIRLRPGGKKKYKLKHIV; Gag p17-p24 (253-284): (SEQ ID NO.: 4)
NPPIPVGEIYKRWIIGLNKIVRMYSPTSILD; or Pol 325-355 (RT 158-188) is:
(SEQ ID NO.: 5) AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY.
In one aspect, the fusion protein peptides are separated by one or
more linkers selected from:
TABLE-US-00026 (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP; (SEQ ID
NO.: 12) PTSTPADSSTITPTATPTATPTIKG; (SEQ ID NO.: 13)
TVTPTATATPSAIVTTITPTATTKP; or (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA.
[0147] Antigenic targets that may be delivered using the
anti-CD40-antigen vaccines of the present invention include genes
encoding antigens such as viral antigens, bacterial antigens,
fungal antigens or parasitic antigens. Pathogens include
trypanosomes, tapeworms, roundworms, helminthes, malaria. Tumor
markers, such as fetal antigen or prostate specific antigen, may be
targeted in this manner. Other examples include: HIV env proteins
and hepatitis B surface antigen. Administration of a vector
according to the present invention for vaccination purposes would
require that the vector-associated antigens be sufficiently
non-immunogenic to enable long-term expression of the transgene,
for which a strong immune response would be desired. In some cases,
vaccination of an individual may only be required infrequently,
such as yearly or biennially, and provide long-term immunologic
protection against the infectious agent. Specific examples of
organisms, allergens and nucleic and amino sequences for use in
vectors and ultimately as antigens with the present invention may
be found in U.S. Pat. No. 6,541,011, relevant portions incorporated
herein by reference, in particular, the tables that match organisms
and specific sequences that may be used with the present
invention.
[0148] Bacterial antigens for use with the anti-CD40-antigen
vaccines disclosed herein include, but are not limited to, e.g.,
bacterial antigens such as pertussis toxin, filamentous
hemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase and other
pertussis bacterial antigen components; diptheria bacterial
antigens such as diptheria toxin or toxoid and other diptheria
bacterial antigen components; tetanus bacterial antigens such as
tetanus toxin or toxoid and other tetanus bacterial antigen
components; streptococcal bacterial antigens such as M proteins and
other streptococcal bacterial antigen components; gram-negative
bacilli bacterial antigens such as lipopolysaccharides and other
gram-negative bacterial antigen components, Mycobacterium
tuberculosis bacterial antigens such as mycolic acid, heat shock
protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A
and other mycobacterial antigen components; Helicobacter pylori
bacterial antigen components; pneumococcal bacterial antigens such
as pneumolysis, pneumococcal capsular polysaccharides and other
pneumococcal bacterial antigen components; haemophilus influenza
bacterial antigens such as capsular polysaccharides and other
haemophilus influenza bacterial antigen components; anthrax
bacterial antigens such as anthrax protective antigen and other
anthrax bacterial antigen components; rickettsiae bacterial
antigens such as rompA and other rickettsiae bacterial antigen
component. Also included with the bacterial antigens described
herein are any other bacterial, mycobacterial, mycoplasmal,
rickettsial, or chlamydial antigens. Partial or whole pathogens may
also be: haemophilus influenza; Plasmodium falciparum; neisseria
meningitidis; streptococcus pneumoniae; neisseria gonorrhoeae;
salmonella serotype typhi; shigella; vibrio cholerae; Dengue Fever;
Encephalitides; Japanese Encephalitis; lyme disease; Yersinia
pestis; west nile virus; yellow fever; tularemia; hepatitis (viral;
bacterial); RSV (respiratory syncytial virus); HPIV 1 and HPIV 3;
adenovirus; small pox; allergies and cancers.
[0149] Fungal antigens for use with compositions and methods of the
invention include, but are not limited to, e.g., candida fungal
antigen components; histoplasma fungal antigens such as heat shock
protein 60 (HSP60) and other histoplasma fungal antigen components;
cryptococcal fungal antigens such as capsular polysaccharides and
other cryptococcal fungal antigen components; coccidiodes fungal
antigens such as spherule antigens and other coccidiodes fungal
antigen components; and tinea fungal antigens such as trichophytin
and other coccidiodes fungal antigen components.
[0150] Examples of protozoal and other parasitic antigens include,
but are not limited to, e.g., plasmodium falciparum antigens such
as merozoite surface antigens, sporozoite surface antigens,
circumsporozoite antigens, gametocyte/gamete surface antigens,
blood-stage antigen pf 155/RESA and other plasmodial antigen
components; toxoplasma antigens such as SAG-1, p30 and other
toxoplasmal antigen components; schistosomae antigens such as
glutathione-S-transferase, paramyosin, and other schistosomal
antigen components; leishmania major and other leishmaniae antigens
such as gp63, lipophosphoglycan and its associated protein and
other leishmanial antigen components; and trypanosoma cruzi
antigens such as the 75-77 kDa antigen, the 56 kDa antigen and
other trypanosomal antigen components.
[0151] Antigen that can be targeted using the anti-CD40-antigen
vaccines of the present invention will generally be selected based
on a number of factors, including: likelihood of internalization,
level of immune cell specificity, type of immune cell targeted,
level of immune cell maturity and/or activation and the like. In
this embodiment, the antibodies may be mono- or bi-specific
antibodies that include one anti-CD40 binding domain and one
binding domain against a second antigen, e.g., cell surface markers
for dendritic cells such as, MHC class I, MHC Class II, B7-2, CD18,
CD29, CD31, CD43, CD44, CD45, CD54, CD58, CD83, CD86, CMRF-44,
CMRF-56, DCIR and/or Dectin-1 and the like; while in some cases
also having the absence of CD2, CD3, CD4, CD8, CD14, CD15, CD16,
CD19, CD20, CD56, and/or CD57. Examples of cell surface markers for
antigen presenting cells include, but are not limited to, MHC class
I, MHC Class II, CD45, B7-1, B7-2, IFN-.gamma. receptor and IL-2
receptor, ICAM-1 and/or Fc.gamma. receptor. Examples of cell
surface markers for T cells include, but are not limited to, CD3,
CD4, CD8, CD14, CD20, CD11b, CD16, CD45 and HLA-DR.
[0152] Target antigens on cell surfaces for delivery include those
characteristic of tumor antigens typically will be derived from the
cell surface, cytoplasm, nucleus, organelles and the like of cells
of tumor tissue. Examples of tumor targets for the antibody portion
of the present invention include, without limitation, hematological
cancers such as leukemias and lymphomas, neurological tumors such
as astrocytomas or glioblastomas, melanoma, breast cancer, lung
cancer, head and neck cancer, gastrointestinal tumors such as
gastric or colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such cervix, uterus, ovarian cancer, vaginal
cancer, testicular cancer, prostate cancer or penile cancer, bone
tumors, vascular tumors, or cancers of the lip, nasopharynx,
pharynx and oral cavity, esophagus, rectum, gall bladder, biliary
tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease,
non-Hodgkin's lymphoma, multiple myeloma and leukemia.
[0153] Examples of antigens that may be delivered alone or in
combination to immune cells for antigen presentation using the
present invention includes tumor proteins, e.g., mutated oncogenes;
viral proteins associated with tumors; and tumor mucins and
glycolipids. The antigens may be viral proteins associated with
tumors would be those from the classes of viruses noted above.
Certain antigens may be characteristic of tumors (one subset being
proteins not usually expressed by a tumor precursor cell), or may
be a protein that is normally expressed in a tumor precursor cell,
but having a mutation characteristic of a tumor. Other antigens
include mutant variant(s) of the normal protein having an altered
activity or subcellular distribution, e.g., mutations of genes
giving rise to tumor antigens.
[0154] Specific non-limiting examples of tumor antigens for use in
an anti-CD40-fusion protein vaccine include, e.g., CEA, prostate
specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12,
MUC (Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides,
ras, myc, tyrosinase, MART (melanoma antigen), Pmel 17(100), GnT-V
intron V sequence (N-acetylglucoaminyltransferase V intron V
sequence), Prostate Ca psm, PRAME (melanoma antigen),
.beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene product),
GAGE (melanoma antigen) 1, MAGE, BAGE (melanoma antigen) 2-10,
c-ERB2 (Her2/neu), DAGE, EBNA (Epstein-Barr Virus nuclear antigen)
1-6, gp75, human papilloma virus (HPV) E6 and E7, p53, lung
resistance protein (LRP), Bcl-2, Ki-67, Cyclin B1, gp100, Survivin,
and NYESO-1
[0155] In addition, the immunogenic molecule can be an autoantigen
involved in the initiation and/or propagation of an autoimmune
disease, the pathology of which is largely due to the activity of
antibodies specific for a molecule expressed by the relevant target
organ, tissue, or cells, e.g., SLE or MG. In such diseases, it can
be desirable to direct an ongoing antibody-mediated (i.e., a
Th2-type) immune response to the relevant autoantigen towards a
cellular (i.e., a Th1-type) immune response. Alternatively, it can
be desirable to prevent onset of or decrease the level of a Th2
response to the autoantigen in a subject not having, but who is
suspected of being susceptible to, the relevant autoimmune disease
by prophylactically inducing a Th1 response to the appropriate
autoantigen. Autoantigens of interest include, without limitation:
(a) with respect to SLE, the Smith protein, RNP ribonucleoprotein,
and the SS-A and SS-B proteins; and (b) with respect to MG, the
acetylcholine receptor. Examples of other miscellaneous antigens
involved in one or more types of autoimmune response include, e.g.,
endogenous hormones such as luteinizing hormone, follicular
stimulating hormone, testosterone, growth hormone, prolactin, and
other hormones.
[0156] Antigens involved in autoimmune diseases, allergy, and graft
rejection can be used in the compositions and methods of the
invention. For example, an antigen involved in any one or more of
the following autoimmune diseases or disorders can be used in the
present invention: diabetes, diabetes mellitus, arthritis
(including rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), multiple sclerosis,
myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), psoriasis, Sjogren's Syndrome, including
keratoconjunctivitis sicca secondary to Sjogren's Syndrome,
alopecia areata, allergic responses due to arthropod bite
reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Crohn's disease, Graves
ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis
posterior, and interstitial lung fibrosis. Examples of antigens
involved in autoimmune disease include glutamic acid decarboxylase
65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid
protein, acetylcholine receptor components, thyroglobulin, and the
thyroid stimulating hormone (TSH) receptor.
[0157] Examples of antigens involved in allergy include pollen
antigens such as Japanese cedar pollen antigens, ragweed pollen
antigens, rye grass pollen antigens, animal derived antigens such
as dust mite antigens and feline antigens, histocompatiblity
antigens, and penicillin and other therapeutic drugs. Examples of
antigens involved in graft rejection include antigenic components
of the graft to be transplanted into the graft recipient such as
heart, lung, liver, pancreas, kidney, and neural graft components.
The antigen may be an altered peptide ligand useful in treating an
autoimmune disease.
[0158] It will be appreciated by those of skill in the art that the
sequence of any protein effector molecule may be altered in a
manner that does not substantially affect the functional advantages
of the effector protein. For example, glycine and alanine are
typically considered to be interchangeable as are aspartic acid and
glutamic acid and asparagine and glutamine. One of skill in the art
will recognize that many different variations of effector sequences
will encode effectors with roughly the same activity as the native
effector. The effector molecule and the antibody may be conjugated
by chemical or by recombinant means as described above. Chemical
modifications include, for example, derivitization for the purpose
of linking the effector molecule and the antibody to each other,
either directly or through a linking compound, by methods that are
well known in the art of protein chemistry. Both covalent and
noncovalent attachment means may be used with the humanized
antibodies of the present invention.
[0159] The procedure for attaching an effector molecule to an
antibody will vary according to the chemical structure of the
moiety to be attached to the antibody. Polypeptides typically
contain a variety of functional groups; e.g., carboxylic acid
(COOH), free amine (--NH.sub.2) or sulfhydryl (--SH) groups, which
are available for reaction with a suitable functional group on an
antibody to result in the binding of the effector molecule.
Alternatively, the antibody can be derivatized to expose or to
attach additional reactive functional groups, e.g., by attachment
of any of a number of linker molecules such as those available from
Pierce Chemical Company, Rockford Ill.
[0160] The linker is capable of forming covalent bonds to both the
antibody and to the effector molecule. Suitable linkers are well
known to those of skill in the art and include, but are not limited
to, straight or branched-chain carbon linkers, heterocyclic carbon
linkers, or peptide linkers. Where the antibody and the effector
molecule are polypeptides, the linkers may be joined to the
constituent amino acids through their side groups (e.g., through a
disulfide linkage to cysteine). However, in a preferred embodiment,
the linkers will be joined to the alpha carbon amino and carboxyl
groups of the terminal amino acids.
[0161] In some circumstances, it is desirable to free the effector
molecule from the antibody when the immunoconjugate has reached its
target site. Therefore, in these circumstances, immunoconjugates
will comprise linkages that are cleavable in the vicinity of the
target site. Cleavage of the linker to release the effector
molecule from the antibody may be prompted by enzymatic activity or
conditions to which the immunoconjugate is subjected either inside
the target cell or in the vicinity of the target site. When the
target site is a tumor, a linker that is cleavable under conditions
present at the tumor site (e.g. when exposed to tumor-associated
enzymes or acidic pH) may be used.
[0162] Exemplary chemical modifications of the effector molecule
and the antibody of the present invention also include
derivitization with polyethylene glycol (PEG) to extend time of
residence in the circulatory system and reduce immunogenicity,
according to well known methods (See for example, Lisi, et al.,
Applied Biochem. 4:19 (1982); Beauchamp, et al., Anal Biochem.
131:25 (1982); and Goodson, et al., Bio/Technology 8:343
(1990)).
[0163] The present invention contemplates vaccines for use in both
active and passive immunization embodiments. Immunogenic
compositions, proposed to be suitable for use as a vaccine, may be
prepared most readily directly from immunogenic T-cell stimulating
peptides prepared in a manner disclosed herein. The final
vaccination material is dialyzed extensively to remove undesired
small molecular weight molecules and/or lyophilized for more ready
formulation into a desired vehicle. In certain embodiment of the
present invention, the compositions and methods of the present
invention are used to manufacture a cellular vaccine, e.g., the
antigen-delivering anti-CD40 binding portion of the antibody is
used to direct the antigen(s) to an antigen presenting cell, which
then "loads" the antigen onto MHC proteins for presentation. The
cellular vaccine is, therefore, the antigen presenting cell that
has been loaded using the compositions of the present invention to
generate antigen-loaded antigen presenting cells.
[0164] When the vaccine is the anti-CD40 binding protein itself,
e.g., a complete antibody or fragments thereof, then these "active
ingredients" can be made into vaccines using methods understood in
the art, e.g., U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;
4,599,230; and 4.578,770, relevant portions incorporated herein by
reference. Typically, such vaccines are prepared as injectables,
e.g., as liquid solutions or suspensions or solid forms suitable
for re-suspension in liquid prior to injection. The preparation may
also be emulsified. The active immunogenic ingredient is often
mixed with excipients that are pharmaceutically acceptable and
compatible with the active ingredient. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol, or the like
and combinations thereof. In addition, if desired, the vaccine may
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, or adjuvants that enhance
the effectiveness of the vaccines.
[0165] The vaccines are administered in a manner compatible with
the dosage formulation, and in such amount as will be
therapeutically effective and immunogenic. The quantity to be
administered depends on the subject to be treated, including, e.g.,
the capacity of the individual's immune system to generate an
immune response. Precise amounts of cells or active ingredient
required to be administered depend on the judgment of the
practitioner. However, suitable dosage ranges are of the order of a
few thousand cells (to millions of cells) for cellular vaccines.
For standard epitope or epitope delivery vaccines then the vaccine
may be several hundred micrograms active ingredient per
vaccination. Suitable regimes for initial administration and
booster shots are also variable, but are typified by an initial
administration followed by subsequent inoculations or other
administrations.
[0166] The manner of application may vary widely, however, certain
embodiments herein will most likely be delivered intravenously or
at the site of a tumor or infection directly. Regardless, any of
the conventional methods for administration of a vaccine are
applicable. The dosage of the vaccine will depend on the route of
administration and will vary according to the size of the host.
[0167] In many instances, it will be desirable to have multiple
administrations of the vaccine, e.g., four to six vaccinations
provided weekly or every other week. A normal vaccination regimen
will often occur in two to twelve week intervals or from three to
six week intervals. Periodic boosters at intervals of 1-5 years,
usually three years, may be desirable to maintain protective levels
of the immune response or upon a likelihood of a remission or
re-infection. The course of the immunization may be followed by
assays for, e.g., T cell activation, cytokine secretion or even
antibody production, most commonly conducted in vitro. These immune
response assays are well known and may be found in a wide variety
of patents and as taught herein.
[0168] The vaccine of the present invention may be provided in one
or more "unit doses" depending on whether the nucleic acid vectors
are used, the final purified proteins, or the final vaccine form is
used. Unit dose is defined as containing a predetermined-quantity
of the therapeutic composition calculated to produce the desired
responses in association with its administration, i.e., the
appropriate route and treatment regimen. The quantity to be
administered, and the particular route and formulation, are within
the skill of those in the clinical arts. The subject to be treated
may also be evaluated, in particular, the state of the subject's
immune system and the protection desired. A unit dose need not be
administered as a single injection but may include continuous
infusion over a set period of time. Unit dose of the present
invention may conveniently be described in terms of DNA/kg (or
protein/Kg) body weight, with ranges between about 0.05, 0.10,
0.15, 0.20, 0.25, 0.5, 1, 10, 50, 100, 1,000 or more mg/DNA or
protein/kg body weight are administered.
[0169] Likewise, the amount of anti-CD40-antigen vaccine delivered
can vary from about 0.2 to about 8.0 mg/kg body weight. Thus, in
particular embodiments, 0.4 mg, 0.5 mg, 0.8 mg, 1.0 mg, 1.5 mg, 2.0
mg, 2.5 mg, 3.0 mg, 4.0 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg
and 7.5 mg of the vaccine may be delivered to an individual in
vivo. The dosage of vaccine to be administered depends to a great
extent on the weight and physical condition of the subject being
treated as well as the route of administration and the frequency of
treatment. A pharmaceutical composition that includes a naked
polynucleotide prebound to a liposomal or viral delivery vector may
be administered in amounts ranging from 1 .mu.g to 1 mg
polynucleotide to 1 .mu.g to 100 mg protein. Thus, particular
compositions may include between about 1 .mu.g, 5 .mu.g, 10 .mu.g,
20 .mu.g, 30 .mu.g, 40 .mu.g, 50 .mu.g, 60 .mu.g, 70 .mu.g, 80
.mu.g, 100 .mu.g, 150 .mu.g, 200 .mu.g, 250 .mu.g, 500 .mu.g, 600
.mu.g, 700 .mu.g, 800 .mu.g, 900 .mu.g or 1,000 .mu.g
polynucleotide or protein that is bound independently to 1 .mu.g, 5
.mu.g, 10 .mu.g, 20 .mu.g, 3.0 .mu.g, 40 .mu.g 50 .mu.g, 60 .mu.g,
70 .mu.g, 80 .mu.g, 100 .mu.g, 150 .mu.g, 200 .mu.g, 250 .mu.g, 500
.mu.g, 600 .mu.g, 700 .mu.g, 800 .mu.g, 900 .mu.g, 1 mg, 1.5 mg, 5
mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg
or 100 mg vector.
[0170] Antibodies of the present invention may optionally be
covalently or non-covalently linked to a detectable label.
Detectable labels suitable for such use include any composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical methods. Useful
labels in the present invention include magnetic beads (e.g.
DYNABEADS), fluorescent dyes (e.g., fluorescein isothiocyanate,
Texas red, rhodamine, green fluorescent protein, and the like),
radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or
.sup.32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric
labels such as colloidal gold or colored glass or plastic (e.g.
polystyrene, polypropylene, latex, etc.) beads.
[0171] Methods of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
illumination Enzymatic labels are typically detected by providing
the enzyme with a substrate and detecting the reaction product
produced by the action of the enzyme on the substrate, and
colorimetric labels are detected by simply visualizing the colored
label.
[0172] The antibody and/or immunoconjugate compositions of this
invention are particularly useful for parenteral administration,
such as intravenous administration or administration into a body
cavity. The compositions for administration will commonly comprise
a solution of the antibody and/or immunoconjugate dissolved in a
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., buffered saline
and the like. These solutions are sterile and generally free of
undesirable matter. These compositions may be sterilized by
conventional, well-known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of fusion protein in these formulations can vary
widely, and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the patient's
needs.
[0173] Thus, a typical pharmaceutical immunoconjugate composition
of the present invention for intravenous administration would be
about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to
about 100 mg per patient per day may be used. Actual methods for
preparing administrable compositions will be known or apparent to
those skilled in the art and are described in more detail in such
publications as REMINGTON'S PHARMACEUTICAL SCIENCE, 19TH ED., Mack
Publishing Company, Easton, Pa. (1995).
[0174] The compositions of the present invention can be
administered for therapeutic treatments. In therapeutic
applications, compositions are administered to a patient suffering
from a disease, in an amount sufficient to cure or at least
partially arrest the disease and its complications. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend upon
the severity of the disease and the general state of the patient's
health. An effective amount of the compound is that which provides
either subjective relief of a symptom(s) or an objectively
identifiable improvement as noted by the clinician or other
qualified observer.
[0175] Single or multiple administrations of the compositions are
administered depending on the dosage and frequency as required and
tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the proteins of this invention to
effectively treat the patient. Preferably, the dosage is
administered once but may be applied periodically until either a
therapeutic result is achieved or until side effects warrant
discontinuation of therapy. Generally, the dose is sufficient to
treat or ameliorate symptoms or signs of disease without producing
unacceptable toxicity to the patient.
[0176] Controlled release parenteral formulations of the
immunoconjugate compositions of the present invention can be made
as implants, oily injections, or as particulate systems. For a
broad overview of protein delivery systems see, Banga, A. J.,
THERAPEUTIC PEPTIDES AND PROTEINS: FORMULATION, PROCESSING, AND
DELIVERY SYSTEMS, Technomic Publishing Company, Inc., Lancaster,
Pa., (1995) incorporated herein by reference. Particulate systems
include microspheres, microparticles, microcapsules, nanocapsules,
nanospheres, and nanoparticles. Microcapsules contain the
therapeutic protein as a central core. In microspheres the
therapeutic is dispersed throughout the particle. Particles,
microspheres, and microcapsules smaller than about 1 .mu.m are
generally referred to as nanoparticles, nanospheres, and
nanocapsules, respectively. Capillaries have a diameter of
approximately 5 .mu.m so that only nanoparticles are administered
intravenously. Microparticles are typically around 100 .mu.m in
diameter and are administered subcutaneously or
intramuscularly.
[0177] Polymers can be used for ion-controlled release of
immunoconjugate compositions of the present invention. Various
degradable and non-degradable polymeric matrices for use in
controlled drug delivery are known in the art (Langer, R., Accounts
Chem. Res. 26:537-542 (1993)). For example, the block copolymer,
poloxamer 407.RTM. exists as a viscous yet mobile liquid at low
temperatures but forms a semisolid gel at body temperature,
hydroxyapatite has been used as a microcarrier for controlled
release of proteins, and/or liposomes may be used for controlled
release as well as drug targeting of the lipid-capsulated drug.
Numerous additional systems for controlled delivery of therapeutic
proteins are known. See, e.g., U.S. Pat. Nos. 5,055,303, 5,188,837,
4,235,871, 4,501,728, 4,837,028, 4,957,735 and 5,019,369,
5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505;
5,506,206, 5,271,961; 5,254,342 and 5,534,496, relevant portions of
each of which are incorporated herein by reference.
[0178] Among various uses of the immunoconjugates of the invention
are included a variety of disease conditions caused by specific
human cells that may be eliminated by the toxic action of the
fusion protein. For example, for the humanized
anti-CD40.sub.--12E12.3F3 (ATCC Accession No. ______),
anti-CD40.sub.--12B4.2C10 (ATCC Accession No. AB13-22.12B4.2C10
(HS446)), and anti-CD40.sub.--11B6.1C3 (ATCC Accession No.
AB13-22.11B6.1C3 (HS440)), antibodies disclosed herein, one
preferred application for immunoconjugates is the treatment of
malignant cells expressing CD40. Exemplary malignant cells include
those of chronic lymphocytic leukemia and hairy cell leukemia.
[0179] In another embodiment, this invention provides kits for the
delivery of antigens, e.g., CD40 or an immunoreactive fragment
thereof, conjugated or in the form of a fusion protein with one or
more T cell or B cell epitopes. A "biological sample" as used
herein is a sample of biological tissue or fluid that contains the
antigen. Such samples include, but are not limited to, tissue from
biopsy, blood, and blood cells (e.g., white cells). Preferably, the
cells are lymphocytes, e.g., dendritic cells. Biological samples
also include sections of tissues, such as frozen sections taken for
histological purposes. A biological sample is typically obtained
from a multicellular eukaryote, preferably a mammal such as rat,
mouse, cow, dog, guinea pig, or rabbit, and more preferably a
primate, such as a macaque, chimpanzee, or human. Most preferably,
the sample is from a human. The antibodies of the invention may
also be used in vivo, for example, as a diagnostic tool for in vivo
imaging.
[0180] Kits will typically comprise a nucleic acid sequence that
encodes an antibody of the present invention (or fragment thereof)
with one or more framework portions or multiple cloning sites at
the carboxy-terminal end into which the coding sequences for one or
more antigens may be inserted. In some embodiments, the antibody
will be a humanized anti-CD40 Fv fragment, such as an scFv or dsFv
fragment. In addition the kits will typically include instructional
materials disclosing methods of use of an antibody of the present
invention (e.g. for loading into dendritic cells prior to
immunization with the dendritic cells, which can be autologous
dendritic cells). The kits may also include additional components
to facilitate the particular application for which the kit is
designed. Thus, for example, the kit may additionally contain
methods of detecting the label (e.g. enzyme substrates for
enzymatic labels, filter sets to detect fluorescent labels,
appropriate secondary labels such as a sheep anti-mouse-HRP, or the
like). The kits may additionally include buffers and other reagents
routinely used for the practice of a particular method. Such kits
and appropriate contents are well known to those of skill in the
art.
[0181] In another set of uses for the invention, immunoconjugates
targeted by antibodies of the invention can be used to purge
targeted cells from a population of cells in a culture. For
example, if a specific population of T cells is preferred, the
immunoconjugates of the present invention may be used to enrich a
population of T cells having the opposite effect of the on-going
immune response. Thus, for example, cells cultured from a patient
having a cancer can be purged of cancer cells by providing the
patient with dendritic cells that were antigen loaded using the
antibodies of the invention as a targeting moiety for the antigens
that will trigger an immune response against the cancer, virus or
other pathogen. Likewise, the immunoconjugates can be used to
increase the population of regulatory T cells or drive the immune
response toward or away from a cytotoxic T cell response or even
drive a B cell response.
Example 1
Anti-CD40-HIV Peptides Vaccine
[0182] Five 19- to 32-amino-acid long sequences were selected from
a multiplicity of cytotoxic T lymphocyte (CTL) epitopes identified
in the HIV-1 Nef, Gag and Env proteins in the context of different
MHC-class I molecules. It has been reported that CTL responses can
be induced efficiently by lipopeptide vaccines in mice, in
primates, and in humans. The five HIV peptides were then modified
in C-terminal position by a (Palm)-NH2 group and the five HIV
peptide sequences have been well described in the scientific
literature [e.g., Characterization of a multi-lipopeptides mixture
used as an HIV-1 vaccine candidate (1999) Klinguer et al., Vaccine,
Volume 18, 259-267] and in a patent application [Cytotoxic T
lymphocyte-inducing lipopeptides and use as vaccines. Gras-Masse H.
et al., Patent No. EP0491628 (1992 Jun. 24); U.S. Pat. No.
5,871,746 (1999 Feb. 16)].
[0183] A very desirable HIV vaccine would be composed of
recombinant anti-dendritic cell receptor antibody fused to the
above HIV peptides. The present invention includes compositions and
methods to efficiently produce proteins and HIV vaccines.
[0184] The sequences shown below are the amino-acid sequences of
the five selected HIV peptides and the amino-acid positions within
each HIV protein are in brackets.
TABLE-US-00027 Nef (66-97) is: (SEQ ID NO.: 1)
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL Nef (116-145) is: (SEQ ID NO.: 2)
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL Gag p17 (17-35) is: (SEQ ID NO.: 3)
EKIRLRPGGKKKYKLKHIV Gag p17-p24 (253-284) is: (SEQ ID NO.: 4)
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD Pol 325-355 (RT 158-188) is: (SEQ
ID NO.: 5) AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY
[0185] The sequence below is a hIgG4 heavy chain (H)-HIV gag17
fusion protein where the Gag p17 (17-35) region is shown in bold.
The underlined AS residues are joining sequences.
TABLE-US-00028 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-gag17] C655 is:
(SEQ ID NO.: 6) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASEKIRLRPGGKKKYKLKHIVAS.
[0186] The sequence below is an H chain-HIV gag253 fusion protein
where the Gag p17-p24 (253-284) region is shown in bold. The
underlined AS residues are joining sequences.
TABLE-US-00029 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-gag253] C656 is:
(SEQ ID NO.: 7) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDAS.
[0187] The sequence below is an H chain-HIV nef116 fusion protein
where the Nef (116-145) region is shown in bold. The underlined AS
residues are joining sequences.
TABLE-US-00030 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-nef116] C680 is:
(SEQ ID NO.: 8) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASHTQGYFPDWQNYTPGPGVRYPLTFGWLYKLAS.
[0188] The sequence below is a H chain-HIV nef66 fusion protein
where the Nef (66-97) region is shown shaded in bold. The
underlined AS residues are joining sequences.
TABLE-US-00031 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-nef66] C679 is:
(SEQ ID NO.: 9) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLAS.
[0189] The sequence below is a H chain-HIV pol158 fusion protein
where the Pol 325-355 (RT 158-188) region is shown in bold. The
underlined AS residues are joining sequences.
TABLE-US-00032 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-pol158] C667 is:
(SEQ ID NO.: 10) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYAS.
[0190] FIG. 1 shows protein A affinity purified recombinant
antibodies fused to various HIV peptides (lanes 1 to 5) secreted
from transfected 293F cells, analyzed by reducing SDS.PAGE and
Coomassie Brilliant Blue staining. Expression vectors for the H
chains fused to various C-terminal HIV peptides coding regions were
co-transfected with the matching light chain (L) plasmid into
transient 293F cells for three days before harvesting the
supernatant for subsequent purification. Cell number and DNA amount
were constant between transfections. Since the protein A affinity
matrix was used in excess, the SDS.PAGE analysis defines both the
production yield and the H chain integrity of the various vaccine
constructs. Lanes 1, 4 and 5 (upper bands) show that the H chains
fused directly to HIV gag17, nef66 and pol158 peptides can be
well-secreted. Lane 2 shows that the H chain fused directly to HIV
gag253 peptide expresses poorly. Lane 3 shows that the H chain
fused directly to HIV nef116 peptide is not expressed at all.
[0191] Surprisingly, it was found that the use of flexible
potentially glycosylated inter-peptide coding region linker
sequences improves the secretion of intact recombinant antibody-HIV
peptides fusion proteins.
[0192] The flexible linker sequences used are derived from
cellulosomal anchoring scaffoldin B precursor [Bacteroides
cellulosolvens] and have been described by the present inventors in
co-pending U.S. Patent Application Ser. No. 61/081,234, relevant
portions incorporated herein by reference.
[0193] The sequences shown below are the 25-amino-acid long
sequences of the four selected peptide linker sequences. The
underlined sequences are predicted N-linked glycosylation
sites.
TABLE-US-00033 Flex1 is: (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP
Flex 2 is: (SEQ ID NO.: 12) PTSTPADSSTITPTATPTATPTIKG Flex 3 is:
(SEQ ID NO.: 13) TVTPTATATPSAIVTTITPTATTKP Flex 4 is: (SEQ ID NO.:
14) TNGSITVAATAPTVTPTVNATPSAA
[0194] These sequences [the linkers shows in bold and underlined
regions obtained from cohesion] are derived from the inter-cohesin
domain spacers of the bacterial protein
>gi|50656899|gb|AAT79550.1| cellulosomal anchoring scaffoldin B
precursor [Bacteroides cellulosolvens]:
TABLE-US-00034 (SEQ ID NO.: 15)
MQSPRLKRKILSVILAVCYIISSFSIQFAATPQVNIIIGSAQGIPGSTV
KVPINLQNVPEIGINNCDFTIKFDSDILDFNSVEAGDIVPLPVASFSSN
NSKDIIKFLFSDATQGNMPINENGLFAVISFKIKDNAQKGISNIKVSSY
GSFSGMSGKEMQSLSPTFFSGSIDVSDVSTSKLDVKVGNVEGIAGTEVN
VPITFENVPDNGINNCNFTLSYDSNALEFLTTEAGNIIPLAIADYSSYR
SMEGKIKFLFSDSSQGTRSIKNDGVFANIKFKIKGNAIRDTYRIDLSEL
GSFSSKQNNNLKSIATQFLSGSVNVKDIESSVSPTTSVHPTPTSVPPTP
TKSSPGNKMKIQIGDVKANQGDTVIVPITFNEVPVMGVNNCNFTLAYDK
NIMEFISADAGDIVTLPMANYSYNMPSDGLVKFLYNDQAQGAMSIKEDG
TFANVKFKIKQSAAFGKYSVGIKAIGSISALSNSKLIPIESIFKDGSIT
VTNKPIVNIEIGKVKVKAGDKIKVPVEIKDIPSIGINNCNFTLKYNSNV
LKYVSNEAGTIVPAPLANLSINKPDEGIIKLLFSDASQGGMPIKDNGIF
VNLEFQAVNDANIGVYGLELDTIGAFSGISSAKMTSIEPQFNNGSIEIF
NSAQTPVPSNTEVQTPTNTISVTPTNNSTPTNNSTPKPNPLYNLNVNIG
EISGEAGGVIEVPIEFKNVPDFGINNCDFSVKYDKSIFEYVTYEAGSIV
KDSIVNLACMENSGIINLLFNDATQSSSPIKNNGVFAKLKFKINSNAAS
GTYQINAEGYGKFSGNLNGKLTSINPIFENGIINIGNVTVKPTSTPADS
STITPTATPTATPTIKGTPTVTPIYWMNVLIGNMNAAIGEEVVVPIEFK
NVPPFGINNCDFKLVYDSNALELKKVEAGDIVPEPLANLSSNKSEGKIQ
FLFNDASQGSMQIENGGVFAKITFKVKSTAASGIYNIRKDSVGSFSGLI
DNKMTSIGPKFTDGSIVVGTVTPTATATPSAIVTTITPTATTKPIATPT
IKGTPTATPMYWMNVVIGKMNAEVGGEVVVPIEFNNVPSFGINNCDFKL
VYDATALELKNVEAGDIIKTPLANFSNNKSEEGKISFLFNDASQGSMQI
ENGGVFAKITFKVKSTTATGVYDLRKDLVGSFSGLKDNKMTSIGAEFTN
GSITVAATAPTVTPTVNATPSAATPTVTPTATATPSVTIPTVTPTATAT
PSVTIPTVTPTATATPSAATPTVTPTATATPSVTIPTVTPTVTATPSDT
IPTVTPTATATPSAIVTTITPTATAKPIATPTIKGTPTATPMYWMNVVI
GKMNAEVGGEVVVPIEFKNVPSFGINNCDFKLVYDATALELKNVEAGDI
IKTPLANFSNNKSEEGKISFLFNDASQGSMQIENGGVSAKITFKVKSTT
AIGVYDIRKDLIGSFSGLKDSKMTSIGAEFTNGSITVATTAPTVTPTAT
ATPSVTIPTVTPTATATPGTATPGTATPTATATPGAATPTETATPSVMI
PTVTPTATATPTATATPTVKGTPTIKPVYKMNVVIGRVNVVAGEEVVVP
VEFKNIPAIGVNNCNFVLEYDANVLEVKKVDAGEIVPDALINFGSNNSD
EGKVYFLFNDALQGRMQIANDGIFANITFKVKSSAAAGIYNIRKDSVGA
FSGLVDKLVPISAEFTDGSISVESAKSTPTATATGTNVTPTVAATVTPT
ATPASTTPTATPTATSTVKGTPTATPLYSMNVIIGKVNAEASGEVVVPV
EFKDVPSIGINNCNFILEYDASALELDSAEAGEIVPVPLGNFSSNNKDE
GKIYFLFSDGTQGRMQIVNDGIFAKIKFKVKSTASDGTYYIRKDSVGAF
SGLIEKKIIKIGAEFTDGSITVRSLTPTPTVTPNVASPTPTKVVAEPTS
NQPAGPGPITGTIPTATTTATATPTKASVATATPTATPIVVVEPTIVRP
GYNKDADLAVFISSDKSRYEESSIITYSIEYKNIGKVNATNVKIAAQIP
KFTKVYDAAKGAVKGSEIVWMIGNLAVGESYTKEYKVKVDSLTKSEEYT
DNTVTISSDQTVDIPENITTGNDDKSTIRVMLYSNRFTPGSHSSYILGY
KDKTFKPKQNVTRAEVAAMFARIMGLTVKDGAKSSYKDVSNKHWALKYI
EAVTKSGIFKGYKDSTFHPNAPITRAELSTVIFNYLHLNNIAPSKVHFT
DINKHWAKNYIEEIYRFKLIQGYSDGSFKPNNNITRAEVVTMINRMLYR
GPLKVKVGSFPDVSPKYWAYGDIEEASRNHKYTRDEKDGSEILIE.
[0195] The sequence below is a heavy chain (H)-HIV
gag17-nef66-nef116 peptides fusion protein where the HIV gag17,
nef66, nef116 peptide sequences are bold. The underlined AS
residues are joining sequences.
TABLE-US-00035 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-gag17-nef66-nef116]
C694 is: (SEQ ID NO.: 16)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREE
QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS
LGKASEKIRLRPGGKKKYKLKHIVASVGFPVTPQVPLRPMTYKAAVDLSH
FLKEKGGLASHTQGYFPDWQNYTPGPGVRYPLTFGWLYKLAS.
[0196] The sequence below is an H chain-HIV gag17-nef116 peptides
fusion protein where the HIV gag17 and nef116 peptide sequences
[italics] are linked via a spacer f1 [shown in bold]. The
underlined AS residues are joining sequences.
TABLE-US-00036 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-gag17-f1-nef116] C692
is: (SEQ ID NO.: 17)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREE
QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS
LGKASEKIRLRPGGKKKYKLKHIVASSSVSPTTSVHPTPTSVPPTPTKSS
PASHTQGYFPDWQNYTPGPGVRYPLTFGWLYKLAS.
[0197] FIG. 2 shows protein A affinity purified recombinant
antibodies fused to various HIV peptides (Lanes 1 and 2) secreted
from transfected 293F cells, then analyzed by reducing SDS.PAGE and
Coomassie Brilliant Blue staining. Expression vectors for the H
chains fused to various C-terminal HIV peptides coding regions were
co-transfected with the matching L chain plasmid into transient
293F cells for three days before harvesting the supernatant for
subsequent purification. Lanes 1 and 2 (upper bands) show that the
H chains fused directly to a HIV peptide string of
gag17-nef66-nef116 can be well-secreted. Also the H chain product
containing a HIV peptide string of gag17 and nef116 separated by
the flexible spacer f1 (Lane 2) is also well expressed. Thus HIV
nef116 peptide, which is not expressed as a secreted product when
directly fused to the H chain alone, can be well-expressed when
appended in certain other peptide and flexible string contexts.
Note that the H chain fused directly to gag17-f1-nef116 [82
residues] migrates slower than H chain with gag17-nef66-nef116 [89
residues] this suggests that the flexible linker f1 is
glycosylated, possibly also enhancing the production of the
secreted gag17-f1-nef116 fusion antibody versus gag17-nef66-nef116
fusion antibody.
[0198] The sequence below is an H chain-HIV peptides string of
gag17-gag253-nef66 fusion protein where each HIV peptide sequence
[shaded in italics] is separated by a inter-peptide spacer f [shown
in bold]. In this case, a 27-amino-acid long linker flex-v1(v1)
[shown in bold italics] derived from cellulosomal anchoring
scaffoldin B precursor [Bacteroides cellulosolvens regions in
bold-italics-underlined] was inserted between the H chain
C-terminus and the HIV peptides-flexible spacers string. The
underlined AS residues are joining sequences.
TABLE-US-00037 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-Pep-gag17-
f1-gag253-f2-nef66] C711 is: (SEQ ID NO.: 18)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREE
QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK
EKIRLRPGGKKKYK LKHIVASSSVSPTTSVHPTPTSVPPTPTKSSPASNPPIPVGEIYKRWIIL
GLNKIVRMYSPTSILDASPTSTPADSSTITPTATPTATPTIKGASVGFPV
TPQVPLRPMTYKAAVDLSHFLKEKGGLAS.
[0199] The sequence below is an H chain-HIV peptides string of
pol158-gag17-nef66-nef116-gag253 fusion protein where peptide
sequences are shaded in grey. The underlined AS residues are
joining sequences.
TABLE-US-00038 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-pol158-gag17-
nef66-nef116-gag253] C713 is: (SEQ ID NO.: 19)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEW
LAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCA
RSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSEST
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK
AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
TQKSLSLSLGKASAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYASEKI
RLRPGGKKKYKLKHIVASVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGG
LASHTQGYFPDWQNYTPGPGVRYPLTFGWLYKLASNPPIPVGEIYKRWI
ILGLNKIVRMYSPTSILDAS.
[0200] FIG. 3 shows protein A affinity purified recombinant
antibodies fused to various HIV peptide strings (Lanes 1 to 5)
secreted from transfected 293F cells, then analyzed by reducing
SDS.PAGE and Coomassie Brilliant Blue staining. Expression vectors
for the H chains fused to various C-terminal HIV peptides coding
regions were co-transfected with the matching L chain plasmid into
transient 293F cells for three days before harvesting the
supernatant for subsequent purification. Lanes 1, 2 and 3 (upper
bands) show that the 4 HIV peptides-flexible spacers fused to H
chain via the flexible linker flex-v1 can be well-secreted.
However, a string of 4 HIV peptides fused directly to H chain is
not expressed at all (Lane 4, upper band). Also, lane 5 (upper
band) shows that a string of 5 HIV peptides fused directly to H
chain is not expressed at all. This result suggests that certain
combinations and contexts of flexible linkers and HIV peptide
coding sequences can enhance secretion of recombinant antibody-HIV
peptide fusion proteins (Lanes 1, 2 and 3).
[0201] The sequence below is for an H chain-HIV peptides string of
gag17-gag253-nef66-nef116-pol158 fusion protein where each HIV
peptide sequence [shaded in italics] is separated by an
inter-peptide spacer f [shown in bold]. The flexible linker flex-v1
(v1) [shown in bold-italics] was inserted between the H chain
C-terminus and the HIV peptides-flexible spacers string. The
underlined AS residues are joining sequences.
TABLE-US-00039 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-hIgG4H-Flex-v1-Pep-
gag17-f1-gag253-f2-nef116-f3-nef66-f4-pol158] C825 is: (SEQ ID NO.:
20) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREE
QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK
ASEKIRLRPGGKKK YKLKHIVASSSVSPTTSVHPTPTSVPPTPTKSSPASNPPIPVGEIYKRWI
ILGLNKIVRMYSPTSILDASPTSTPADSSTITPTATPTATPTIKGASHTQ
GYFPDWQNYTPGPGVRYPLTFGWLYKLASTVTPTATATPSAIVTTITPTA
TTKPASVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLASTNGSITVAAT
APTVTPTVNATPSAAASAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYAS.
[0202] FIG. 4 shows protein A affinity purified recombinant
antibodies fused to various HIV peptide strings (Lanes 1 to 6)
secreted from transfected 293F cells, then analyzed by reducing
SDS.PAGE and Coomassie Brilliant Blue staining. Expression vectors
for the H chains fused to various C-terminal HIV peptides coding
regions were co-transfected with the matching L chain plasmid into
transient 293F cells for three days before harvesting the
supernatant for subsequent purification. Lanes 1, 3 and 5 (upper
bands) show that the string of 4 HIV peptides-flexible spacers
fused to H chain via the flexible linker flex-v1 can be
well-secreted. Lanes 2 and 6 (upper bands) show that the string of
5 HIV peptides-flexible spacers fused to H chain via the flexible
linker flex-v1 expresses poorly. However certain combinations and
contexts of HIV peptide coding sequences enhance secretion of
recombinant antibody-HIV peptide fusion proteins (Lanes 3 and 4).
Thus H chain fused to a string of 5 HIV peptides-flexible spacers
via the flexible linker flex-v1 can be well-expressed when appended
in certain other peptide and flexible string contexts (Lane 4).
[0203] The present invention includes compositions and methods for
flexible potentially glycosylated inter-peptide coding region
linker sequences and combinations of such HIV peptide coding
regions that are particularly favorable to efficient secretion of
recombinant anti-DC receptor antibody-HIV peptide fusion
vaccines.
[0204] The use of inter-structural domain linker sequences derived
from cellulose-degrading bacteria as preferred inter-domain linker
sequences in protein engineering--particularly those with highly
predicted glycosylation sites. Desirable properties of these
sequences are i) inherent flexibility, thereby facilitating
separation of linked domains which should greatly help their
correct folding and maintaining B cell receptor access to
conformationally-dependent antigen epitopes; ii) glycosylation,
thereby helping secretion and solubility of the intact produced
fusion protein, and also protecting of the linker sequences from
culture medium proteases.
[0205] Certain combinations of HIV peptide coding regions favor
secretion and that particular flexible linker sequences inserted
between the HIV peptide coding sequences can also help secretion of
intact HIV peptide string vaccines--principles that can also be
applied to solve similar issues for other preferred peptide
antigens.
[0206] DNA sequences of preferred linker and antigen coding
sequences. Joining sequence codons and stop codons are in bold:
TABLE-US-00040 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-gag17] C655 is:
(SEQ ID NO.: 21) GCTAGTGAGAAGATCCGGCTGCGGCCCGGCGGCAAGAAGAAGTACAAGC
TGAAGCACATCGTGGCTAGCTGA [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-nef66
C679 is: (SEQ ID NO.: 22)
GCTAGTGTGGGCTTCCCCGTGACCCCCCAGGTGCCCCTGCGGCCCATGA
CCTACAAGGCCGCCGTGGACCTGAGCCACTTCCTGAAGGAGAAGGGCGG CCTGGCTAGCTGA
[mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-pol158] C667 is: (SEQ ID NO.: 23)
GCTAGTGCCATCTTCCAGAGCAGCATGACCAAGATCCTGGAGCCCTTCC
GGAAGCAGAACCCCGACATCGTGATCTACCAGTACATGGACGACCTGTA CGCTAGCTGA
[mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-Pep-gag253] C681 is: (SEQ ID
NO.: 24) GCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACA
GCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTAACCC
CCCCATCCCCGTGGGCGAGATCTACAAGCGGTGGATCATCCTGGGCCTG
AACAAGATCGTGCGGATGTACAGCCCCACCAGCATCCTGGACGCTAGCT GA
[mAnti-DCIR_9E8_H-LV-hIgG4H-Flex-v1-Pep-gag17- nef116] C686 is:
(SEQ ID NO.: 25) GCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACA
GCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTGAGAA
GATCCGGCTGCGGCCCGGCGGCAAGAAGAAGTACAAGCTGAAGCACATC
GTGGCTAGTCACACCCAGGGCTACTTCCCCGACTGGCAGAACTACACCC
CCGGCCCCGGCGTGCGGTACCCCCTGACCTTCGGCTGGCTGTACAAGCT GGCTAGCTGA
[mAnti-DCIR_9E8_H-LV-hIgG4H-C-hIgG4H-Flex-v1-Pep-
gag17-f1-gag253-f2-nef116-f3-nef66-f4- pol158] C825 is: (SEQ ID
NO.: 26) GCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACA
GCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTGAGAA
GATCCGGCTGCGGCCCGGCGGCAAGAAGAAGTACAAGCTGAAGCACATC
GTGGCTAGTAGCAGCGTGAGCCCCACCACCAGCGTGCACCCCACCCCCA
CCAGCGTGCCCCCCACCCCCACCAAGAGCAGCCCCGCTAGTAACCCCCC
CATCCCCGTGGGCGAGATCTACAAGCGGTGGATCATCCTGGGCCTGAAC
AAGATCGTGCGGATGTACAGCCCCACCAGCATCCTGGACGCTAGTCCCA
CCAGCACCCCCGCCGACAGCAGCACCATCACCCCCACCGCCACCCCCAC
CGCCACCCCCACCATCAAGGGCGCTAGTCACACCCAGGGCTACTTCCCC
GACTGGCAGAACTACACCCCCGGCCCCGGCGTGCGGTACCCCCTGACCT
TCGGCTGGCTGTACAAGCTGGCTAGTACCGTGACCCCCACCGCCACCGC
CACCCCCAGCGCCATCGTGACCACCATCACCCCCACCGCCACCACCAAG
CCCGCTAGTGTGGGCTTCCCCGTGACCCCCCAGGTGCCCCTGCGGCCCA
TGACCTACAAGGCCGCCGTGGACCTGAGCCACTTCCTGAAGGAGAAGGG
CGGCCTGGCTAGTACCAACGGCAGCATCACCGTGGCCGCCACCGCCCCC
ACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTAGTGCCA
TCTTCCAGAGCAGCATGACCAAGATCCTGGAGCCCTTCCGGAAGCAGAA
CCCCGACATCGTGATCTACCAGTACATGGACGACCTGTACGCTAGCTGA.
[0207] DNA sequences of preferred linker and antigen coding
sequences. Joining sequence codons are in bold:
TABLE-US-00041 Nef (66-97) is: (SEQ ID NO.: 27)
GCTAGTGTGGGCTTCCCCGTGACCCCCCAGGTGCCCCTGCGGCCCATGAC
CTACAAGGCCGCCGTGGACCTGAGCCACTTCCTGAAGGAGAAGGGCGGCC TGGCTAGC Nef
(116-145) is: (SEQ ID NO.: 28)
GCTAGTCACACCCAGGGCTACTTCCCCGACTGGCAGAACTACACCCCCGG
CCCCGGCGTGCGGTACCCCCTGACCTTCGGCTGGCTGTACAAGCTGGCTA GC Gag p17
(17-35) is: (SEQ ID NO.: 29)
GCTAGTGAGAAGATCCGGCTGCGGCCCGGCGGCAAGAAGAAGTACAAGCT
GAAGCACATCGTGGCTAGC Gag p17-p24 (253-284) is: (SEQ ID NO.: 30)
GCTAGTAACCCCCCCATCCCCGTGGGCGAGATCTACAAGCGGTGGATCAT
CCTGGGCCTGAACAAGATCGTGCGGATGTACAGCCCCACCAGCATCCTGG ACGCTAGC Pol
325-355 (RT 158-188) is: (SEQ ID NO.: 31)
GCTAGTGCCATCTTCCAGAGCAGCATGACCAAGATCCTGGAGCCCTTCCG
GAAGCAGAACCCCGACATCGTGATCTACCAGTACATGGACGACCTGTACG CTAGC Flex1 is:
(SEQ ID NO.: 32) GCTAGTAGCAGCGTGAGCCCCACCACCAGCGTGCACCCCACCCCCACCAG
CGTGCCCCCCACCCCCACCAAGAGCAGCCCCGCTAGC Flex2 is: (SEQ ID NO.: 33)
GCTAGTCCCACCAGCACCCCCGCCGACAGCAGCACCATCACCCCCACCGC
CACCCCCACCGCCACCCCCACCATCAAGGGCGCTAGC Flex3 is: (SEQ ID NO.: 34)
GCTAGTACCGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGAC
CACCATCACCCCCACCGCCACCACCAAGCCCGCTAGC Flex4 is: (SEQ ID NO.: 35)
GCTAGTACCAACGGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGAC
CCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTAGC
[0208] The present invention includes compositions and methods for
assembling constructs encoding HIV peptides and Flexible linker
sequences. The H chain expression vectors typically have a Nhe I
site [g|ctagc] appended to the H chain C-terminal residue codon, or
[for flex-v1 vectors] to the C-terminal codon of the flex-v1
sequence. Flexible linker sequences or HIV peptide sequences have
an Spe I site [a|ctagt] preceding the N-terminal flexible linker or
HIV peptide codon, a Nhe I site appended to the C-terminal flexible
linker or HIV peptide codon, followed by a TGA stop codon, followed
by a Eco RI site, followed by a Not I site. Such flexible linker or
HIV peptide Spe I-Not I fragments are inserted into the H chain
vector prepared with Nhe I-Not I digestion. Nhe I and Spe I are
compatible sites, but when ligated [glctagt] is no longer either a
Nhe I or Spe I site. Thus additional Spe I-Not I flexible linker or
HIV peptide fragments can be inserted into the new Nhe I-Not I
interval distal to the initial flexible linker or HIV peptide. In
this way, strings of HIV peptide and/or flexible linker coding
regions can be appended to the expression vector H chain coding
region.
Example 2
HIV Peptides Vaccine--In Vitro Antigen-Targeting Biology
[0209] Anti-CD40.LIPO5 HIV peptides vaccine tests on HIV patients
in vitro. To study the ability of .alpha.CD40.LIPO5 HIV peptide
fusion recombinant antibody (.alpha.CD40.LIPO5 rAb) to mediate
antigen presentation, the fusion rAb was added to blood cells from
HIV-infected individuals and measured cytokine production form
peripheral blood mononuclear cells (PBMCs).
[0210] FIG. 5 describes the protocol used in vitro to assay the
potency of .alpha.CD40.LIPO5 HIV peptide fusion recombinant
antibody (.alpha.CD40.LIPO5 rAb) to elicit the expansion of
antigen-specific T cells in the context of a PBMC culture. Briefly,
PBMCs (2.times.10.sup.6 cells/ml) from apheresis of HIV patients
are incubated with a dose range of .alpha.CD40.LIPO5 HIV peptide
vaccine. On day 2, 100 U/ml IL-2 are added to the culture and then,
the media is refreshed every 2 days with 100 U/ml IL-2. On day 10,
the expanded cells are challenged for 48 h with the individual long
peptides corresponding to the 5 HIV peptide sequences incorporated
in the .alpha.CD40.LIPO5 HIV peptide fusion rAb. Then, culture
supernatants are harvested and assessed for cytokine production (by
the T cells with T cell receptor [TCR] specificities for peptide
sequences) using multiplex beads assay (Luminex). Antigen-specific
cytokine production detected in such an assay, if it depends on the
presence of the anti-CD40.LIPO5 HIV peptide vaccine, reflects
vaccine uptake by antigen presenting cells [APC] in the culture,
and processing [proteolytic degradation] and presentation of
peptides on MHC. The antigen-MHC complexes are recognized by T
cells with TCR that recognize only the particular HIV antigen-MHC
complex. In a HIV patient, such cells are likely to be memory T
cells that expanded in the patient in response to the HIV
infection.
[0211] Epitopes from all 5 HIV peptide regions of the vaccine can
be presented by APCs. The scheme in FIG. 5 was used to assay the in
vitro expansion of HIV peptide-specific T cells in response to
anti-CD40.LIPO5 peptide vaccine. Results from 7 individuals are
shown in FIG. 6A-C and indicate that the .alpha.CD40.LIPO5 HIV
peptide fusion rAb elicited HIV peptide-specific IFN.gamma.
responses in all of the patients studied. Thus, the
.alpha.-CD40.LIPO5 HIV peptide fusion rAb allows DCs to
cross-present at least 1 or 2 different peptides out of the 5
peptides within the vaccine to the T cells of each individual.
However, the set of HIV peptides that stimulated IFN.gamma.
production was different for each patient--most likely reflecting
different pools of memory T cells for HIV specificity.
[0212] FIG. 6A-C shows the HIV peptide-specific IFN' production in
PBMCs from HIV patients incubated with various concentrations of
anti-CD40.LIPO5 peptide string vaccine. C is the control group,
which received no vaccine, and defines the baseline response of the
culture to each peptide.
[0213] FIG. 7 is a summary of .alpha.CD40.LIPO5 peptide vaccine
responses against the 5 peptide regions from 8 HIV patients. The
data are based on peptide-specific IFN.gamma. production. FIG. 7
shows that the antigen-specific responses observed in 8 HIV
patients. The data demonstrate that all HIV peptide regions on the
vaccine have the capacity to be processed and presented to T
cells'assuming the likely situation that responses to these
peptides will only be observed if the appropriate TCR-bearing cells
are present. Thus, each patient has a characteristic spectrum of
such cells.
[0214] The .alpha.CD40.LIPO5 peptide vaccine can evoke the
proliferation of antigen-specific T cells capable of secreting a
wide spectrum of cytokines
[0215] FIG. 8A-B shows that .alpha.CD40.LIPO5 HIV peptide vaccine
elicits expansion of HIV peptide-specific T cells capable of
secreting multiple cytokines--a desirable feature in a vaccine. In
FIG. 8A-B .alpha.CD40.LIPO5 HIV peptide vaccine elicits gag253,
nef66, nef116 and pol325 peptide-specific responses characterized
by production of multiple cytokines. This is patient A5.
[0216] Anti-CD40.LIPO5 HIV Peptide Vaccination of Ex Vivo DCs.
[0217] FIG. 9 shows the protocol for testing .alpha.CD40.LIPO5 HIV
peptide vaccine for its ability to direct the expansion of
antigen-specific T cells resulting from targeted uptake by DCs and
presentation of peptide epitopes on their surface MHC complex.
Briefly, HIV patient monocytes are differentiated into DCs by
culture for 2 days with IFN.alpha. and GM-CSF. Different doses
.alpha.CD40.LIPO5 HIV peptide vaccine or a mix of the 5 peptides
are then added for 18 h. Autologous T cells were added to the
co-culture (at a ratio of 1:20) on day 3. On day 5, 100 U/ml IL-2
are added to the culture and then, the media is refreshed every 2
days with 100 U/ml IL-2. On day 10, the expanded cells are
rechallenged for 48 h with the individual long peptides
corresponding to the 5 HIV peptide sequences incorporated in the
.alpha.CD40.LIPO5 HIV peptide fusion rAb. Then, culture
supernatants are harvested and assessed for cytokine production
using Luminex.
[0218] FIG. 10A-B shows the cytokine secretion in response to HIV
peptides from DC-T cell co-cultures treated with various doses of
.alpha.CD40.LIPO5 HIV peptide vaccine. This is patient A10. The
results in the patient A10 shown in FIG. 10 demonstrate expansion
of antigen-specific T cells corresponding to epitopes within the
gag17, gag253, and pol325 HIV peptide regions. In most instances,
there is concordance of responses between .alpha.CD40.LIPO5 HIV
peptide vaccine and non-LIPO5 vaccine [mixture of 5 non-lipidated
HIV peptides with sequences corresponding to those in the
.alpha.CD40.LIPO5 HIV peptide vaccine]. Thus, the .alpha.CD40.LIPO5
HIV peptide vaccine functions well in this in vitro setting where
cultured DCs effectively process and present the HIV antigens to T
cells. This exemplifies use of the .alpha.CD40.LIPO5 HIV peptide
vaccine for ex vivo vaccination, whereby the `vaccinated DCs` would
be cryopreserved for future re-injection into the same patient.
[0219] .alpha.CD40.LIPO5 HIV peptide vaccine--possible immune
effect of the flexible linker regions. It is possible that the
flexible linker sequences interspersing the HIV peptide sequences
within the .alpha.CD40.LIPO5 HIV peptide vaccine themselves contain
T cell epitopes. FIG. 11A-B shows that patient A4 does not appear
to have a significant pool of memory T cells with specificities to
the five flexible linker sequences within .alpha.CD40.LIPO5 HIV
peptide vaccine. In FIG. 11A-B, PBMCs from patient A4 treated with
the .alpha.CD40.LIPO5 HIV peptide vaccine elicit expansion of
antigen-specific T cells with specificity to the gag253 region, but
not to the flexible linker sequences. The protocol describe in FIG.
9 was used, with the flexible linker long peptides corresponding in
sequence to the bold areas, the HIV peptides are in bold-italics,
shown in the sequence below.
[0220] .alpha.CD40.LIPO5 HIV peptide vaccine heavy chain sequence
showing flexible linker regions in bold, joining sequences
underlined and HIV peptide regions shaded in bold italics.
TABLE-US-00042 (SEQ ID NO.: 36)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREE
QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS
LGKASQTPTNTISVTPTNNSTPTNNSNPKPNPAS ASSSVSPTTSVHPTPTSVPPTPTKSSPAS
ASPTSTPADSSTITPTATP TATPTIKGAS ASTV TPTATATPSAIVTTITPTATTKPAS
ASTNGSITVAATAPTVTPTVNATPSAAAS AS..
[0221] In FIG. 12A, the PBMCs from patient A3 treated with the
.alpha.CD40.LIPO5 HIV peptide vaccine elicit expansion of
antigen-specific T cells with specificities to the gag253, nef66,
and nef116 regions, but not to the flexible linker sequences. The
protocol described in FIG. 1 was used, with the flexible linker
long peptides corresponding in sequence to the bold areas shown in
FIG. 8A-B.
[0222] FIGS. 12B-1 and 12B-2 shows HIV antigen-specific T cell
responses evoked from HIV patient A17 PBMCs incubated with 30 nM of
three different HIV5 peptide DC targeting vaccines. Cells were
cultured for 10 days with IL-2 and then stimulated with individual
long peptides corresponding to the 5 HIV peptide sequences
encompassed within the DC-targeting vaccines. After 1 hr brefeldin
A was added and incubation continued for a further 5 hrs before
staining for FACS analysis. The FACS plots show IFN.gamma. and CD8
staining on CD3+ T cells. Circles indicate significant
vaccine-evoked expansion of IFN.gamma.+ cells compared to cells
from PBMCs cultured without vaccine. CD8- cells are CD4+ T cells.
The data show that that anti-CD40.HIV5pep vaccine evokes a strong
expansion of nef66 (N66)-specific CD8+ T cells which is not seen
with the other DC targeting vehicles.
[0223] These are data based on the LIPO5 HIV peptide string. For
example the anti-CD40 H chain is
anti-CD40.sub.--12E12.3F3_H-LV-hIgG4H-C-Flex-v1-Pep-gag17-f1-gag253-f2-ne-
f116-f3-nef66-f4-pol158] with sequence:
TABLE-US-00043 (SEQ ID NO.: 37)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVA
YINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCAR
RGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREE
QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL
SLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASEKIRLRPGGKKK
YKLKHIVASSSVSPTTSVHPTPTSVPPTPTKSSPASNPPIPVGEIYKRW
IILGLNKIVRMYSPTSILDASPTSTPADSSTITPTATPTATPTIKGASH
TQGYFPDWQNYTPGPGVRYPLTFGWLYKLASTVTPTATATPSAIVTTIT
PTATTKPASVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLASTNGSIT
VAATAPTVTPTVNATPSAAASAIFQSSMTKILEPFRKQNPDIVIYQYM DDLYAS.
[0224] FIGS. 12C-1 and 12C-2 is a similar study to that show in
FIGS. 12B-1 and 12B-2, except that the PBMCs are from a different
HIV patient (A2). The data show antigen-specific CD4+ and CD8+ T
cell responses evoked by anti-CD40.HIV5pep but not the other
DC-targeting vaccines, or by a mixture of the peptides
themselves.
[0225] FIG. 12D shows that, based on analysis of 15 different HIV
peptide responses [5 peptide regions sampled in 3 patients],
anti-CD40.HIV5pep vaccine is clearly superior to anti-DCIR.HIV5pep,
anti-LOX-1.HIV5pep and non-LIPO5 mix for eliciting a broad range of
HIV peptide-specific CD8+ and CD4+ T responses.
[0226] The immunogenicity of the flexible linker sequences is of
concern for the .alpha.CD40.LIPO5 HIV peptide vaccine design. The
limited datasets shown above, testing recall of T cells with
specificities for epitopes within the flexible linker sequences,
suggest that the human repertoire against these sequences is
variable. Also, the ability of these sequences to prime responses
de novo is untested. Responses to the .alpha.CD40.LIPO5 HIV peptide
vaccine in monkeys can be tested using the present invention. If
necessary, certain less desirable epitopes within these regions can
be identified by a combination of predictive computational means
and peptide stimulation scans, and then eliminated by introducing
mutational changes that abrogate the TCR interaction.
[0227] The anti-CD40 binding molecule includes a light chain having
the following amino acid sequence (SEQ ID NO. 38). The variable
region of the antibody light chain is underlined and the CDRs are
bolded (SEQ ID NOS.: 42, 43 and 44, respectively).
TABLE-US-00044 (SEQ ID NO.: 38)
MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCSASQGIS
NYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTIGNLEP
EDIATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
[0228] The anti-CD40 binding molecule includes a heavy chain having
the following sequence. The variable region of the antibody light
chain is underlined and the CDRs are bolded (SEQ ID NOS.: 45, 46
and 47, respectively).
TABLE-US-00045 (SEQ ID NO.: 39)
MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFSD
YYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYL
QMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLA
PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDG
VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS
IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA
LHNHYTQKSLSLSLGKAS.
[0229] In one aspect the nucleic acid that encodes the light chain
comprises the SEQ ID NO. The variable region of the antibody light
chain nucleic acid sequence is underlined and the CDRs are
bolded.
TABLE-US-00046 (SEQ ID NO.: 40)
ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAG
GTACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGC
CTCTCTAGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATT
AGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAAC
TCCTGATCTATTACACATCAATTTTACACTCAGGAGTCCCATCAAGGTT
CAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCGGCAACCTG
GAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTTTAATAAGCTTC
CTCCGACGTTCGGTGGAGGCACCAAACTCGAGATCAAACGAACTGTGGC
TGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG
CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA
GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC
AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATG
CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTT
CAACAGGGGAGAGTGTTAG.
[0230] In one aspect the nucleic acid that encodes the heavy chain
comprises the SEQ ID NO.:40. The variable region of the antibody
heavy chain nucleic acid sequence is underlined and the CDRs are
bolded.
TABLE-US-00047 (SEQ ID NO.: 41)
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTG
TCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCC
TGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGT
GACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGT
GGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACAC
TGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTG
TACCTGCAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACT
GTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGG
AACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTC
CCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGG
GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAA
CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG
TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA
GCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCA
CCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCC
CCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCAC
GTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAAC
TGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGG
AGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGAT
GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCC
AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT
ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTA
CAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGA
GCCTCTCCCTGTCTCTGGGTAAAGCTAGCTGA.
[0231] A humanized antibody includes the heavy chain variable
region (V.sub.H) and a light chain variable region (V.sub.L),
wherein the framework regions of the heavy chain and light chain
variable regions are from a donor human antibody, and wherein the
light chain complementarity determining regions (CDRs) have at
least 80%, 90%, 95% or higher identity to CDR1.sub.L having the
amino acid sequence SASQGISNYLN (SEQ ID NO.:41), the CDR2.sub.L
having the amino acid sequence YTSILHS (SEQ ID NO.:42) and the
CDR3.sub.L having the amino acid sequence QQFNKLPPT (SEQ ID
NO.:43); and wherein the heavy chain complementarity determining
regions comprise at least 80%, 90%, 95% or higher identity to the
CDR1.sub.H, CDR2.sub.H and CDR3.sub.H, the CDR1.sub.H having the
amino acid sequence GFTFSDYYMY (SEQ ID NO.:45), the CDR2.sub.H
having the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:46),
and the CDR3.sub.H having the amino acid sequence RGLPFHAMDY (SEQ
ID NO.:47). For example, the humanized antibody may comprise a VL
framework having at least 95% identity to the framework of SEQ ID
NO.:38 and a VH framework that has at least 95% identity to the
framework of SEQ ID NO.:39. In another aspect, the donor CDR
sequences are from ANTI-CD40.sub.--12E12.3F3 and further, wherein
the antibody or fragment thereof specifically binds to CD40.
Example 3
Prostate-Specific Antigen (PSA), Cycline D1, MART-1, Influenza
Viral Nucleoprotein (NP) and HA1 Subunit of Influenza Viral
Hemagglutinin (H1N1, PR8) and Peptide Screen
[0232] Internalization of anti-CD40 mAb. 1.times.10.sup.6 IL-4DCs
were incubated for 1 h in ice with 3 mg/ml human gamma globulin in
PBS containing 3% BSA to block non-specific binding. Cells were
pulsed for 30 minutes on ice with Alexa 568 labeled anti-CD40 mAb
(all at 20 ng/ml final concentration in non-specific block). Cells
were then washed and allowed to internalize surface bound
antibodies for different times, between 0 and 90 minutes, at
37.degree. C. Following internalization, cells were washed twice
with ice-cold PBS containing 1% BSA and 0.05% sodium azide (PBA)
and fixed in ice-cold 1% methanol-free formaldehyde (MFF) in PBS
overnight at 4.degree. C. Cells were permeablized in PBS 3% BSA
containing 0.5% saponin (PBAS) for 20 minutes at 4.degree. C., and
transferred to a 96-well round bottom polypropylene microtiter
plate. After washing twice with ice-cold PBAS, cells were incubated
for 1 h on ice with 3 mg/ml human gamma globulin in PBAS.
BODIPY-phalloidin diluted in PBAS and incubated with cells for 1
hour in ice. Cells were further stained with TOPRO-II, as a nuclear
counterstain. Slides were imaged on a Leica SP1 confocal
microscope.
[0233] Cells. Monoclonal antibodies for cell surface staining were
purchased from BD Biosciences (CA). Monocytes (1.times.10.sup.6/ml)
from healthy donors were cultured in Cellgenics media (France)
containing GM-CSF (100 ng/ml) and IL-4 (50 ng/ml) or GM-CSF (100
ng/ml) and IFN.alpha. (500 Units/ml) (R&D, CA). For IFNDCs,
cells were fed on day 1 with IFN.alpha. and GM-CSF. For IL-4DCs,
the same amounts of cytokines were supplemented into the media on
day one and day three. PBMCs were isolated from Buffy coats using
Percoll.TM. gradients (GE Healthcare, Buckinghamshire, UK) by
density gradient centrifugation. Total CD4+ and CD8+ T cells were
purified by using StemCell kits (CA).
[0234] Peptides. 15-mers (11 amino acid overlapping) for
prostate-specific antigen (PSA), Cycline D1, MART-1, influenza
viral nucleoprotein (NP) and HA1 subunit of influenza viral
hemagglutinin (H1N1, PR8), were synthesized (Mimotopes).
[0235] DCs and T cell co-culture and cytokine expressions.
5.times.10.sup.3 DCs loaded with recombinant fusion proteins
(anti-CD40-HA1, Control Ig-HA1, anti-CD40-PSA, anti-CD40-Cyclin D1,
anti-CD40-MART-1, anti-MARCO-MART-1, and control Ig-MART-1) were
co-cultured with 2.times.10.sup.5 CFSE-labeled CD4+ T cells for 8
days. Proliferation was tested by measuring CFSE dilution after
staining cells with anti-CD4 antibody labeled with APC.
[0236] For measuring the expression of intracellular IFN.gamma.,
CD4+ T cells were restimulated with 1-5 uM of indicated peptides
for 5 h in the presence of Brefeldin A. In separate experiments,
CD4+ T cells were restimulated with peptides indicated for 36 h,
and then cytokines secreted by CD4+ T cells were measured by the
Luminex.
[0237] CD8+ T cells were co-cultured with DCs for 10 days in the
presence of 20 units/ml IL-2 and 20 units/ml IL-7. On day 10 of the
culture, CD8+ T cells were stained with anti-CD8 and tetramers
indicated.
[0238] CTL assay. On day 10 of the culture, a 5-h .sup.51Cr release
assay was performed. T2 cells pulsed with .sup.51Cr first and then
labeled with 10 uM HLA-A2 epitope of MART-1 or 1 nM epitope of
influenza viral M1. T2 cells without peptide were used as control.
The mean of triplicate samples was calculated, and the percentage
of specific lysis was determined using the following formula:
percentage of specific lysis=100.times.(experimental .sup.51Cr
release-control .sup.51Cr release)/(maximum .sup.51Cr
release-control .sup.51Cr release). The maximum release refers to
counts from targets in 2.5% Triton X-100.
[0239] Preparation of mAbs specific for human CD40. Receptor
ectodomain.hIgG (human IgG1Fc) and AP (human placental alkaline
phosphatase) fusion proteins were produced for immunizing mice and
screening mAbs, respectively. A mammalian vector for human IgFc
fusion proteins was engineered as described [J. Immunol. 163:
1973-1983 (1999)]. The mammalian expression vector for receptor
ectodomain.AP proteins was generated using PCR to amplify cDNA for
AP resides 133-1581 (gb|BC009647|) while adding a proximal in-frame
Xho I site and a distal 6C-terminal His residues followed by a TGA
stop codon and Not I site. This Xho I-Not I fragment replaced the
human IgG Fc coding sequence in the above ectodomain.IgG vector.
Fusion proteins were produced using the FreeStyle.TM. 293
Expression System (Invitrogen, CA) according to the manufacturer's
protocol (1 mg total plasmid DNA with 1.3 ml 293Fectin reagent/L of
transfection). Receptor ectodomain.hIgG was purified by 1 ml HiTrap
protein A affinity chromatography (GE Healthcare, CA) eluted with
0.1 M glycine, pH 2.7. Fractions were neutralized with 2M Tris, and
then dialyzed against PBS.
[0240] Mouse mAbs were generated by conventional technology.
Briefly, six-week-old BALB/c mice were immunized i.p. with 20 .mu.g
of receptor ectodomain.hIgGFc fusion protein with Ribi adjuvant,
then boosted with 20 .mu.g antigen ten days and fifteen days later.
After three months, the mice were boosted again three days prior to
taking the spleens. Three to four days after a final boosting,
draining lymph nodes (LN) were harvested. B cells from spleen or LN
cells were fused with SP2/O-Ag 14 cells (ATCC). Hybridoma
supernatants were screened to analyze mAbs specific to the receptor
ectodomain fusion protein compared to the fusion partner alone, or
to the receptor ectodomain fused to alkaline phosphatase [J.
Immunol. 163: 1973-1983 (1999)]. Positive wells were then screened
in FACS using 293F cells transiently transfected with expression
plasmids encoding full-length receptor cDNAs. Selected hybridomas
were single cell cloned and expanded in CELLine flasks (Integra,
CA). Hybridoma supernatants were mixed with an equal volume of 1.5
M glycine, 3 M NaCl, 1.times.PBS, pH 7.8 (binding buffer) and
tumbled with MabSelect resin (GE Healthcare, CA) (800 .mu.l/5 ml
supernatant). The resin was washed with binding buffer and eluted
with 0.1 M glycine, pH 2.7. Following neutralization with 2 M Tris,
mAbs were dialyzed against PBS.
[0241] Expression and purification of recombinant mAbs. Total RNA
was prepared from hybridoma cells using RNeasy kit (Qiagen, CA) and
used for cDNA synthesis and PCR (SMART RACE kit, BD Biosciences)
using supplied 5' primers and gene specific 3' primers
(mIgG.kappa., 5'ggatggtgggaagatggatacagttggtgcagcatc3' (SEQ ID
NO.:48); mIgG2a, 5'ccaggcatcctagagtcaccgaggagccagt3') (SEQ ID
NO.:49). PCR products were then cloned (pCR2.1 TA kit, Invitrogen)
and characterized by DNA sequencing (MC Lab, CA). Using the derived
sequences for the mouse heavy (H) and light (L) chain variable
(V)-region cDNAs, specific primers were used to PCR amplify the
signal peptide and V-regions while incorporating flanking
restriction sites for cloning into expression vectors encoding
downstream human IgG.kappa. or IgG4H regions. The vector for
expression of chimeric mV.kappa.-hIg.kappa. was built by amplifying
residues 401-731 (gi|63101937|) flanked by Xho I and Not I sites
and inserting this into the Xho I-Not I interval of pIRES2-DsRed2
(BD Biosciences). PCR was used to amplify the mAb V.kappa. region
from the initiator codon, appending a Nhe I or Spe I site then
CACC, to the region encoding (e.g., residue 126 of gi|76779294|),
appending a distal Xho I site. The PCR fragment was then cloned
into the Nhe I-Not I interval of the above vector. The control
human IgG.kappa. sequence corresponds to gi|49257887| residues
26-85 and gi|21669402| residues 67-709. The control human IgG4H
vector corresponds to residues 12-1473 of gi|19684072| with S229P
and L236E substitutions, which stabilize a disulphide bond and
abrogate residual FcR interaction [J. Immunol. 164: 1925-1933
(2000)], inserted between the Bgl II and Not I sites of
pIRES2-DsRed2 while adding the sequence 5'gctagctgattaattaa 3'
instead of the stop codon. PCR was used to amplify the mAb VH
region from the initiator codon, appending CACC then a Bgl II site,
to the region encoding residue 473 of gi|19684072|. The PCR
fragment was then cloned into the Bgl II-Apa I interval of the
above vector.
[0242] Expression and purification of Flu HA1 fusion protein. The
Flu HA1 antigen coding sequence is a CipA protein [Clostridium.
thermocellum] gi|479126| residues 147-160 preceding hemagglutinin
[Influenza A virus (A/Puerto Rico/8/34(H1N1))] gi|12659927| 1
residues 18-331 with a P321L change and with 6 C-terminal His
residues was inserted between the H chain vector Nhe I and Not I
sites to encode recombinant antibody-HA1 fusion proteins (rAb.HA1).
Similarly, recombinant antibody-PSA fusion proteins (rAb.PSA) were
encoded by inserting gi|34784812| prostate specific antigen
residues 101-832 with proximal sequence
GCTAGCGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACACTTCTAGCGC
(SEQ ID NO.:50) (Nhe I site and CipA spacer) and a distal Not I
site into the same H chain vector. Recombinant antibody proteins
were expressed and purified as described above for hFc fusion
proteins. In some cases the rAb.antigen coding region and the
corresponding L chain coding region were transferred to separate
cetHS-puro UCOE vectors (Millipore, CA). The use of UCOE vectors in
combination with a preadapted serum free, suspension cell line
allowed for rapid production of large quantities of protein
[Cytotechnology 38, 43-46 (2002).] CHO-S cells grown in CD-CHO with
GlutaMAX and HT media supplement (Invitrogen) were seeded at
5.times.10.sup.5 ml 24 h prior to transfection in 500 ml Corning
Ehrlenmyer flasks and incubated in 8% CO.sub.2 at 125 rpm. On the
day of transfection, 1.2.times.10.sup.7 cells with viability at
least 95% were added to a final volume of 30 ml in a 125 ml flask
in CD-CHO with GlutaMAX. 48 .mu.l of FreeStyle Max reagent
(Invitrogen) in 0.6 ml of OptiPRO SFM (Invitrogen) was added with
gentle mixing to 24 .mu.g of Sce I-linearized light chain vector
and 24 .mu.g of Sce I-linearized H chain vector mixed and sterile
filtered in 0.6 ml of OptiPRO SFM. After 20 min, the DNA-lipid
complex was slowly added to the 125 ml CHO-S culture flask with
swirling. Cells were incubated 24 h before adding 30 ml of a
combined media solution of CD-CHO with CHO-M5 (Sigma, C0363
component of CHO Kit 1) containing 5 .mu.g/ml of puromycin (A.G.
Scientific, CA), 2.times. GlutaMAX and 0.25.times. Pen/Strep
(Invitrogen). At day 2, another 5 .mu.g/ml of puromycin was added
directly to the culture and selection was allowed to proceed
.about.10-14 days while following cell viability from six days post
transfection. The viable cell count dropped and when the viable
density is .about.2-3.times.10.sup.6/ml, the cells were transferred
to fresh selection medium (CD CHO-S+CHO M5 with 2.times. GlutaMAX,
0.25.times. Pen/Strep, 10 .mu.g/ml Puromycin) at 1E6/ml. Frozen
cell stocks were prepared when viability reached >90%. Cells
were split in selection medium when cell density exceeded
2.times.10.sup.6/ml until scaled to 4.times.250 ml in 500 ml
flasks. Supernatant was harvested when cell viability dropped below
80% with a maximum final cell density .about.7.times.10.sup.6/ml.
Endotoxin levels were less than 0.2 units/ml.
[0243] Expression and purification of recombinant Flu M1 and MART-1
proteins. PCR was used to amplify the ORF of Influenza A/Puerto
Rico/8/34/Mount Sinai (H1N1) M1 gene while incorporating an Nhe I
site distal to the initiator codon and a Not I site distal to the
stop codon. The digested fragment was cloned into pET-28b(+)
(Novagen), placing the M1 ORF in-frame with a His6 tag, thus
encoding His.Flu M1 protein. A pET28b (+) derivative encoding an
N-terminal 169 residue cohesin domain from C. thermocellum
(unpublished) inserted between the Nco I and Nhe I sites expressed
Coh.His. For expression of Cohesin-Flex-hMART-1-PeptideA-His, the
sequence
GACACCACCGAGGCCCGCCACCCCCACCCCCCCGTGACCACCCCCACCACCACCGACCGGAAG
GGCACCACCGCCGAGGAGCTGGCCGGCATCGGCATCCTGACCGTGATCCTGGGCGGCAAGCG
GACCAACAACAGCACCCCCACCAAGGGCGAATTCTGCAGATATCCATCACACTGGCGGCCG (SEQ
ID NO.:51) (encoding
DTTEARHPHPPVTTPTTDRKGTTAEELAGIGILTVILGGKRTNNSTPTKGEFCRYPSHWRP (SEQ
ID NO.:52)--the italicized residues are the immunodominant
HLA-A2-restricted peptide and the underlined residues surrounding
the peptide are from MART-1) was inserted between the Nhe I and Xho
I sites of the above vector. The proteins were expressed in E. coli
strain BL21 (DE3) (Novagen) or T7 Express (NEB), grown in LB at
37.degree. C. with selection for kanamycin resistance (40 .mu.g/ml)
and shaking at 200 rounds/min to mid log phase growth when 120 mg/L
IPTG was added. After three hours, the cells were harvested by
centrifugation and stored at -80.degree. C. E. coli cells from each
1 L fermentation were resuspended in 30 ml ice-cold 50 mM Tris, 1
mM EDTA pH 8.0 (buffer B) with 0.1 ml of protease inhibitor
Cocktail II (Calbiochem, CA). The cells were sonicated on ice
2.times.5 min at setting 18 (Fisher Sonic Dismembrator 60) with a 5
min rest period and then spun at 17,000 r.p.m. (Sorvall SA-600) for
20 min at 4.degree. C. For His.Flu M1 purification the 50 ml cell
lysate supernatant fraction was passed through 5 ml Q Sepharose
beads and 6.25 ml 160 mM Tris, 40 mM imidazole, 4 M NaCl pH 7.9 was
added to the Q Sepharose flow through. This was loaded at 4 ml/min
onto a 5 ml HiTrap chelating HP column charged with Ni++. The
column-bound protein was washed with 20 mM NaPO.sub.4, 300 mM NaCl
pH 7.6 (buffer D) followed by another wash with 100 mM H.sub.3COONa
pH 4.0. Bound protein was eluted with 100 mM H.sub.3COONa pH 4.0.
The peak fractions were pooled and loaded at 4 ml/min onto a 5 ml
HiTrap S column equilibrated with 100 mM H.sub.3COONa pH 5.5, and
washed with the equilibration buffer followed by elution with a
gradient from 0-1 M NaCl in 50 mM NaPO.sub.4 pH 5.5. Peak fractions
eluting at about 500 mM NaCl were pooled. For Coh.Flu M1.His
purification, cells from 2 L of culture were lysed as above. After
centrifugation, 2.5 ml of Triton X114 was added to the supernatant
with incubation on ice for 5 min. After further incubation at
25.degree. C. for 5 min, the supernatant was separated from the
Triton X114 following centrifugation at 25.degree. C. The
extraction was repeated and the supernatant was passed through 5 ml
of Q Sepharose beads and 6.25 ml 160 mM Tris, 40 mM imidazole, 4 M
NaCl pH 7.9 was added to the Q Sepharose flow through. The protein
was then purified by Ni.sup.++ chelating chromatography as
described above and eluted with 0-500 mM imidazole in buffer D.
[0244] FIG. 13 shows the internalization of anti-CD40 mAb:IL-4DC.
IL-4DCs were treated with 500 ng/ml of anti-CD40-Alexa 568.
[0245] FIG. 14 shows CD4 and CD8 T cell proliferation by DCs
targeted with anti-CD40-HA1. 5.times.10e3 IFNDCs loaded with 2
ug/ml of anti-CD40-HA or control Ig-HA1 were co-cultured with
CFSE-labeled autologous CD4+ or CD8+ T cells (2.times.10e5) for 7
days. Cells were then stained with anti-CD4 or anti-CD8 antibodies.
Cell proliferation was tested by measuring CFSE-dilution.
[0246] FIG. 15 shows a titration of HA1.sub.-- fusion protein on
CD4+ T proliferation. IFNDCs (5K) loaded with fusion proteins were
co-cultured with CFSE-labeled CD4+ T cells (200K) for 7 days.
[0247] FIG. 16 shows IFNDCs targeted with anti-CD40-HA1 activate
HA1-specific CD4+ T cells. CD4+ T cells were restimulated with DCs
loaded with 5 uM of indicated peptides, and then intracellular
IFN.gamma. was stained.
[0248] FIG. 17 shows IFNDCs targeted with anti-CD40-HA1 activate
HA1-specific CD4+ T cells. CD4+ T cells were restimulated with DCs
loaded with indicated peptides for 36 h, and then culture
supernatant was analyzed for measuring IFN.gamma..
[0249] FIG. 18 shows that targeting CD40 results in enhanced
cross-priming of MART-1 specific CD8+ T cells. IFNDCs (5K/well)
loaded with fusion proteins were co-cultured with purified CD8+ T
cells for 10 days. Cells were stained with anti-CD8 and tetramer.
Cells are from healthy donors (HLA-A*0201+).
[0250] FIG. 19 shows targeting CD40 results in enhanced
cross-priming of MART-1 specific CD8+ T cells (Summary of
8-repeated experiments using cells from different healthy
donors).
[0251] FIG. 20 shows CD8+ CTL induced with IFNDCs targeted with
anti-CD40-MART-1 are functional. CD8+ T cells co-cultured with
IFNDCs targeted with fusion proteins were mixed with T2 cells
loaded with 10 uM peptide epitope.
[0252] FIG. 21 shows CD8+ CTL induced with IFNDCs targeted with
anti-CD40-Flu M1 are functional. CD8+ T cells co-cultured with
IFNDCs targeted with fusion proteins were mixed with T2 cells
loaded with 1.0 nM peptide epitope.
[0253] FIG. 22 shows an outline of protocol to test the ability a
vaccine composed of anti-CD4012E12 linked to PSA (prostate specific
antigen) to elicit the expansion from a naive T cell population.
PSA-specific CD4+ T cells corresponding to a broad array of PSA
epitopes. Briefly, DCs derived by culture with IFN.alpha. and
GM-CSF of monocytes from a healthy donor are incubated with the
vaccine. The next day, cells are placed in fresh medium and pure
CD4+ T cells from the same donor are added. Several days later, PSA
peptides are added and, after four hours, secreted gamma-IFN levels
in the culture supernatants are determined.
[0254] FIG. 23 shows that many PSA peptides elicit potent
gamma-IFN-production responses indicating that anti-CD4012E12 and
similar anti-CD40 agents can efficiently deliver antigen to DCs,
resulting in the priming of immune responses against multiple
epitopes of the antigen. The peptide mapping of PSA antigens.
5.times.10e3 IFNDCs loaded with 2 ug/ml of anti-CD40-PSA were
co-cultured with purified autologous CD4+ T cells (2.times.10e5)
for 8 days. Cells were then restimulated with 5 uM of individual
peptides derived from PSA for 36 h. The amount of IFN' was measured
by Luminex. Cells are from healthy donors.
[0255] FIG. 24 shows DCs targeted with anti-CD40-PSA induce
PSA-specific CD8+ T cell responses. IFNDCs were targeted with 1 ug
mAb fusion protein with PSA. Purified autologous CD8+ T cells were
co-cultured for 10 days. Cells were stained with anti-CD8 and PSA
(KLQCVDLHV)-tetramer. Cells are from a HLA-A*0201 positive healthy
donor. The results demonstrate that anti-CD40 effectively deliver
PSA to the DCs, which in turn elicit the expansion of PSA-specific
CD8+ T cells. Briefly, 5.times.10e3 IFNDCs loaded with 2 ug/ml of
anti-CD40-PSA were co-cultured with purified autologous CD8+ T
cells (2.times.10e5) for 10 days. Cells were then stained with
tetramer. Cells are from HLA-0*201 positive healthy donor.
[0256] FIG. 25 a scheme (left) and the IFN' production by T cells
of the pools of peptides and control for Donor 2. 5.times.10e3
IFNDCs loaded with 2 ug/ml of anti-CD40-Cyclin D1 were co-cultured
with purified autologous CD4+ T cells (2.times.10e5) for 8 days.
Cells were then restimulated with 5 uM of individual peptides
derived from CyclinD1 for 5 h in the presence of Brefeldin A. Cells
were stained for measuring intracellular IFN.gamma. expression.
[0257] FIG. 26 shows a peptide scan and IFN.gamma. production by T
cells obtained from the pools of peptides shown in FIG. 25 and
control for Donor 2. 5.times.10e3 IFNDCs loaded with 2 ug/ml of
anti-CD40-Cyclin D1 were co-cultured with purified autologous CD4+
T cells (2.times.10e5) for 8 days. Cells were then restimulated
with 5 uM of individual peptides derived from CyclinD1 for 5 h in
the presence of Brefeldin A. Cells were stained for measuring
intracellular IFN.gamma. expression.
[0258] In conclusion, delivering antigens to DCs, the most potent
antigen presenting cells, via CD40 is an efficient way to induce
and activate antigen specific both CD4+ and CD8+ T cell-mediated
immunity. Thus, vaccines made of anti-CD40 mAb will induce potent
immunity against cancer and infections.
[0259] Peptide Information:
TABLE-US-00048 HA1 sequences: (SEQ ID NO.: 53)
MKANLLVLLCALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL EDSHNGKLCR (SEQ
ID NO.: 54) LKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPG
DFIDYEELRE (SEQ ID NO.: 55)
QLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEK EGSYPKLKNS (SEQ
ID NO.: 56) YVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEI
AERPKVRDQA (SEQ ID NO.: 57)
GRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHE CNTKCQTPLG (SEQ
ID NO.: 58) AINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSI
[0260] Sequences of Peptides in FIG. 17
TABLE-US-00049 Peptide 22: (SEQ ID NO.: 59) SSFERFEIFPKESSWPN
Peptide 45: (SEQ ID NO.: 60) GNLIAPWYAFALSRGFG Peptide 46: (SEQ ID
NO.: 61) WYAFALSRGFGSGIITS NP sequences: (SEQ ID NO.: 62)
MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKL SDYEGRLIQNS (SEQ
ID NO.: 63) LTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELIL
YDKEEIRRIW (SEQ ID NO.: 64)
RQANNGDDATAGLTHMMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGS TLPRRSGAAG (SEQ
ID NO.: 65) AAVKGVGTMVMELVRMIKRGINDRNFWRGENGRKTRIAYERMCNILKGKF
QTAAQKAMMD (SEQ ID NO.: 66)
QVRESRNPGNAEFEDLTFLARSALILRGSVAHKSCLPACVYGPAVASGYD FEREGYSLVG (SEQ
ID NO.: 67) IDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGT
KVLPRGKLST (SEQ ID NO.: 68)
RGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISIQP TFSVQRNLPF (SEQ
ID NO.: 69) DRTTIMAAFNGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEK
AASPIVPSFD (SEQ ID NO.: 70) MSNEGSYFFGDNAEEYDN
[0261] Sequences of Peptides in FIG. 23
TABLE-US-00050 (SEQ ID NO.: 71) Peptide 22: GKWVRELVLYDKEEIRR (SEQ
ID NO.: 72) Peptide 33: RTGMDPRMCSLMQGSTL (SEQ ID NO.: 73) Peptide
46: MCNILKGKFQTAAQKAM
[0262] Prostate Specific Antigen (PSA) Sequence
TABLE-US-00051 (SEQ ID NO.: 74)
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAV CGGVLVHPQWV (SEQ
ID NO.: 75) LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNR
FLRPGDDSSHD (SEQ ID NO.: 76)
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPK KLQCVDLHVIS (SEQ
ID NO.: 77) NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSW
GSEPCALPERP (SEQ ID NO.: 78) SLYTKVVHYRKWIKDTIVANP
[0263] Sequences of Peptides in FIG. 23
TABLE-US-00052 (SEQ ID NO.: 79) Peptide 1: APLILSRIVGGWECE (SEQ ID
NO.: 80) Peptide 4: ECEKHSQPWQVLVAS (SEQ ID NO.: 81) Peptide 25:
GDDSSHDLMLLRLSE (SEQ ID NO.: 82) Peptide 26: SHDLMLLRLSEPAEL (SEQ
ID NO.: 83) Peptide 49: SGDSGGPLVCNGVLQ (SEQ ID NO.: 84) Peptide
54: GSEPCALPERPSLYT (SEQ ID NO.: 85) Peptide 56: ERPSLYTKVVHYRKW
(SEQ ID NO.: 86) Peptide 58: VVHYRKWIKDTIVAN
[0264] Cyclin D1 Sequence
TABLE-US-00053 (SEQ ID NO.: 87)
MRSYRFSDYLHMSVSFSNDMDLFCGEDSGVFSGESTVDFSSSEVDSWPG DSIACFIEDER (SEQ
ID NO.: 88) HFVPGHDYLSRFQTRSLDASAREDSVAWILKVQAYYNFQPLTAYLAVNY
MDRFLYARRLP (SEQ ID NO.: 89)
ETSGWPMQLLAVACLSLAAKMEEILVPSLFDFQVAGVKYLFEAKTIKRM ELLVLSVLDWR (SEQ
ID NO.: 90) LRSVTPFDFISFFAYKIDPSGTFLGFFISHATEIILSNIKEASFLEYWP
SSIAAAAILCV (SEQ ID NO.: 91)
ANELPSLSSVVNPHESPETWCDGLSKEKIVRCYRLMKAMAIENNRLNTP KVIAKLRVSVR (SEQ
ID NO.: 92) ASSTLTRPSDESSFSSSSPCKRRKLSGYSWVGDETSTSN
[0265] Sequences of Peptides in FIG. 26.
TABLE-US-00054 (SEQ ID NO.: 93) Peptide 7: DRVLRAMLKAEETCA (SEQ ID
NO.: 94) Peptide 8: RAMLKAEETCAPSVS (SEQ ID NO.: 95) Peptide 10:
TCAPSVSYFKCVQKE
[0266] MART-1 Antigen. MART-1 is a tumor-associated melanocytic
differentiation antigen. Vaccination with MART-1 antigen may
stimulate a host cytotoxic T-cell response against tumor cells
expressing the melanocytic differentiation antigen, resulting in
tumor cell lysis.
[0267] FIG. 27 shows the expression and construct design for
anti-CD40-MART-1 peptide antibodies. FIG. 28 is a summary of the
CD4.sup.+ and CD8.sup.+ immunodominant epitopes for MART-1. FIGS.
27 and 28 show the use of the flexible linker technology to permit
the successful expression of recombinant anti-DC receptor targeting
antibodies fused to significant (.about.2/3) parts of human MART-1.
Recombinant antibody fused at the H chain C-terminus to the entire
MART-1 coding region is not at all secreted from production
mammalian cells [not shown]. The Flex-v1-hMART-1-Pep-3-f4-Pep-1
adduct is particularly well expressed and is one preferred
embodiment of a MART-1-targeting vaccine, as is the
Flex-v1-hMART-1-Pep-3-f4-Pep-1-f3-Pep-2 adduct which bears a
maximum load of MART-1 epitopes. Slide 2 of the MART-1 powerpoint
presentation shows that these adducts can be successfully appended
to multiple anti-DC receptor vehicles.
[0268] The sequence below is a H chain-hMART-1 peptides string of
pep3-pep1-pep2 fusion protein where each hMART1 peptide sequence
[bold-italics] is separated by a inter-peptide spacer f [shown in
bold]. In this case, a 27-amino-acid long linker flex-v1(v1)
[italics] derived from cellulosomal anchoring scaffoldin B
precursor [Bacteroides cellulosolvens--described in the gag-nef
vaccine invention disclosure] was inserted between the H chain
C-terminus and the hMART1 peptides-flexible spaces string. The
underlined AS residues are joining sequences.
TABLE-US-00055
[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3-f4-Pep-1]
C981 is: (SEQ ID NO.: 96)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRF
TISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRS
TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV
DHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV
QFNVVYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP
AS ITVAATATPTVTPTVNAT PSAAAS
[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3-f4-Pep-1-
f3-Pep-2] C978 is: (SEQ ID NO.: 97)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRF
TISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRS
TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV
DHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV
QFNVVYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP
AS ASTNGSITVAATAPTVTPTVNAT PSAAAS ASTVTPTATATPSAIVTTITPT ATTKPAS
[mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3-f4-Pep-1] C1012
is: (SEQ ID NO.: 98)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSR
LTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPL
APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK
TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
QEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIET
ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSN
PNPNPAS ASTNGSITVAATAPTVTP TVNATPSAAAS AS
[mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-344-Pep-1-f3-Pep-
2] C1013 is: (SEQ ID NO.: 99)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSR
LTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPL
APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK
TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
QPEVQFNVVYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI
SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNP
KPNPAS ASTNGSITVAATAPTVTPT VNATPSAAAS ASTVTPTATATPSAIVTT
ITPTATTKPAS AS
[0269] MART-1 DNA Sequence:
[0270] MART-1 constructs with 3 peptides, Start/stop sites are
underlined, peptide 1 is bold, peptide 2 is bold-italics and
peptide 3 is bold-underlined:
TABLE-US-00056 (SEQ ID NO.: 100)
AACACCGACAACAACAGATGATCTGGATGCAGCTAGTGGGTTTGATCATCGGGACAGCAAAGT
GTCTCTTCAAGAGAAAAACTGTGAACCTGTGGTTCCCAATGCTCCACCTGCTTATGAGAAACT
CTCTGCAGAACAGTCACCACCACCTTATTCACCTGCTAGTACCAACGGCAGCATCACCGTGGC
CGCCACCGCCCCCACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTAGT GCTAGTAC
CGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGACCACCATCACCCCCACCGCCAC
CACCAAGCCCGCTAGTGTCTTACTGCTCATCGGCTGTTGGTATTGTAGAAGACGAAATGGATA
CAGAGCCTTGATGGATAAAAGTCTTCATGTTGGCACTCAATGTGCCTTAACAAGAAGATGCCC
ACAAGAAGGGtgaGCGGCCGCATCGAAGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTG
CAGGCATGC
[0271] Peptide 3 is bold followed by the Flex-4 amino acid
sequence--underlined.
TABLE-US-00057 (SEQ ID NO.: 101)
GFDHRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPPYSPASTNGSITV AATAPTVTPT
[0272] Peptide 1 is bold followed by the Flex-3 amino acid
sequence--underlined.
TABLE-US-00058 (SEQ ID NO.: 102)
VNATPSAAASMPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILG ASTVTPTATATP
[0273] Peptide 3 is bold.
TABLE-US-00059 (SEQ ID NO.: 103)
SAIVTTITPTATTKPASVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALT RRCPQEG
[0274] MART1-Peptide 3, the italicized portion is the CD4+
immunodominant epitope.
TABLE-US-00060 (SEQ ID NO.: 104)
GFDHRDSKVSLQEKNCEPWPNAPPAYEKLSAEQSPPPYSP
[0275] Flex-4
TABLE-US-00061 (SEQ ID NO.: 105) ASTNGSITVAATAPTVTPTVNATPSAAAS
[0276] MART1-Peptide 1 the italicized portion is the CD4+
immunodominant epitope and the underlined-italicized portion is the
CD8+ immunodominant epitope
TABLE-US-00062 (SEQ ID NO.: 106)
MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILG
[0277] Flex-3:
TABLE-US-00063 (SEQ ID NO.: 107) ASTVTPTATATPSAIVTTITPTATTKPAS
[0278] MART1-Peptide 2 the italicized portion is the CD4+
immunodominant epitope.
TABLE-US-00064 (SEQ ID NO.: 108)
VLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEG
[0279] MART1 Constructs with Two Peptides:
[0280] Peptide 3 is bold-italics-underlined, flex-4 is bold and
Peptide 1 is bold-italics-underlined:
TABLE-US-00065 (SEQ ID NO.: 109) ASTNGSITVAATAPTVTPTVNATPS AS
[0281] Protein Sequence: C978.
rAB-cetHS-puro[manti-CD40.sub.--12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-P-
ep-3 (bold-italics-underlined)-f4 (bold)-Pep-1 (bold-italics)-f3
(italics)-Pep-2 (bold-underlined)]
TABLE-US-00066 (SEQ ID NO.: 110)
MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFS
DYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTL
YLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVF
PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN
VVYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSN PKPNPAS
ASTNGSITVAATAPTVTPTVNATPSAAA ASTVTPT
ATATPSAIVTTTTPTATTKPASVLLLIGCWYCRRRNGYRALMDKSLHVG
TQCALTRRCPQEGAS
[0282] Protein Sequence: C981.
rAB-cetHS-puro[manti-CD40.sub.--12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-P-
ep-3 (bold-italics-underlined)-f4-(bold)-Pep-1]
(bold-underlined)
TABLE-US-00067 (SEQ ID NO.: 111)
MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTF
SDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKN
TLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGP
SVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY
GPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED
PEVQFNVVYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD
KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPT NNSTPTNNSNPKPNPAS
ASTNGSITVAATAPTVTPTVNATPSAAASMPREDAHF
IYGYPKKGHGHSYTTAEEAAGIGILTVILGAS
[0283] GP100 Antigen. GP100 antigen is a melanoma-associated
antigen. When administered in a vaccine formulation, gp100 antigen
may stimulate a cytotoxic T cell HLA-A2.1-restricted immune
response against tumors that express this antigen, which may result
in a reduction in tumor size.
[0284] GP100 ectodomain coding region fused to recombinant antibody
H chain coding region is not at all secreted by production
mammalian cells [not shown]. The total sequence is shown
below--italics residues are the leader sequence and the
transmembrane domain, the peptides are in bold-italics and the
transmembrane domain is italics-underlined.
TABLE-US-00068 (SEQ ID NO.: 112)
MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQL
YPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKV
LPDGQVIWYNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWS QKRSFVYVW
LGGPVSGLSIGTGRAMLGTHTMEVTVYHRR GSRSYVPLAHSSSAFT
SVSQLRALDGGNKHFLRNQPLTF ALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHT
QVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTT
PGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEV
MGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELP
IPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRY
GSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEI
SSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNS
LAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRLMKQD
FSVPQLPHSSSHWLRLPRIFCSCPIGENSPLLSGQQV
[0285] Known HLA-A0201 restricted peptides sequences are: GP100 M:
209-217 (2M): IMDQVPFSV (SEQ ID NO.:113); 209-217 WT: ITDQVPFSV
(SEQ ID NO.:114) GP100 M: 280-288 (9V): YLEPGPVTV (SEQ ID NO.:115)
280-288 WT: YLEPGPVTA (SEQ ID NO.:116) GP100 WT: 154-162: KTWGQYWQV
(SEQ ID NO.:117)
[0286] FIG. 29-33 show the gp100 adducts which were successfully
expressed as secreted anti-DC receptor targeting vaccines. These
employed the use of the flexible linker sequences and fragmentation
and shuffling of the gp100 ectodomain coding region. Preferred
embodiments of gp100 vaccine adducts are described.
[0287] FIG. 29 shows the expression and construct design for
anti-CD40-gp100 peptide antibodies. FIG. 30 shows the design for
additional anti-CD40-gp100 peptide antibodies. FIG. 31 shows the
expression and construct design for additional anti-CD40-gp100
peptide antibodies. FIG. 32 is a summary of the CD4.sup.+ and
CD8.sup.+ immunodominant epitopes for gp100. FIG. 33 shows the
expression and construct design for additional anti-CD40-gp100
peptide antibodies.
[0288]
rAB-cetHS-puro[manti-CD40.sub.--12E12.3F3_H-LV-hIgG4H-C-Flex-hgp100-
-Pep-1-f4-Pep-3-f3-Pep-4-f4-Pep-5-f3-Pep-2] C1285, the peptides are
bold-italics, flexible linkers are bold and the underlined AS
residues are joining sequences:
TABLE-US-00069 (SEQ ID NO.: 118)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTI
SRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSES
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN
TKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVD
GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMHEALHNHYTQKSLSLSLGKAS ASTNGSITV AATAPTVTPTVNATPSAAAS
ASTVTPTATATPSAIVTTITPTATTKP TNGSITVAATAPTVTPTVNATPSAAAS
TVTPTATATPSAIVTTITPTATT KPAS AS
[0289]
rAB-cetHS-puro[hIgG4H-C-Flex-hgp100-Pep-1-f4-Pep-3-f3-Pep-4-f4-Pep--
5-f3-Pep-2] C1286:
TABLE-US-00070 (SEQ ID NO.: 119)
RLQLQESGPGLLKPSVTLSLTCTVSGDSVASSSYYWGWVRQPPGKGLEWIGTINFSGNMYYSPSLRSRVTMSAD-
MSE
NSFYLKLDSVTAADTAVYYCAAGHLVMGFGAHWGQGKLVSVSPASTKGPSVFPLAPCSRSTSESTAALGCLVKD-
YFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP-
CPA
PEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTYRVVS-
VLT
VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW-
ESN
GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS
TNGSITVAATAPTVTPTVNATPSAAAS ASTVTPTATATPSAIVTTITPTATTKP
ASTNGSITVAATAPTVTPTVNATPSAAAS ASTVTPTATATPSAIVTTITPTATTKPAS AS
[0290] gp100:--Nucleic Acid Sequence. Peptide 1-underlined, Peptide
2-italics, Peptide 3-bold, Peptide 4-bold-underlined, Peptide 5
bold-italics.
TABLE-US-00071 (SEQ ID NO.: 120)
GATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAAAAGTACCCAGAAA
CCAGGACTGGCTTGGTGTCTCAAGGCAACTCAGAACCAAAGCCTGGAACAGGCAGCTGTATCCAGAGT
GGACAGAAGCCCAGAGACTTGACTGCTGGAGAGGTGGTCAAGTGTCCCTCAAGGTCAGTAATGATGGG
CCTACACTGATTGGTGCAAATGCCTCCTTCTCTATTGCCTTGAACTTCCCTGGAAGCCAAAAGGTATT
GCCAGATGGGCAGGTTATCTGGGTCAACAATACCATCATCAATGGGAGCCAGGTGTGGGGAGGACAGC
CAGTGTATCCCCAGGAAACTGACGATGCCTGCATCTTCCCTGATGGTGGACCTTGCCCATCTGGCTCT
TGGTCTCAGAAGAGAAGCTTTGTTTATGTCTGGAAGACCTGGGGCCAATACTGGCAAGTTCTAGGGGG
CCCAGTGTCTGGGCTGAGCATTGGGACAGGCAGGGCAATGCTGGGCACACACACCATGGAAGTGACTG
TCTACCATCGCCGGGGATCCCAGAGCTATGTGCCTCTTGCTCATTCCAGCTCAGCCTTCACCATTACT
GACCAGGTGCCTTTCTCCGTGAGCGTGTCCCAGTTGCGGGCCTTGGATGGAGGGAACAAGCACTTCCT
GAGAAATCAGGCTAGTACCAACGGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGACCCCCACCG
TGAACGCCACCCCCAGCGCCGCCGCTAGTGGCACCACAGATGGGCACAGGCCAACTGCAGAGGCCCCT
AACACCACAGCTGGCCAAGTGCCTACTACAGAAGTTGTGGGTACTACACCTGGTCAGGCGCCAACTGC
AGAGCCCTCTGGAACCACATCTGTGCAGGTGCCAACCACTGAAGTCATAAGCACTGCACCTGTGCAGA
TGCCAACTGCAGAGAGCACAGGTATGACACCTGAGAAGGTGCCAGTTTCAGAGGTCATGGGTACCACA
CTGGCAGAGATGTCAACTCCAGAGGCTACAGGTATGACACCTGCAGAGGTATCAATTGTGGTGCTTTC
TGGAACCACAGCTGCAGCTAGTACCGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGACCA
CCATCACCCCCACCGCCACCACCAAGCCCGCTAGTCAGGTAACAACTACAGAGTGGGTGGAGACCACA
GCTAGAGAGCTACCTATCCCTGAGCCTGAAGGTCCAGATGCCAGCTCAATCATGTCTACGGAAAGTAT
TACAGGTTCCCTGGGCCCCCTGCTGGATGGTACAGCCACCTTAAGGCTGGTGAAGAGACAAGTCCCCC
TGGATTGTGTTCTGTATCGATATGGTTCCTTTTCCGTCACCCTGGACATTGTCCAGGCTAGTACCAAC
GGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGC
CGCTAGTGGTATTGAAAGTGCCGAGATCCTGCAGGCTGTGCCGTCCGGTGAGGGGGATGCATTTGAGC
TGACTGTGTCCTGCCAAGGCGGGCTGCCCAAGGAAGCCTGCATGGAGATCTCATCGCCAGGGTGCCAG
CCCCCTGCCCAGCGGCTGTGCCAGCCTGTGCTACCCAGCCCAGCCTGCCAGCTGGTTCTGCACCAGAT
ACTGAAGGGTGGCTCGGGGACATACTGCCTCAATGTGTCTCTGGCTGATACCAACAGCCTGGCAGTGG
TCAGCACCCAGCTTATCGTGCCTGGGATTCTTCTCACAGGTCAAGAAGCAGGCCTTGGGCAGTAAGCT
AGTACCGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGACCACCATCACCCCCACCGCCAC
CACCAAGCCCGCTAGT GCTAGCTGA
[0291] GP100-Peptide 1--Nucleic Acid Sequence.
TABLE-US-00072 (SEQ ID NO.: 121)
GATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAAC
AACAAAAGTACCCAGAAACCAGGACTGGCTTGGTGTCTCAAGGCAACTCA
GAACCAAAGCCTGGAACAGGCAGCTGTATCCAGAGTGGACAGAAGCCCAG
AGACTTGACTGCTGGAGAGGTGGTCAAGTGTCCCTCAAGGTCAGTAATGA
TGGGCCTACACTGATTGGTGCAAATGCCTCCTTCTCTATTGCCTTGAACT
TCCCTGGAAGCCAAAAGGTATTGCCAGATGGGCAGGTTATCTGGGTCAAC
AATACCATCATCAATGGGAGCCAGGTGTGGGGAGGACAGCCAGTGTATCC
CCAGGAAACTGACGATGCCTGCATCTTCCCTGATGGTGGACCTTGCCCAT
CTGGCTCTTGGTCTCAGAAGAGAAGCTTTGTTTATGTCTGGAAGACCTGG
GGCCAATACTGGCAAGTTCTAGGGGGCCCAGTGTCTGGGCTGAGCATTGG
GACAGGCAGGGCAATGCTGGGCACACACACCATGGAAGTGACTGTCTACC
ATCGCCGGGGATCCCAGAGCTATGTGCCTCTTGCTCATTCCAGCTCAGCC
TTCACCATTACTGACCAGGTGCCTTTCTCCGTGAGCGTGTCCCAGTTGCG
GGCCTTGGATGGAGGGAACAAGCACTTCCTGAGAAATCAG
[0292] Protein Sequence:
TABLE-US-00073 (SEQ ID NO.: 122)
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEA
QRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIW
VNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVW
KTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPLA
HSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQ
[0293] GP100-Peptide 3
TABLE-US-00074 (SEQ ID NO.: 123)
GGCACCACAGATGGGCACAGGCCAACTGCAGAGGCCCCTAACACCACAG
CTGGCCAAGTGCCTACTACAGAAGTTGTGGGTACTACACCTGGTCAGGC
GCCAACTGCAGAGCCCTCTGGAACCACATCTGTGCAGGTGCCAACCACT
GAAGTCATAAGCACTGCACCTGTGCAGATGCCAACTGCAGAGAGCACAG
GTATGACACCTGAGAAGGTGCCAGTTTCAGAGGTCATGGGTACCACACT
GGCAGAGATGTCAACTCCAGAGGCTACAGGTATGACACCTGCAGAGGTA
TCAATTGTGGTGCTTTCTGGAACCACAGCTGCA
[0294] Protein Sequence:
TABLE-US-00075 (SEQ ID NO.: 124)
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTT
EVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEV SIVVLSGTTAA
[0295] GP100-Peptide 4:
TABLE-US-00076 (SEQ ID NO.: 125)
CAGGTAACAACTACAGAGTGGGTGGAGACCACAGCTAGAGAGCTACCTA
TCCCTGAGCCTGAAGGTCCAGATGCCAGCTCAATCATGTCTACGGAAAG
TATTACAGGTTCCCTGGGCCCCCTGCTGGATGGTACAGCCACCTTAAGG
CTGGTGAAGAGACAAGTCCCCCTGGATTGTGTTCTGTATCGATATGGTT
CCTTTTCCGTCACCCTGGACATTGTCCAG
[0296] Protein Sequence:
TABLE-US-00077 (SEQ ID NO.: 126)
QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLR
LVKRQVPLDCVLYRYGSFSVTLDIVQ
[0297] GP100-Peptide 5
TABLE-US-00078 (SEQ ID NO.: 127)
GGTATTGAAAGTGCCGAGATCCTGCAGGCTGTGCCGTCCGGTGAGGGGG
ATGCATTTGAGCTGACTGTGTCCTGCCAAGGCGGGCTGCCCAAGGAAGC
CTGCATGGAGATCTCATCGCCAGGGTGCCAGCCCCCTGCCCAGCGGCTG
TGCCAGCCTGTGCTACCCAGCCCAGCCTGCCAGCTGGTTCTGCACCAGA
TACTGAAGGGTGGCTCGGGGACATACTGCCTCAATGTGTCTCTGGCTGA
TACCAACAGCCTGGCAGTGGTCAGCACCCAGCTTATCGTGCCTGGGATT
CTTCTCACAGGTCAAGAAGCAGGCCTTGGGCAG
[0298] Protein Sequence:
TABLE-US-00079 (SEQ ID NO.: 128)
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRL
CQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIVPGI LLTGQEAGLGQ
[0299] GP100-Peptide 2
TABLE-US-00080 (SEQ ID NO.: 129)
CCTCTGACCTTTGCCCTCCAGCTCCATGACCCTAGTGGCTATCTGGCTG
AAGCTGACCTCTCCTACACCTGGGACTTTGGAGACAGTAGTGGAACCCT
GATCTCTCGGGCACYTGTGGTCACTCATACTTACCTGGAGCCTGGCCCA
GTCACTGCCCAGGTGGTCCTGCAGGCTGCCATTCCTCTCACCTCCTGTG
GCTCCTCCCCAGTTCCAGCTAGC
[0300] Protein Sequence:
TABLE-US-00081 (SEQ ID NO.: 130)
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGP
VTAQVVLQAAIPLTSCGSSPVPAS
[0301] Cyclin B1 Antigen. Cyclin B1, also known as CCNB1, is a
human gene that encodes a regulatory protein involved in mitosis.
Cyclin B1 complexes with p34(cdc2) to form the maturation-promoting
factor (MPF). Two alternative transcripts are known that are the
result of alternative transcription initiation sites. A first
transcript encodes a constitutively expressed transcript. The
second transcript is a cell cycle-regulated transcript expressed
predominantly during G2/M phase.
[0302] FIG. 34A shows that full-length Cyclin B1 fused to the
C-terminus of either antibody H chain or cohesion fail to be
secreted from mammalian 293F cells. FIG. 34B shows that full-length
Cyclin B1 fused to the C-terminus of either antibody H chain or
cohesion fail to be secreted from mammalian 293F cells.
[0303] The data are anti-human Fc and anti-cohesin ELISA on serial
dilutions of transfection supernatants. rAb.Cyclin B1 and
Coh.Cyclin B1 proteins are poorly expressed as products secreted
from mammalian cells.
[0304] The following amino acid sequence is human cyclin B1. Two
peptide regions known to contain T cell epitopes are highlighted in
bold-underlined and italics-underlined.
TABLE-US-00082 (SEQ ID NO.: 131)
MALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTALGDIGNKVSEQLQAKMPMKK
EAKPSATGKVIDKKLPKPLEKVPMLVPVPVSEPVPEPEPEPEPEPVKEEKLSPEPILVDTASPS
PMETSGCAPAEEDLCQAFSDVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQLEEEQAVRPKYL
LGREVTGNMRAILIDWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKKMLQLVGVTAMFIAS
KYEEMYPPEIGDFAFVTDNTYTKHQIRQ
DMVHFPPSQIAAGAFCLALKILDNGEWTPTLQHYLSYTEESLLPVMQHLAK
NVVMVNQGLTKHMTVKNKYATSKHAKISTLPQLNSALVQDLAKAVAKVHHHHHH Peptide-1
(SEQ ID NO.: 132)
MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDY Peptide-2 (SEQ
ID NO.: 133) DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK
[0305] FIG. 35 shows a summary of relative expression levels of
prototype Cyclin B1 vaccines secreted from transfected mammalian
293F cells. The flexible linker sequences facilitate secretion.
[0306] C1189
rAB-cetHS-puro[manti-CD40.sub.--12E12.3F3_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinB1-Peptide-2(italics)-Peptide-1 (bold-italics)-f4
(bold)] [AS linkers--underlined]
TABLE-US-00083 (SEQ ID NO.: 134)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAY
INSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRG
LPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY
TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQ
TPTNTISVTPTNNSTPTNNSNPKPNPASDWLVQVQMKFRLLQETMYMTVS
IIDRFMQNNCVPKKASMEMKILRALNFGLGRPLPLHFLRRAS ASTNDSI
TVAATAPTVTPTVNATPSAAAS
[0307] Above is the sequence of the mature secreted H chain for one
form of anti-CD4012E12-cyclin B1 vaccine. The AS residues are from
joining restriction sites. The DNA coding sequence is shown below,
and this includes the signal peptide.
TABLE-US-00084 (SEQ ID NO.: 135)
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGT
CCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCCG
GAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGAC
TATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGT
CGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAA
AGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTG
CAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAG
ACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCTCAG
TCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCG
CCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAA
GACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACA
AGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCT
GAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA
CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG
TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTG
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTA
CACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCA
GGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAG
TCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCC
CCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTGACTGGCTAGTA
CAGGTTCAAATGAAATTCAGGTTGTTGCAGGAGACCATGTACATGACTGT
CTCCATTATTGATCGGTTCATGCAGAATAATTGTGTGCCCAAGAAGGCTA
GTATGGAAATGAAGATTCTAAGAGCTTTAAACTTTGGTCTGGGTCGGCCT
CTACCTTTGCACTTCCTTCGGAGAGCATCTAAGATTGGAGAGGTTGATGT
CGAGCAACATACTTTGGCCAAATACCTGATGGAACTAACTATGTTGGACT
ATGCTAGTACCAACGACAGCATCACCGTGGCCGCCACCGCCCCCACCGTG
ACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTAGCTGA
[0308] C1143
rAB-cetHS-puro[manti-CD40.sub.--12E12.3F3_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinB1-Peptide-2(italics)-f3 (bold)] [AS
linkers--underlined].
TABLE-US-00085 (SEQ ID NO.: 136)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAY
INSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRG
LPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY
TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL
PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQT
PTNTISVTPTNNSTPTNNSNPKPNPASDWLVQVQMKERLLQETMYMTVSI
IDRFMQNNCVPKKASTVTPTATATPSAIVTTITPTATTKPAS
[0309] Above is the sequence of the mature secreted H chain for one
form of anti-CD4012E12-cyclin B1 vaccine. The AS residues are from
joining restriction sites. The DNA coding sequence is shown below,
and this includes the signal peptide.
TABLE-US-00086 (SEQ ID NO.: 137)
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGT
CCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCCG
GAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGAC
TATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGT
CGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAA
AGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTG
CAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAG
ACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCTCAG
TCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCG
CCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAA
GACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACA
AGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCT
GAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA
CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG
TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTG
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTA
CACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCA
GGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAG
TCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCC
CCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTGACTGGCTAGTA
CAGGTTCAAATGAAATTCAGGTTGTTGCAGGAGACCATGTACATGACTGT
CTCCATTATTGATCGGTTCATGCAGAATAATTGTGTGCCCAAGAAGGCTA
GTACCGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGACCACC
ATCACCCCCACCGCCACCACCAAGCCCGCTAGCTGA
[0310] C911
rAB-cetHS-puro[manti-CD40.sub.--12E12.3F3_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinB1-Peptide-1 (italics)-f4 (bold)]
TABLE-US-00087 (SEQ ID NO.: 138)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAY
INSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRG
LPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY
TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQ
TPTNTISVTPTNNSTPTNNSNPKPNPASMEMKILRALNFGLGRPLPLHFL
RRASKIGEVDVEQHTLAKYLMELTMLDYASTNGSITVAATAPTVTPTVNA TPSAAAS
[0311] C911
rAB-cetHS-puro[manti-CD40.sub.--12E12.3F3_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinB1-Peptide-1 (italics)-f4 (bold)] nucleic acid
sequence.
TABLE-US-00088 (SEQ ID NO.: 139)
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGT
CCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCCG
GAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGAC
TATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGT
CGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAA
AGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTG
CAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAG
ACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCTCAG
TCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCG
CCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAA
GACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACA
AGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCT
GAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA
CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG
TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTG
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTA
CACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCA
GGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAG
TCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCC
CCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTATGGAAATGAAG
ATTCTAAGAGCTTTAAACTTTGGTCTGGGTCGGCCTCTACCTTTGCACTT
CCTTCGGAGAGCATCTAAGATTGGAGAGGTTGATGTCGAGCAACATACTT
TGGCCAAATACCTGATGGAACTAACTATGTTGGACTATGCTAGTACCAAC
GGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGACCCCCACCGTGAA
CGCCACCCCCAGCGCCGCCGCTAGCTGA
[0312] D-type Cyclin Antigen. D-type cyclins are predominantly
expressed in the G1 phase of the cell cycle. The expression pattern
of cyclin D1 has been extensively studied in certain cancer types
including lymphoma and non-small cell lung cancer. Approximately 30
percent of breast carcinomas are Cyclin D1 positive. Over
expression of Cyclin D1 is now a well established criterion for the
diagnosis of Mantle Cell Lymphoma, a malignant, non-Hodgkin's
lymphoma which is characterized by a unique chromosomal
translocation t(11;14).
[0313] Cyclin D1--Peptide 1-bold, Peptide 2-bold-underlined,
Peptide-3 italics, Peptide 4-underlined.
TABLE-US-00089 (SEQ ID NO.: 140)
MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCVQK
EVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQ
LLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELLLVNKLKW
NLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPP
SMVAAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQ
IEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI
[0314] Pep-1
TABLE-US-00090 (SEQ ID NO.: 141)
MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV
[0315] Pep-2
TABLE-US-00091 (SEQ ID NO.: 142)
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSR
LQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELL
[0316] Pep-3
TABLE-US-00092 (SEQ ID NO.: 143)
LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDV KFISNPPSMV
[0317] Pep-4
TABLE-US-00093 (SEQ ID NO.: 144)
AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEA
LLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI
[0318] Flex-4 sequence
TABLE-US-00094 (SEQ ID NO.: 14) TNGSITVAATAPTVTPTVNATPSAA
[0319] Flex-3 sequence
TABLE-US-00095 (SEQ ID NO.: 13) TVTPTATATPSAIVTTITPTATTKP
[0320] Flex-var1
TABLE-US-00096 (SEQ ID NO.: 145) QTPTNTISVTPTNNSTPTNNSNPKPNP
[0321] FIG. 35 shows Cyclin B1 segmentation strategy based on known
or predicted structural domain regions.
[0322] FIG. 36 shows that Cyclin D1 segments p1, p3, and p4, but
not p2 express well as direct fusions to the H chain C-terminus.
These are transient transfections of the H chain vectors
co-transfected with the L chain expression vector into 293F cells
and the supernatants harvested after 48-72 hours of expression. The
Cyclin D1 p3+p4 segments joined together at the H chain C-terminus
also express well, but various other combinations, with and without
interspersed flex segments do not express, or express very
poorly.
[0323] FIG. 37 shows the relative expression levels of various
Cyclin D1 segments as direct fusions to the H chain C-terminus in
various combinations with flexible linker sequences. These are
transient transfections of the H chain vectors co-transfected with
the L chain expression vector into 293F cells and the supernatants
harvested after 48-72 hours of expression. The Cyclin D1
p2+p3+p4+f4 segments joined together at the H chain C-terminus also
express well enough for vaccine production.
[0324] Sequences of useful anti-DCIR 9E8-cyclin D1H chain fusion
proteins are below.
[0325] 1082 is
rAB-pIRES2[mAnti-DCIR.sub.--9E8_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinD1-Pep-1 (italics)-f4 (bold)-]
TABLE-US-00097 (SEQ ID NO.: 146)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEW
LAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCA
RSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSEST
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAK
TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS
KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH
YTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASMEHQLL
CCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCVASTNGSI
TVAATAPTVTPTVNATPSAAAS
[0326] C1086 is
rAB-pIRES2[mAnti-DCIR.sub.--9E8_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinD1-Pep-2-(bold)-Pep-3(bold-underlined)-Pep-4
(italics)-f4)(bold)]
TABLE-US-00098 (SEQ ID NO.: 147)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDGVEVHNAKTKPREE
QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS
LGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASQKEVLPSMRKIVATWM
LEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVASKMK
ETIPLTAEKLCIYTDNSIRPEELLQMELLLVNKLKWNLAAMTPHDFIEHF
LSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPPSMVAAGSVVAAVQG
LNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQ
NMDPKAAEEEEEEEEEVDLACTPTDVRDVDIASTNGSITVAATAPTVTPT VNATPSAAAS
[0327] FIG. 38 show a summary of various H chain-Cyclin D1 segment
constructs and their relative expressibility as vaccines.
[0328] FIG. 39 above shows that full-length Cyclin D1 fused to the
C-terminus of a DC targeting antibody H chain is very poorly
expressed as a secreted recombinant antibody.
[0329] anti-CD40.sub.--12E12.3F3
[0330] anti-CD40.sub.--12E12.3F3_H-V-hIgG4H-C--underlined region
shows the Heavy chain V region amino acid sequence:
TABLE-US-00099 (SEQ ID NO.: 148)
MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFSD
YYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYL
QMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLA
PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNVVYVDG
VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS
IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA
LHNHYTQKSLSLSLGKAS
[0331] anti-CD40.sub.--12E12.3F3_K-V-hIgGK-C--underlined region
shows the Light chain V region amino acid sequence
TABLE-US-00100 (SEQ ID NO.: 149)
MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCSASQGIS
NYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTIGNLEP
EDIATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-
[0332] anti-CD40.sub.--12B4.2C10
[0333] anti-CD40.sub.--12B4.2C10 Heavy Chain:
TABLE-US-00101 (SEQ ID No.: 150)
MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTD
YVLHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYM
ELSSLTSEDSAVYYCARGYPAYSGYAMDYWGQGTSVTVSSAKTTPPSVYP
LAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQKG EFV
[0334] anti-CD40.sub.--12B4.2C10 Light Chain:
TABLE-US-00102 (SEQ ID No.: 151)
MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRASQDIS
NYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQ
EDIATYFCHHGNTLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGA
SVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT
LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
[0335] anti-CD40.sub.--12B4.2C10 Light Chain--alternative clone
(17K6)
TABLE-US-00103 (SEQ ID No.: 152)
MDFQVQIFSFLLISASVIMSRGQIVLTQSPAILSASPGEKVTMTCSASSS
VSYMYRYQQKPGSSPKPWIYGTSNLASGVPARFSGSGSGTSYSLTISSME
AEDAATYYCQQYHSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGG
ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTL
TLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
[0336] anti-CD40.sub.--11B6.1C3
[0337] anti-CD40.sub.--11B6.1C3 Heavy Chain:
TABLE-US-00104 (SEQ ID No.: 153)
MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKASGYSFTG
YYMHWVKQSHVKSLEWIGRINPYNGATSYNQNFKDKASLTVDKSSSTAYM
ELHSLTSEDSAVYYCAREDYVYWGQGTTLTVSSAKTTPPSVYPLAPGSAA
QTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQKGEFV
[0338] anti-CD40.sub.--11B6.1C3 Light Chain:
TABLE-US-00105 (SEQ ID No.: 154)
MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVH
SNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFALKIS
RVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLT
SGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMS
STLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
[0339] [anti-CD40.sub.--12E12.3F3_K-V-hIgGK-C]--underlined region
shows the Light chain V region sequence
TABLE-US-00106 (SEQ ID NO.: 155)
ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGG
TACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCT
CTCTAGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGC
AATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCT
GATCTATTACACATCAATTTTACACTCAGGAGTCCCATCAAGGTTCAGTG
GCAGTGGGTCTGGGACAGATTATTCTCTCACCATCGGCAACCTGGAACCT
GAAGATATTGCCACTTACTATTGTCAGCAGTTTAATAAGCTTCCTCCGAC
GTTCGGTGGAGGCACCAAACTCGAGATCAAACGAACTGTGGCTGCACCAT
CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC
TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACA
GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG
CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCAC
CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG
[0340] [anti-CD40.sub.--12E12.3F3_H-V-hIgG4H-C]--underlined region
shows the Heavy chain V region sequence:
TABLE-US-00107 (SEQ ID NO.: 156)
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGT
CCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTG
GAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGAC
TATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGT
CGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAA
AGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTG
CAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAG
ACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCTCAG
TCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCG
CCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAA
GACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACA
AGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCT
GAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA
CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG
TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTG
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTA
CACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCA
GGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAG CTGA
[0341] anti-CD40.sub.--12B4.2C10_H-V-hIgG4H-C heavy chain
TABLE-US-00108 (SEQ ID NO.: 157)
ATGGAATGGAGTTGGATATTTCTCTTTCTTCTGTCAGGAACTGCAGGTGT
CCACTCTGAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTG
GGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTGAC
TATGTTTTGCACTGGGTGAAACAGAAGCCTGGGCAGGGCCTTGAGTGGAT
TGGATATATTAATCCTTACAATGATGGTACTAAGTACAATGAGAAGTTCA
AAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATG
GAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAG
GGGCTATCCGGCCTACTCTGGGTATGCTATGGACTACTGGGGTCAAGGAA
CCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCC
CTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTG
CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG
GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA
GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG
CACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGG
TGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCA
GCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACC
CAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGG
TGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAA
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCC
TCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACA
GGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCA
GCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG
TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT
GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACA
AGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG
GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAA AGCTAGCTGA
[0342] anti-CD40.sub.--12B4.2C10_K-V-hIgGK-C (variant 1) light
chain
TABLE-US-00109 (SEQ ID NO.: 158)
ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGT
CATAATGTCCAGGGGACAAATTGTTCTCACCCAGTCTCCAGCAATCCTGT
CTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGT
GTAAGTTACATGTACAGGTACCAGCAGAAGCCAGGATCCTCACCCAAACC
CTGGATTTATGGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCA
GTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAG
GCTGAAGATGCTGCCACTTATTACTGCCAGCAATATCATAGTTACCCGCT
CACGTTCGGTGCTGGGACCAAGCTCGAGATCAAACGAACTGTGGCTGCAC
CATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACT
GCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGT
ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG
ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGT
CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGTTAG
[0343] anti-CD40.sub.--12B4.2C10_K-V-hIgGK-C (Variant 2) light
chain
TABLE-US-00110 (SEQ ID NO.: 159)
ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGG
TACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCT
CTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGC
AATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCT
GATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTG
GCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAA
GAAGATATTGCCACTTACTTTTGCCATCATGGTAATACGCTTCCGTGGAC
GTTCGGTGGAGGCACCAAGCTCGAGATCAAACGAACTGTGGCTGCACCAT
CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC
TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACA
GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG
CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCAC
CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG
[0344] anti-CD40.sub.--11B6.1C3_H-V-hIgG4H-C heavy chain
TABLE-US-00111 (SEQ ID NO.: 160)
ATGGGATGGAGCTGGATCTTTCTCTTTCTCCTGTCAGGAACTGCAGGTGT
CCTCTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTG
GGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGC
TACTACATGCACTGGGTGAAGCAAAGCCATGTAAAGAGCCTTGAGTGGAT
TGGACGTATTAATCCTTACAATGGTGCTACTAGCTACAACCAGAATTTCA
AGGACAAGGCCAGCTTGACTGTAGATAAGTCCTCCAGCACAGCCTACATG
GAGCTCCACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAG
AGAGGACTACGTCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG
CCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGC
ACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC
ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAA
CGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCA
AATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGA
CCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTC
CCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACC
CCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAG
CGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCC
AAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATC
CCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG
GCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG
GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTT
CTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGA
ATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACA
CAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCTGA
[0345] anti-CD40.sub.--11B6.1C3_K-V-hIgGK-C light chain
TABLE-US-00112 (SEQ ID NO.: 161)
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTC
CAGCAGTGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTC
TTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACAC
AGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTC
TCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAG
ACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCGCACTCAAGATCAGT
AGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACA
TGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTCGAGATCAAACGAACTG
TGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA
TCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA
GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCC
AGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC
AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGC
CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA
ACAGGGGAGAGTGTTAG
[0346] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method, kit,
reagent, or composition of the invention, and vice versa.
Furthermore, compositions of the invention can be used to achieve
methods of the invention.
[0347] It will be understood that particular embodiments described
herein are shown by way of illustration and not as limitations of
the invention. The principal features of this invention can be
employed in various embodiments without departing from the scope of
the invention. Those skilled in the art will recognize, or be able
to ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the claims.
[0348] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0349] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one." The use of
the term "or" in the claims is used to mean "and/or" unless
explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0350] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0351] The term "or combinations thereof" as used herein refers to
all permutations and combinations of the listed items preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, CBA,
BCA, ACB, BAC, or CAB. Continuing with this example, expressly
included are combinations that contain repeats of one or more item
or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so
forth. The skilled artisan will understand that typically there is
no limit on the number of items or terms in any combination, unless
otherwise apparent from the context.
[0352] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
Sequence CWU 1
1
161132PRTArtificial SequenceSynthetic peptide. 1Val Gly Phe Pro Val
Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr 1 5 10 15 Lys Ala Ala
Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu 20 25 30
230PRTArtificial SequenceSynthetic peptide. 2His Thr Gln Gly Tyr
Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro 1 5 10 15 Gly Val Arg
Tyr Pro Leu Thr Phe Gly Trp Leu Tyr Lys Leu 20 25 30
319PRTArtificial SequenceSynthetic peptide. 3Glu Lys Ile Arg Leu
Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 1 5 10 15 His Ile Val
432PRTArtificial SequenceSynthetic peptide. 4Asn Pro Pro Ile Pro
Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu 1 5 10 15 Gly Leu Asn
Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp 20 25 30
531PRTArtificial SequenceSynthetic peptide. 5Ala Ile Phe Gln Ser
Ser Met Thr Lys Ile Leu Glu Pro Phe Arg Lys 1 5 10 15 Gln Asn Pro
Asp Ile Val Ile Tyr Gln Tyr Met Asp Asp Leu Tyr 20 25 30
6475PRTArtificial SequenceSynthetic peptide. 6Gln Val Thr Leu Lys
Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly
Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40
45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser
50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn
Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp Ala
Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr
Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala Gly Thr Thr
Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170
175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295
300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420
425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu 435 440 445 Ser Leu Gly Lys Ala Ser Glu Lys Ile Arg Leu Arg Pro
Gly Gly Lys 450 455 460 Lys Lys Tyr Lys Leu Lys His Ile Val Ala Ser
465 470 475 7488PRTArtificial SequenceSynthetic peptide. 7Gln Val
Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu
Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr
Ala Asp Ala Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr
Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala
Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150
155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395
400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys Ala Ser Asn Pro Pro Ile
Pro Val Gly Glu Ile Tyr 450 455 460 Lys Arg Trp Ile Ile Leu Gly Leu
Asn Lys Ile Val Arg Met Tyr Ser 465 470 475 480 Pro Thr Ser Ile Leu
Asp Ala Ser 485 8486PRTArtificial SequenceSynthetic peptide. 8Gln
Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10
15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30 Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly
Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg
Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp
Thr Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val Asp
Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His
Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly
Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145
150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265
270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390
395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys Ala Ser His Thr Gln
Gly Tyr Phe Pro Asp Trp Gln 450 455 460 Asn Tyr Thr Pro Gly Pro Gly
Val Arg Tyr Pro Leu Thr Phe Gly Trp 465 470 475 480 Leu Tyr Lys Leu
Ala Ser 485 9488PRTArtificial SequenceSynthetic peptide. 9Gln Val
Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu
Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr
Ala Asp Ala Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr
Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala
Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150
155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395
400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys Ala Ser Val Gly Phe Pro
Val Thr Pro Gln Val Pro 450 455 460 Leu Arg Pro Met Thr Tyr Lys Ala
Ala Val Asp Leu Ser His Phe Leu 465 470 475 480 Lys Glu Lys Gly Gly
Leu Ala Ser 485 10487PRTArtificial SequenceSynthetic peptide. 10Gln
Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10
15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30 Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly
Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg
Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp
Thr Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val Asp
Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His
Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly
Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145
150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245
250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys Ala Ser
Ala Ile Phe Gln Ser Ser Met Thr Lys Ile 450 455 460 Leu Glu Pro Phe
Arg Lys Gln Asn Pro Asp Ile Val Ile Tyr Gln Tyr 465 470 475 480 Met
Asp Asp Leu Tyr Ala Ser 485 1125PRTArtificial SequenceSynthetic
peptide. 11Ser Ser Val Ser Pro Thr Thr Ser Val His Pro Thr Pro Thr
Ser Val 1 5 10 15 Pro Pro Thr Pro Thr Lys Ser Ser Pro 20 25
1225PRTArtificial SequenceSynthetic peptide. 12Pro Thr Ser Thr Pro
Ala Asp Ser Ser Thr Ile Thr Pro Thr Ala Thr 1 5 10 15 Pro Thr Ala
Thr Pro Thr Ile Lys Gly 20 25 1325PRTArtificial SequenceSynthetic
peptide. 13Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val
Thr Thr 1 5 10 15 Ile Thr Pro Thr Ala Thr Thr Lys Pro 20 25
1425PRTArtificial SequenceSynthetic peptide. 14Thr Asn Gly Ser Ile
Thr Val Ala Ala Thr Ala Pro Thr Val Thr Pro 1 5 10 15 Thr Val Asn
Ala Thr Pro Ser Ala Ala 20 25 152299PRTBacteroides cellulosolvens
15Met Gln Ser Pro Arg Leu Lys Arg Lys Ile Leu Ser Val Ile Leu Ala 1
5 10 15 Val Cys Tyr Ile Ile Ser Ser Phe Ser Ile Gln Phe Ala Ala Thr
Pro 20 25 30 Gln Val Asn Ile Ile Ile Gly Ser Ala Gln Gly Ile Pro
Gly Ser Thr 35 40 45 Val Lys Val Pro Ile Asn Leu Gln Asn Val Pro
Glu Ile Gly Ile Asn 50 55 60 Asn Cys Asp Phe Thr Ile Lys Phe Asp
Ser Asp Ile Leu Asp Phe Asn 65 70 75 80 Ser Val Glu Ala Gly Asp Ile
Val Pro Leu Pro Val Ala Ser Phe Ser 85 90 95 Ser Asn Asn Ser Lys
Asp Ile Ile Lys Phe Leu Phe Ser Asp Ala Thr 100 105 110 Gln Gly Asn
Met Pro Ile Asn Glu Asn Gly Leu Phe Ala Val Ile Ser 115 120 125 Phe
Lys Ile Lys Asp Asn Ala Gln Lys Gly Ile Ser Asn Ile Lys Val 130 135
140 Ser Ser Tyr Gly Ser Phe Ser Gly Met Ser Gly Lys Glu Met Gln Ser
145 150 155 160 Leu Ser Pro Thr Phe Phe Ser Gly Ser Ile Asp Val Ser
Asp Val Ser 165 170 175 Thr Ser Lys Leu Asp Val Lys Val Gly Asn Val
Glu Gly Ile Ala Gly 180 185 190 Thr Glu Val Asn Val Pro Ile Thr Phe
Glu Asn Val Pro Asp Asn Gly 195 200 205 Ile Asn Asn Cys Asn Phe Thr
Leu Ser Tyr Asp Ser Asn Ala Leu Glu 210 215 220 Phe Leu Thr Thr Glu
Ala Gly Asn Ile Ile Pro Leu Ala Ile Ala Asp 225 230 235 240 Tyr Ser
Ser Tyr Arg Ser Met Glu Gly Lys Ile Lys Phe Leu Phe Ser 245 250 255
Asp Ser Ser Gln Gly Thr Arg Ser Ile Lys Asn Asp Gly Val Phe Ala 260
265 270 Asn Ile Lys Phe Lys Ile Lys Gly Asn Ala Ile Arg Asp Thr Tyr
Arg 275 280 285 Ile Asp Leu Ser Glu Leu Gly Ser Phe Ser Ser Lys Gln
Asn Asn Asn 290 295 300 Leu Lys Ser Ile Ala Thr Gln Phe Leu Ser Gly
Ser Val Asn Val Lys 305 310 315 320 Asp Ile Glu Ser Ser Val Ser Pro
Thr Thr Ser Val His Pro Thr Pro 325 330 335 Thr Ser Val Pro Pro Thr
Pro Thr Lys Ser Ser Pro Gly Asn Lys Met 340 345 350 Lys Ile Gln Ile
Gly Asp Val Lys Ala Asn Gln Gly Asp Thr Val Ile 355 360 365 Val Pro
Ile Thr Phe Asn Glu Val Pro Val Met Gly Val Asn Asn Cys 370 375 380
Asn Phe Thr Leu Ala Tyr Asp Lys Asn Ile Met Glu Phe Ile Ser Ala 385
390 395 400 Asp Ala Gly Asp Ile Val Thr Leu Pro Met Ala Asn Tyr Ser
Tyr Asn 405 410 415 Met Pro Ser Asp Gly Leu Val Lys Phe Leu Tyr Asn
Asp Gln Ala Gln 420 425 430 Gly Ala Met Ser Ile Lys Glu Asp Gly Thr
Phe Ala Asn Val Lys Phe 435 440 445 Lys Ile Lys Gln Ser Ala Ala Phe
Gly Lys Tyr Ser Val Gly Ile Lys 450 455 460 Ala Ile Gly Ser Ile Ser
Ala Leu Ser Asn Ser Lys Leu Ile Pro Ile 465 470 475 480 Glu Ser Ile
Phe Lys Asp Gly Ser Ile Thr Val Thr Asn Lys Pro Ile 485 490 495 Val
Asn Ile Glu Ile Gly Lys Val Lys Val Lys Ala Gly Asp Lys Ile 500 505
510 Lys Val Pro Val Glu Ile Lys Asp Ile Pro Ser Ile Gly Ile Asn Asn
515 520 525 Cys Asn Phe Thr Leu Lys Tyr Asn Ser Asn Val Leu Lys Tyr
Val Ser 530 535 540 Asn Glu Ala Gly Thr Ile Val Pro Ala Pro Leu Ala
Asn Leu Ser Ile 545 550 555 560 Asn Lys Pro Asp Glu Gly Ile Ile Lys
Leu Leu Phe Ser Asp Ala Ser 565 570 575 Gln Gly Gly Met Pro Ile Lys
Asp Asn Gly Ile Phe Val Asn Leu Glu 580 585 590 Phe Gln Ala Val Asn
Asp Ala Asn Ile Gly Val Tyr Gly Leu Glu Leu 595 600 605 Asp Thr Ile
Gly Ala Phe Ser Gly Ile Ser Ser Ala Lys Met Thr Ser 610 615 620 Ile
Glu Pro Gln Phe Asn Asn Gly Ser Ile Glu Ile Phe Asn Ser Ala 625 630
635 640 Gln Thr Pro Val Pro Ser Asn Thr Glu Val Gln Thr Pro Thr Asn
Thr 645 650 655 Ile Ser Val Thr Pro Thr Asn Asn Ser Thr Pro Thr Asn
Asn Ser Thr 660 665 670 Pro Lys Pro Asn Pro Leu Tyr Asn Leu Asn Val
Asn Ile Gly Glu Ile 675 680 685 Ser Gly Glu Ala Gly Gly Val Ile Glu
Val Pro Ile Glu Phe Lys Asn 690 695 700 Val Pro Asp Phe Gly Ile Asn
Asn Cys Asp Phe Ser Val Lys Tyr Asp 705 710 715 720 Lys Ser Ile Phe
Glu Tyr Val Thr Tyr Glu Ala Gly Ser Ile Val Lys 725 730 735 Asp Ser
Ile Val Asn Leu Ala Cys Met Glu Asn Ser Gly Ile Ile Asn 740 745 750
Leu Leu Phe Asn Asp Ala Thr Gln Ser Ser Ser Pro Ile Lys Asn Asn 755
760 765 Gly Val Phe Ala Lys Leu Lys Phe Lys Ile Asn Ser Asn Ala Ala
Ser 770 775 780 Gly Thr Tyr Gln Ile Asn Ala Glu Gly Tyr Gly Lys Phe
Ser Gly Asn 785 790 795 800 Leu Asn Gly Lys Leu Thr Ser Ile Asn Pro
Ile Phe Glu Asn Gly Ile 805 810 815 Ile Asn Ile Gly Asn Val Thr Val
Lys Pro Thr Ser Thr Pro Ala Asp 820 825 830 Ser Ser Thr Ile Thr Pro
Thr Ala Thr Pro Thr Ala Thr Pro Thr Ile 835 840 845 Lys Gly Thr Pro
Thr Val Thr Pro Ile Tyr Trp Met Asn Val Leu Ile 850 855 860 Gly Asn
Met Asn Ala Ala Ile Gly Glu Glu Val Val Val Pro Ile Glu 865 870 875
880 Phe Lys Asn Val Pro Pro Phe Gly Ile Asn Asn Cys Asp Phe Lys Leu
885 890 895 Val Tyr Asp Ser Asn Ala Leu Glu Leu Lys Lys Val Glu Ala
Gly Asp 900 905 910 Ile Val Pro Glu Pro Leu Ala Asn Leu Ser Ser Asn
Lys Ser Glu Gly 915 920 925 Lys Ile Gln Phe Leu Phe Asn Asp Ala Ser
Gln Gly Ser Met Gln Ile 930 935 940 Glu Asn Gly Gly Val Phe Ala Lys
Ile Thr Phe Lys Val Lys Ser Thr 945 950 955 960 Ala Ala Ser Gly Ile
Tyr Asn Ile Arg Lys Asp Ser Val Gly Ser Phe 965 970 975 Ser Gly Leu
Ile Asp Asn Lys Met Thr Ser Ile Gly Pro Lys Phe Thr 980 985 990 Asp
Gly Ser Ile Val Val Gly Thr Val Thr Pro Thr Ala Thr Ala Thr 995
1000 1005 Pro Ser Ala Ile Val Thr Thr Ile Thr Pro Thr Ala Thr Thr
Lys 1010 1015 1020 Pro Ile Ala Thr Pro Thr Ile Lys Gly Thr Pro Thr
Ala Thr Pro 1025 1030 1035 Met Tyr Trp Met Asn Val Val Ile Gly Lys
Met Asn Ala Glu Val 1040 1045 1050 Gly Gly Glu Val Val Val Pro Ile
Glu Phe Asn Asn Val Pro Ser 1055 1060 1065 Phe Gly Ile Asn Asn Cys
Asp Phe Lys Leu Val Tyr Asp Ala Thr 1070 1075 1080 Ala Leu Glu Leu
Lys Asn Val Glu Ala Gly Asp Ile Ile Lys Thr 1085 1090 1095 Pro Leu
Ala Asn Phe Ser Asn Asn Lys Ser Glu Glu Gly Lys Ile 1100 1105 1110
Ser Phe Leu Phe Asn Asp Ala Ser Gln Gly Ser Met Gln Ile Glu 1115
1120 1125 Asn Gly Gly Val Phe Ala Lys Ile Thr Phe Lys Val Lys Ser
Thr 1130 1135 1140 Thr Ala Thr Gly Val Tyr Asp Leu Arg Lys Asp Leu
Val Gly Ser 1145 1150 1155 Phe Ser Gly Leu Lys Asp Asn Lys Met Thr
Ser Ile Gly Ala Glu 1160 1165 1170 Phe Thr Asn Gly Ser Ile Thr Val
Ala Ala Thr Ala Pro Thr Val 1175 1180 1185 Thr Pro Thr Val Asn Ala
Thr Pro Ser Ala Ala Thr Pro Thr Val 1190 1195 1200 Thr Pro Thr Ala
Thr Ala Thr Pro Ser Val Thr Ile Pro Thr Val 1205 1210 1215 Thr Pro
Thr Ala Thr Ala Thr Pro Ser Val Thr Ile Pro Thr Val 1220 1225 1230
Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ala Thr Pro Thr Val 1235
1240 1245 Thr Pro Thr Ala Thr Ala Thr Pro Ser Val Thr Ile Pro Thr
Val 1250 1255 1260 Thr Pro Thr Val Thr Ala Thr Pro Ser Asp Thr Ile
Pro Thr Val 1265 1270 1275 Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala
Ile Val Thr Thr Ile 1280 1285 1290 Thr Pro Thr Ala Thr Ala Lys Pro
Ile Ala Thr Pro Thr Ile Lys 1295 1300 1305 Gly Thr Pro Thr Ala Thr
Pro Met Tyr Trp Met Asn Val Val Ile 1310 1315 1320 Gly Lys Met Asn
Ala Glu Val Gly Gly Glu Val Val Val Pro Ile 1325 1330 1335 Glu Phe
Lys Asn Val Pro Ser Phe Gly Ile Asn Asn Cys Asp Phe 1340 1345 1350
Lys Leu Val Tyr Asp Ala Thr Ala Leu Glu Leu Lys Asn Val Glu 1355
1360 1365 Ala Gly Asp Ile Ile Lys Thr Pro Leu Ala Asn Phe Ser Asn
Asn 1370 1375 1380 Lys Ser Glu Glu Gly Lys Ile Ser Phe Leu Phe Asn
Asp Ala Ser 1385 1390 1395 Gln Gly Ser Met Gln Ile Glu Asn Gly Gly
Val Ser Ala Lys Ile 1400 1405 1410 Thr Phe Lys Val Lys Ser Thr Thr
Ala Ile Gly Val Tyr Asp Ile 1415 1420 1425 Arg Lys Asp Leu Ile Gly
Ser Phe Ser Gly Leu Lys Asp Ser Lys 1430 1435 1440 Met Thr Ser Ile
Gly Ala Glu Phe Thr Asn Gly Ser Ile Thr Val 1445 1450 1455 Ala Thr
Thr Ala Pro Thr Val Thr Pro Thr Ala Thr Ala Thr Pro 1460 1465 1470
Ser Val Thr Ile Pro Thr Val Thr Pro Thr Ala Thr Ala Thr Pro 1475
1480 1485 Gly Thr Ala Thr Pro Gly Thr Ala Thr Pro Thr Ala Thr Ala
Thr 1490 1495 1500 Pro Gly Ala Ala Thr Pro Thr Glu Thr Ala Thr Pro
Ser Val Met 1505 1510 1515 Ile Pro Thr Val Thr Pro Thr Ala Thr Ala
Thr Pro Thr Ala Thr 1520 1525 1530 Ala Thr Pro Thr Val Lys Gly Thr
Pro Thr Ile Lys Pro Val Tyr 1535 1540 1545 Lys Met Asn Val Val Ile
Gly Arg Val Asn Val Val Ala Gly Glu 1550 1555 1560 Glu Val Val Val
Pro Val Glu Phe Lys Asn Ile Pro Ala Ile Gly 1565 1570 1575 Val Asn
Asn Cys Asn Phe Val Leu Glu Tyr Asp Ala Asn Val Leu 1580 1585 1590
Glu Val Lys Lys Val Asp Ala Gly Glu Ile Val Pro Asp Ala Leu 1595
1600 1605 Ile Asn Phe Gly Ser Asn Asn Ser Asp Glu Gly Lys Val Tyr
Phe 1610 1615 1620 Leu Phe Asn Asp Ala Leu Gln Gly Arg Met Gln Ile
Ala Asn Asp 1625 1630 1635 Gly Ile Phe Ala Asn Ile Thr Phe Lys Val
Lys Ser Ser Ala Ala 1640 1645 1650 Ala Gly Ile Tyr Asn Ile Arg Lys
Asp Ser Val Gly Ala Phe Ser 1655 1660 1665 Gly Leu Val Asp Lys Leu
Val Pro Ile Ser Ala Glu Phe Thr Asp 1670 1675 1680 Gly Ser Ile Ser
Val Glu Ser Ala Lys Ser Thr Pro Thr Ala Thr 1685 1690 1695 Ala Thr
Gly Thr Asn Val Thr Pro Thr Val Ala Ala Thr Val Thr 1700 1705 1710
Pro Thr Ala Thr Pro Ala Ser Thr Thr Pro Thr Ala Thr Pro Thr 1715
1720 1725 Ala Thr Ser Thr Val Lys Gly Thr Pro Thr Ala Thr Pro Leu
Tyr 1730 1735 1740 Ser Met Asn Val Ile Ile Gly Lys Val Asn Ala Glu
Ala Ser Gly 1745 1750 1755 Glu Val Val Val Pro Val Glu Phe Lys Asp
Val Pro Ser Ile Gly 1760 1765 1770 Ile Asn Asn Cys Asn Phe Ile Leu
Glu Tyr Asp Ala Ser Ala Leu 1775 1780 1785 Glu Leu Asp Ser Ala Glu
Ala Gly Glu Ile Val Pro Val Pro Leu 1790 1795 1800 Gly Asn Phe Ser
Ser Asn Asn Lys Asp Glu Gly Lys Ile Tyr Phe 1805 1810
1815 Leu Phe Ser Asp Gly Thr Gln Gly Arg Met Gln Ile Val Asn Asp
1820 1825 1830 Gly Ile Phe Ala Lys Ile Lys Phe Lys Val Lys Ser Thr
Ala Ser 1835 1840 1845 Asp Gly Thr Tyr Tyr Ile Arg Lys Asp Ser Val
Gly Ala Phe Ser 1850 1855 1860 Gly Leu Ile Glu Lys Lys Ile Ile Lys
Ile Gly Ala Glu Phe Thr 1865 1870 1875 Asp Gly Ser Ile Thr Val Arg
Ser Leu Thr Pro Thr Pro Thr Val 1880 1885 1890 Thr Pro Asn Val Ala
Ser Pro Thr Pro Thr Lys Val Val Ala Glu 1895 1900 1905 Pro Thr Ser
Asn Gln Pro Ala Gly Pro Gly Pro Ile Thr Gly Thr 1910 1915 1920 Ile
Pro Thr Ala Thr Thr Thr Ala Thr Ala Thr Pro Thr Lys Ala 1925 1930
1935 Ser Val Ala Thr Ala Thr Pro Thr Ala Thr Pro Ile Val Val Val
1940 1945 1950 Glu Pro Thr Ile Val Arg Pro Gly Tyr Asn Lys Asp Ala
Asp Leu 1955 1960 1965 Ala Val Phe Ile Ser Ser Asp Lys Ser Arg Tyr
Glu Glu Ser Ser 1970 1975 1980 Ile Ile Thr Tyr Ser Ile Glu Tyr Lys
Asn Ile Gly Lys Val Asn 1985 1990 1995 Ala Thr Asn Val Lys Ile Ala
Ala Gln Ile Pro Lys Phe Thr Lys 2000 2005 2010 Val Tyr Asp Ala Ala
Lys Gly Ala Val Lys Gly Ser Glu Ile Val 2015 2020 2025 Trp Met Ile
Gly Asn Leu Ala Val Gly Glu Ser Tyr Thr Lys Glu 2030 2035 2040 Tyr
Lys Val Lys Val Asp Ser Leu Thr Lys Ser Glu Glu Tyr Thr 2045 2050
2055 Asp Asn Thr Val Thr Ile Ser Ser Asp Gln Thr Val Asp Ile Pro
2060 2065 2070 Glu Asn Ile Thr Thr Gly Asn Asp Asp Lys Ser Thr Ile
Arg Val 2075 2080 2085 Met Leu Tyr Ser Asn Arg Phe Thr Pro Gly Ser
His Ser Ser Tyr 2090 2095 2100 Ile Leu Gly Tyr Lys Asp Lys Thr Phe
Lys Pro Lys Gln Asn Val 2105 2110 2115 Thr Arg Ala Glu Val Ala Ala
Met Phe Ala Arg Ile Met Gly Leu 2120 2125 2130 Thr Val Lys Asp Gly
Ala Lys Ser Ser Tyr Lys Asp Val Ser Asn 2135 2140 2145 Lys His Trp
Ala Leu Lys Tyr Ile Glu Ala Val Thr Lys Ser Gly 2150 2155 2160 Ile
Phe Lys Gly Tyr Lys Asp Ser Thr Phe His Pro Asn Ala Pro 2165 2170
2175 Ile Thr Arg Ala Glu Leu Ser Thr Val Ile Phe Asn Tyr Leu His
2180 2185 2190 Leu Asn Asn Ile Ala Pro Ser Lys Val His Phe Thr Asp
Ile Asn 2195 2200 2205 Lys His Trp Ala Lys Asn Tyr Ile Glu Glu Ile
Tyr Arg Phe Lys 2210 2215 2220 Leu Ile Gln Gly Tyr Ser Asp Gly Ser
Phe Lys Pro Asn Asn Asn 2225 2230 2235 Ile Thr Arg Ala Glu Val Val
Thr Met Ile Asn Arg Met Leu Tyr 2240 2245 2250 Arg Gly Pro Leu Lys
Val Lys Val Gly Ser Phe Pro Asp Val Ser 2255 2260 2265 Pro Lys Tyr
Trp Ala Tyr Gly Asp Ile Glu Glu Ala Ser Arg Asn 2270 2275 2280 His
Lys Tyr Thr Arg Asp Glu Lys Asp Gly Ser Glu Ile Leu Ile 2285 2290
2295 Glu 16541PRTArtificial SequenceSynthetic peptide. 16Gln Val
Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu
Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr
Ala Asp Ala Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr
Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala
Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150
155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395
400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys Ala Ser Glu Lys Ile Arg
Leu Arg Pro Gly Gly Lys 450 455 460 Lys Lys Tyr Lys Leu Lys His Ile
Val Ala Ser Val Gly Phe Pro Val 465 470 475 480 Thr Pro Gln Val Pro
Leu Arg Pro Met Thr Tyr Lys Ala Ala Val Asp 485 490 495 Leu Ser His
Phe Leu Lys Glu Lys Gly Gly Leu Ala Ser His Thr Gln 500 505 510 Gly
Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val Arg 515 520
525 Tyr Pro Leu Thr Phe Gly Trp Leu Tyr Lys Leu Ala Ser 530 535 540
17534PRTArtificial SequenceSynthetic peptide. 17Gln Val Thr Leu Lys
Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly
Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40
45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser
50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn
Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp Ala
Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr
Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala Gly Thr Thr
Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170
175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295
300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420
425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu 435 440 445 Ser Leu Gly Lys Ala Ser Glu Lys Ile Arg Leu Arg Pro
Gly Gly Lys 450 455 460 Lys Lys Tyr Lys Leu Lys His Ile Val Ala Ser
Ser Ser Val Ser Pro 465 470 475 480 Thr Thr Ser Val His Pro Thr Pro
Thr Ser Val Pro Pro Thr Pro Thr 485 490 495 Lys Ser Ser Pro Ala Ser
His Thr Gln Gly Tyr Phe Pro Asp Trp Gln 500 505 510 Asn Tyr Thr Pro
Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp 515 520 525 Leu Tyr
Lys Leu Ala Ser 530 18626PRTArtificial SequenceSynthetic peptide.
18Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1
5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr
Ser 20 25 30 Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys
Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys
Arg Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys
Asp Thr Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val
Asp Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser
His Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp
Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135
140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385
390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys Ala Ser Gln Thr
Pro Thr Asn Thr Ile Ser Val Thr 450 455 460 Pro Thr Asn Asn Ser Thr
Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn 465 470 475 480 Pro Ala Ser
Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr 485 490 495 Lys
Leu Lys His Ile Val Ala Ser Ser Ser Val Ser Pro Thr Thr Ser 500 505
510 Val His Pro Thr Pro Thr Ser Val Pro Pro Thr Pro Thr Lys Ser Ser
515 520 525 Pro Ala Ser Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys
Arg Trp 530 535 540 Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr
Ser Pro Thr Ser 545 550 555 560 Ile Leu Asp Ala Ser Pro Thr Ser Thr
Pro Ala Asp Ser Ser Thr Ile 565 570 575 Thr Pro Thr Ala Thr Pro Thr
Ala Thr Pro Thr Ile Lys Gly Ala Ser 580 585 590 Val Gly Phe Pro Val
Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr 595 600 605 Lys Ala Ala
Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu 610 615 620 Ala
Ser 625 19608PRTArtificial SequenceSynthetic peptide. 19Gln Val Thr
Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr
Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25
30 Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu
35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn
Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser
Ser Asn Gln Val 65
70 75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp Ala Ala Thr
Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr Gly Tyr
Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala Gly Thr Thr Val Thr
Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185
190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300 Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310
315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415 Val Asp
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435
440 445 Ser Leu Gly Lys Ala Ser Ala Ile Phe Gln Ser Ser Met Thr Lys
Ile 450 455 460 Leu Glu Pro Phe Arg Lys Gln Asn Pro Asp Ile Val Ile
Tyr Gln Tyr 465 470 475 480 Met Asp Asp Leu Tyr Ala Ser Glu Lys Ile
Arg Leu Arg Pro Gly Gly 485 490 495 Lys Lys Lys Tyr Lys Leu Lys His
Ile Val Ala Ser Val Gly Phe Pro 500 505 510 Val Thr Pro Gln Val Pro
Leu Arg Pro Met Thr Tyr Lys Ala Ala Val 515 520 525 Asp Leu Ser His
Phe Leu Lys Glu Lys Gly Gly Leu Ala Ser His Thr 530 535 540 Gln Gly
Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val 545 550 555
560 Arg Tyr Pro Leu Thr Phe Gly Trp Leu Tyr Lys Leu Ala Ser Asn Pro
565 570 575 Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu
Gly Leu 580 585 590 Asn Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile
Leu Asp Ala Ser 595 600 605 20745PRTArtificial SequenceSynthetic
peptide. 20Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro
Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser
Leu Ser Thr Ser 20 25 30 Gly Met Gly Leu Ser Trp Ile Arg Gln Pro
Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp
Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile
Thr Ile Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95 Cys Ala
Arg Ser Ser His Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110
Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115
120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235
240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360
365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Arg Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly
Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys Ala
Ser Gln Thr Pro Thr Asn Thr Ile Ser Val Thr 450 455 460 Pro Thr Asn
Asn Ser Thr Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn 465 470 475 480
Pro Ala Ser Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr 485
490 495 Lys Leu Lys His Ile Val Ala Ser Ser Ser Val Ser Pro Thr Thr
Ser 500 505 510 Val His Pro Thr Pro Thr Ser Val Pro Pro Thr Pro Thr
Lys Ser Ser 515 520 525 Pro Ala Ser Asn Pro Pro Ile Pro Val Gly Glu
Ile Tyr Lys Arg Trp 530 535 540 Ile Ile Leu Gly Leu Asn Lys Ile Val
Arg Met Tyr Ser Pro Thr Ser 545 550 555 560 Ile Leu Asp Ala Ser Pro
Thr Ser Thr Pro Ala Asp Ser Ser Thr Ile 565 570 575 Thr Pro Thr Ala
Thr Pro Thr Ala Thr Pro Thr Ile Lys Gly Ala Ser 580 585 590 His Thr
Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro 595 600 605
Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Leu Tyr Lys Leu Ala Ser 610
615 620 Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val Thr
Thr 625 630 635 640 Ile Thr Pro Thr Ala Thr Thr Lys Pro Ala Ser Val
Gly Phe Pro Val 645 650 655 Thr Pro Gln Val Pro Leu Arg Pro Met Thr
Tyr Lys Ala Ala Val Asp 660 665 670 Leu Ser His Phe Leu Lys Glu Lys
Gly Gly Leu Ala Ser Thr Asn Gly 675 680 685 Ser Ile Thr Val Ala Ala
Thr Ala Pro Thr Val Thr Pro Thr Val Asn 690 695 700 Ala Thr Pro Ser
Ala Ala Ala Ser Ala Ile Phe Gln Ser Ser Met Thr 705 710 715 720 Lys
Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro Asp Ile Val Ile Tyr 725 730
735 Gln Tyr Met Asp Asp Leu Tyr Ala Ser 740 745 2172DNAArtificial
SequenceSynthetic oligonucleotide. 21gctagtgaga agatccggct
gcggcccggc ggcaagaaga agtacaagct gaagcacatc 60gtggctagct ga
7222111DNAArtificial SequenceSynthetic oligonucleotide.
22gctagtgtgg gcttccccgt gaccccccag gtgcccctgc ggcccatgac ctacaaggcc
60gccgtggacc tgagccactt cctgaaggag aagggcggcc tggctagctg a
11123108DNAArtificial SequenceSynthetic oligonucleotide.
23gctagtgcca tcttccagag cagcatgacc aagatcctgg agcccttccg gaagcagaac
60cccgacatcg tgatctacca gtacatggac gacctgtacg ctagctga
10824198DNAArtificial SequenceSynthetic oligonucleotide.
24gctagtcaga cccccaccaa caccatcagc gtgaccccca ccaacaacag cacccccacc
60aacaacagca accccaagcc caaccccgct agtaaccccc ccatccccgt gggcgagatc
120tacaagcggt ggatcatcct gggcctgaac aagatcgtgc ggatgtacag
ccccaccagc 180atcctggacg ctagctga 19825255DNAArtificial
SequenceSynthetic oligonucleotide. 25gctagtcaga cccccaccaa
caccatcagc gtgaccccca ccaacaacag cacccccacc 60aacaacagca accccaagcc
caaccccgct agtgagaaga tccggctgcg gcccggcggc 120aagaagaagt
acaagctgaa gcacatcgtg gctagtcaca cccagggcta cttccccgac
180tggcagaact acacccccgg ccccggcgtg cggtaccccc tgaccttcgg
ctggctgtac 240aagctggcta gctga 25526882DNAArtificial
SequenceSynthetic oligonucleotide. 26gctagtcaga cccccaccaa
caccatcagc gtgaccccca ccaacaacag cacccccacc 60aacaacagca accccaagcc
caaccccgct agtgagaaga tccggctgcg gcccggcggc 120aagaagaagt
acaagctgaa gcacatcgtg gctagtagca gcgtgagccc caccaccagc
180gtgcacccca cccccaccag cgtgcccccc acccccacca agagcagccc
cgctagtaac 240ccccccatcc ccgtgggcga gatctacaag cggtggatca
tcctgggcct gaacaagatc 300gtgcggatgt acagccccac cagcatcctg
gacgctagtc ccaccagcac ccccgccgac 360agcagcacca tcacccccac
cgccaccccc accgccaccc ccaccatcaa gggcgctagt 420cacacccagg
gctacttccc cgactggcag aactacaccc ccggccccgg cgtgcggtac
480cccctgacct tcggctggct gtacaagctg gctagtaccg tgacccccac
cgccaccgcc 540acccccagcg ccatcgtgac caccatcacc cccaccgcca
ccaccaagcc cgctagtgtg 600ggcttccccg tgacccccca ggtgcccctg
cggcccatga cctacaaggc cgccgtggac 660ctgagccact tcctgaagga
gaagggcggc ctggctagta ccaacggcag catcaccgtg 720gccgccaccg
cccccaccgt gacccccacc gtgaacgcca cccccagcgc cgccgctagt
780gccatcttcc agagcagcat gaccaagatc ctggagccct tccggaagca
gaaccccgac 840atcgtgatct accagtacat ggacgacctg tacgctagct ga
88227108DNAArtificial SequenceSynthetic oligonucleotide.
27gctagtgtgg gcttccccgt gaccccccag gtgcccctgc ggcccatgac ctacaaggcc
60gccgtggacc tgagccactt cctgaaggag aagggcggcc tggctagc
10828102DNAArtificial SequenceSynthetic oligonucleotide.
28gctagtcaca cccagggcta cttccccgac tggcagaact acacccccgg ccccggcgtg
60cggtaccccc tgaccttcgg ctggctgtac aagctggcta gc
1022969DNAArtificial SequenceSynthetic oligonucleotide.
29gctagtgaga agatccggct gcggcccggc ggcaagaaga agtacaagct gaagcacatc
60gtggctagc 6930108DNAArtificial SequenceSynthetic oligonucleotide.
30gctagtaacc cccccatccc cgtgggcgag atctacaagc ggtggatcat cctgggcctg
60aacaagatcg tgcggatgta cagccccacc agcatcctgg acgctagc
10831105DNAArtificial SequenceSynthetic oligonucleotide.
31gctagtgcca tcttccagag cagcatgacc aagatcctgg agcccttccg gaagcagaac
60cccgacatcg tgatctacca gtacatggac gacctgtacg ctagc
1053287DNAArtificial SequenceSynthetic oligonucleotide.
32gctagtagca gcgtgagccc caccaccagc gtgcacccca cccccaccag cgtgcccccc
60acccccacca agagcagccc cgctagc 873387DNAArtificial
SequenceSynthetic oligonucleotide. 33gctagtccca ccagcacccc
cgccgacagc agcaccatca cccccaccgc cacccccacc 60gccaccccca ccatcaaggg
cgctagc 873487DNAArtificial SequenceSynthetic oligonucleotide.
34gctagtaccg tgacccccac cgccaccgcc acccccagcg ccatcgtgac caccatcacc
60cccaccgcca ccaccaagcc cgctagc 873587DNAArtificial
SequenceSynthetic oligonucleotide. 35gctagtacca acggcagcat
caccgtggcc gccaccgccc ccaccgtgac ccccaccgtg 60aacgccaccc ccagcgccgc
cgctagc 8736745PRTArtificial SequenceSynthetic peptide. 36Gln Val
Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu
Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr
Ala Asp Ala Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr
Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala
Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150
155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395
400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 435 440
445 Ser Leu Gly Lys Ala Ser Gln Thr Pro Thr Asn Thr Ile Ser Val Thr
450 455 460 Pro Thr Asn Asn Ser Thr Pro Thr Asn Asn Ser Asn Pro Lys
Pro Asn 465 470 475 480 Pro Ala Ser Glu Lys Ile Arg Leu Arg Pro Gly
Gly Lys Lys Lys Tyr 485 490 495 Lys Leu Lys His Ile Val Ala Ser Ser
Ser Val Ser Pro Thr Thr Ser 500 505 510 Val His Pro Thr Pro Thr Ser
Val Pro Pro Thr Pro Thr Lys Ser Ser 515 520 525 Pro Ala Ser Asn Pro
Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp 530 535 540 Ile Ile Leu
Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr Ser 545 550 555 560
Ile Leu Asp Ala Ser Pro Thr Ser Thr Pro Ala Asp Ser Ser Thr Ile 565
570 575 Thr Pro Thr Ala Thr Pro Thr Ala Thr Pro Thr Ile Lys Gly Ala
Ser 580 585 590 His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr
Pro Gly Pro 595 600 605 Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Leu
Tyr Lys Leu Ala Ser 610 615 620 Thr Val Thr Pro Thr Ala Thr Ala Thr
Pro Ser Ala Ile Val Thr Thr 625 630 635 640 Ile Thr Pro Thr Ala Thr
Thr Lys Pro Ala Ser Val Gly Phe Pro Val 645 650 655 Thr Pro Gln Val
Pro Leu Arg Pro Met Thr Tyr Lys Ala Ala Val Asp 660 665 670 Leu Ser
His Phe Leu Lys Glu Lys Gly Gly Leu Ala Ser Thr Asn Gly 675 680 685
Ser Ile Thr Val Ala Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn 690
695 700 Ala Thr Pro Ser Ala Ala Ala Ser Ala Ile Phe Gln Ser Ser Met
Thr 705 710 715 720 Lys Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro Asp
Ile Val Ile Tyr 725 730 735 Gln Tyr Met Asp Asp Leu Tyr Ala Ser 740
745 37739PRTArtificial SequenceSynthetic peptide. 37Glu Val Lys Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30
Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35
40 45 Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Leu Pro Phe His Ala
Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser
Ala Lys Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165
170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe
Glu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290
295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410
415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
Ala Ser 435 440 445 Gln Thr Pro Thr Asn Thr Ile Ser Val Thr Pro Thr
Asn Asn Ser Thr 450 455 460 Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn
Pro Ala Ser Glu Lys Ile 465 470 475 480 Arg Leu Arg Pro Gly Gly Lys
Lys Lys Tyr Lys Leu Lys His Ile Val 485 490 495 Ala Ser Ser Ser Val
Ser Pro Thr Thr Ser Val His Pro Thr Pro Thr 500 505 510 Ser Val Pro
Pro Thr Pro Thr Lys Ser Ser Pro Ala Ser Asn Pro Pro 515 520 525 Ile
Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn 530 535
540 Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ala Ser Pro
545 550 555 560 Thr Ser Thr Pro Ala Asp Ser Ser Thr Ile Thr Pro Thr
Ala Thr Pro 565 570 575 Thr Ala Thr Pro Thr Ile Lys Gly Ala Ser His
Thr Gln Gly Tyr Phe 580 585 590 Pro Asp Trp Gln Asn Tyr Thr Pro Gly
Pro Gly Val Arg Tyr Pro Leu 595 600 605 Thr Phe Gly Trp Leu Tyr Lys
Leu Ala Ser Thr Val Thr Pro Thr Ala 610 615 620 Thr Ala Thr Pro Ser
Ala Ile Val Thr Thr Ile Thr Pro Thr Ala Thr 625 630 635 640 Thr Lys
Pro Ala Ser Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu 645 650 655
Arg Pro Met Thr Tyr Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys 660
665 670 Glu Lys Gly Gly Leu Ala Ser Thr Asn Gly Ser Ile Thr Val Ala
Ala 675 680 685 Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr Pro
Ser Ala Ala 690 695 700 Ala Ser Ala Ile Phe Gln Ser Ser Met Thr Lys
Ile Leu Glu Pro Phe 705 710 715 720 Arg Lys Gln Asn Pro Asp Ile Val
Ile Tyr Gln Tyr Met Asp Asp Leu 725 730 735 Tyr Ala Ser
38234PRTArtificial SequenceSynthetic peptide. 38Met Met Ser Ser Ala
Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln 1 5 10 15 Gly Thr Arg
Cys Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser 20 25 30 Ala
Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly 35 40
45 Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val
50 55 60 Lys Leu Leu Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val
Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
Leu Thr Ile Gly 85 90 95 Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr
Tyr Cys Gln Gln Phe Asn 100 105 110 Lys Leu Pro Pro Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170
175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 39467PRTArtificial SequenceSynthetic peptide. 39Met Asn Leu
Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val
Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25
30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe
35 40 45 Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys
Arg Leu 50 55 60 Glu Trp Val Ala Tyr Ile Asn Ser Gly Gly Gly Ser
Thr Tyr Tyr Pro 65 70 75 80 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Arg
Leu Lys Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr Cys Ala Arg Arg
Gly Leu Pro Phe His Ala Met Asp Tyr Trp 115 120 125 Gly Gln Gly Thr
Ser Val Thr Val Ser Ser Ala Lys Thr Lys Gly Pro 130 135 140 Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 145 150 155
160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp 210 215 220 His Lys Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr 225 230 235 240 Gly Pro Pro Cys Pro
Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 245 250 255 Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 275 280
285 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys 340 345 350 Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365 Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375 380 Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405
410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys 420 425 430 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu Gly 450 455 460 Lys Ala Ser 465 40705DNAArtificial
SequenceSynthetic oligonucleotide. 40atgatgtcct ctgctcagtt
ccttggtctc ctgttgctct gttttcaagg taccagatgt 60gatatccaga tgacacagac
tacatcctcc ctgtctgcct ctctaggaga cagagtcacc 120atcagttgca
gtgcaagtca gggcattagc aattatttaa actggtatca gcagaaacca
180gatggaactg ttaaactcct gatctattac acatcaattt tacactcagg
agtcccatca 240aggttcagtg gcagtgggtc tgggacagat tattctctca
ccatcggcaa cctggaacct 300gaagatattg ccacttacta ttgtcagcag
tttaataagc ttcctccgac gttcggtgga 360ggcaccaaac tcgagatcaa
acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
480cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 540gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 600ctgagcaaag cagactacga gaaacacaaa
gtctatgcct gcgaagtcac ccatcagggc 660ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gttag 705411404DNAArtificial
SequenceSynthetic oligonucleotide. 41atgaacttgg ggctcagctt
gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60gtgaagctgg tggagtctgg
gggaggctta gtgcagcctg gagggtccct gaaactctcc 120tgtgcaacct
ctggattcac tttcagtgac tattacatgt attgggttcg ccagactcca
180gagaagaggc tggagtgggt cgcatacatt aattctggtg gtggtagcac
ctattatcca 240gacactgtaa agggccgatt caccatctcc agagacaatg
ccaagaacac cctgtacctg 300caaatgagcc ggctgaagtc tgaggacaca
gccatgtatt actgtgcaag acgggggtta 360ccgttccatg ctatggacta
ttggggtcaa ggaacctcag tcaccgtctc ctcagccaaa 420acgaagggcc
catccgtctt ccccctggcg ccctgctcca ggagcacctc cgagagcaca
480gccgccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt
gtcgtggaac 540tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg
tcctacagtc ctcaggactc 600tactccctca gcagcgtggt gaccgtgccc
tccagcagct tgggcacgaa gacctacacc 660tgcaacgtag atcacaagcc
cagcaacacc aaggtggaca agagagttga gtccaaatat 720ggtcccccat
gcccaccctg cccagcacct gagttcgaag ggggaccatc agtcttcctg
780ttccccccaa aacccaagga cactctcatg atctcccgga cccctgaggt
cacgtgcgtg 840gtggtggacg tgagccagga agaccccgag gtccagttca
actggtacgt ggatggcgtg 900gaggtgcata atgccaagac aaagccgcgg
gaggagcagt tcaacagcac gtaccgtgtg 960gtcagcgtcc tcaccgtcct
gcaccaggac tggctgaacg gcaaggagta caagtgcaag 1020gtctccaaca
aaggcctccc gtcctccatc gagaaaacca tctccaaagc caaagggcag
1080ccccgagagc cacaggtgta caccctgccc ccatcccagg aggagatgac
caagaaccag 1140gtcagcctga cctgcctggt caaaggcttc taccccagcg
acatcgccgt ggagtgggag 1200agcaatgggc agccggagaa caactacaag
accacgcctc ccgtgctgga ctccgacggc 1260tccttcttcc tctacagcag
gctaaccgtg gacaagagca ggtggcagga ggggaatgtc 1320ttctcatgct
ccgtgatgca tgaggctctg cacaaccact acacacagaa gagcctctcc
1380ctgtctctgg gtaaagctag ctga 1404427PRTArtificial
SequenceSynthetic peptide. 42Tyr Thr Ser Ile Leu His Ser 1 5
439PRTArtificial SequenceSynthetic peptide. 43Gln Gln Phe Asn Lys
Leu Pro Pro Thr 1 5 4410PRTArtificial SequenceSynthetic peptide.
44Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr 1 5 10 4517PRTArtificial
SequenceSynthetic peptide. 45Tyr Ile Asn Ser Gly Gly Gly Ser Thr
Tyr Tyr Pro Asp Thr Val Lys 1 5 10 15 Gly 4610PRTArtificial
SequenceSynthetic peptide. 46Arg Gly Leu Pro Phe His Ala Met Asp
Tyr 1 5 10 4710PRTArtificial SequenceSynthetic peptide. 47Arg Gly
Leu Pro Phe His Ala Met Asp Tyr 1 5 10 4836DNAArtificial
SequenceSynthetic oligonucleotide. 48ggatggtggg aagatggata
cagttggtgc agcatc 364931DNAArtificial SequenceSynthetic
oligonucleotide. 49ccaggcatcc tagagtcacc gaggagccag t
315070DNAArtificial SequenceSynthetic oligonucleotide. 50gctagcgata
caacagaacc tgcaacacct acaacacctg taacaacacc gacaacaaca 60cttctagcgc
7051186DNAArtificial SequenceSynthetic oligonucleotide.
51gacaccaccg aggcccgcca cccccacccc cccgtgacca cccccaccac caccgaccgg
60aagggcacca ccgccgagga gctggccggc atcggcatcc tgaccgtgat cctgggcggc
120aagcggacca acaacagcac ccccaccaag ggcgaattct gcagatatcc
atcacactgg 180cggccg
1865261PRTArtificial SequenceSynthetic peptide. 52Asp Thr Thr Glu
Ala Arg His Pro His Pro Pro Val Thr Thr Pro Thr 1 5 10 15 Thr Asp
Arg Lys Gly Thr Thr Ala Glu Glu Leu Ala Gly Ile Gly Ile 20 25 30
Leu Thr Val Ile Leu Gly Gly Lys Arg Thr Asn Asn Ser Thr Pro Thr 35
40 45 Lys Gly Glu Phe Cys Arg Tyr Pro Ser His Trp Arg Pro 50 55 60
5360PRTArtificial SequenceSynthetic peptide. 53Met Lys Ala Asn Leu
Leu Val Leu Leu Cys Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr
Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val
Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40
45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg 50 55 60
5460PRTArtificial SequenceSynthetic peptide. 54Leu Lys Gly Ile Ala
Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly 1 5 10 15 Trp Leu Leu
Gly Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser 20 25 30 Trp
Ser Tyr Ile Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr 35 40
45 Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu 50 55 60
5560PRTArtificial SequenceSynthetic peptide. 55Gln Leu Ser Ser Val
Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys 1 5 10 15 Glu Ser Ser
Trp Pro Asn His Asn Thr Asn Gly Val Thr Ala Ala Cys 20 25 30 Ser
His Glu Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr 35 40
45 Glu Lys Glu Gly Ser Tyr Pro Lys Leu Lys Asn Ser 50 55 60
5660PRTArtificial SequenceSynthetic peptide. 56Tyr Val Asn Lys Lys
Gly Lys Glu Val Leu Val Leu Trp Gly Ile His 1 5 10 15 His Pro Pro
Asn Ser Lys Glu Gln Gln Asn Leu Tyr Gln Asn Glu Asn 20 25 30 Ala
Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg Arg Phe Thr Pro 35 40
45 Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 50 55 60
5760PRTArtificial SequenceSynthetic peptide. 57Gly Arg Met Asn Tyr
Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 1 5 10 15 Ile Phe Glu
Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 20 25 30 Leu
Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser Met 35 40
45 His Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly 50 55 60
5840PRTArtificial SequenceSynthetic peptide. 58Ala Ile Asn Ser Ser
Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile 1 5 10 15 Gly Glu Cys
Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr 20 25 30 Gly
Leu Arg Asn Ile Pro Ser Ile 35 40 5917PRTArtificial
SequenceSynthetic peptide. 59Ser Ser Phe Glu Arg Phe Glu Ile Phe
Pro Lys Glu Ser Ser Trp Pro 1 5 10 15 Asn 6017PRTArtificial
SequenceSynthetic peptide. 60Gly Asn Leu Ile Ala Pro Trp Tyr Ala
Phe Ala Leu Ser Arg Gly Phe 1 5 10 15 Gly 6117PRTArtificial
SequenceSynthetic peptide. 61Trp Tyr Ala Phe Ala Leu Ser Arg Gly
Phe Gly Ser Gly Ile Ile Thr 1 5 10 15 Ser 6260PRTArtificial
SequenceSynthetic peptide. 62Met Ala Ser Gln Gly Thr Lys Arg Ser
Tyr Glu Gln Met Glu Thr Asp 1 5 10 15 Gly Glu Arg Gln Asn Ala Thr
Glu Ile Arg Ala Ser Val Gly Lys Met 20 25 30 Ile Gly Gly Ile Gly
Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys 35 40 45 Leu Ser Asp
Tyr Glu Gly Arg Leu Ile Gln Asn Ser 50 55 60 6360PRTArtificial
SequenceSynthetic peptide. 63Leu Thr Ile Glu Arg Met Val Leu Ser
Ala Phe Asp Glu Arg Arg Asn 1 5 10 15 Lys Tyr Leu Glu Glu His Pro
Ser Ala Gly Lys Asp Pro Lys Lys Thr 20 25 30 Gly Gly Pro Ile Tyr
Arg Arg Val Asn Gly Lys Trp Met Arg Glu Leu 35 40 45 Ile Leu Tyr
Asp Lys Glu Glu Ile Arg Arg Ile Trp 50 55 60 6458PRTArtificial
SequenceSynthetic peptide. 64Arg Gln Ala Asn Asn Gly Asp Asp Ala
Thr Ala Gly Leu Thr His Met 1 5 10 15 Met Ile Trp His Ser Asn Leu
Asn Asp Ala Thr Tyr Gln Arg Thr Arg 20 25 30 Ala Leu Val Arg Thr
Gly Met Asp Pro Arg Met Cys Ser Leu Met Gln 35 40 45 Gly Ser Thr
Leu Pro Arg Arg Ser Gly Ala 50 55 6560PRTArtificial
SequenceSynthetic peptide. 65Ala Ala Val Lys Gly Val Gly Thr Met
Val Met Glu Leu Val Arg Met 1 5 10 15 Ile Lys Arg Gly Ile Asn Asp
Arg Asn Phe Trp Arg Gly Glu Asn Gly 20 25 30 Arg Lys Thr Arg Ile
Ala Tyr Glu Arg Met Cys Asn Ile Leu Lys Gly 35 40 45 Lys Phe Gln
Thr Ala Ala Gln Lys Ala Met Met Asp 50 55 60 6660PRTArtificial
SequenceSynthetic peptide. 66Gln Val Arg Glu Ser Arg Asn Pro Gly
Asn Ala Glu Phe Glu Asp Leu 1 5 10 15 Thr Phe Leu Ala Arg Ser Ala
Leu Ile Leu Arg Gly Ser Val Ala His 20 25 30 Lys Ser Cys Leu Pro
Ala Cys Val Tyr Gly Pro Ala Val Ala Ser Gly 35 40 45 Tyr Asp Phe
Glu Arg Glu Gly Tyr Ser Leu Val Gly 50 55 60 6760PRTArtificial
SequenceSynthetic peptide. 67Ile Asp Pro Phe Arg Leu Leu Gln Asn
Ser Gln Val Tyr Ser Leu Ile 1 5 10 15 Arg Pro Asn Glu Asn Pro Ala
His Lys Ser Gln Leu Val Trp Met Ala 20 25 30 Cys His Ser Ala Ala
Phe Glu Asp Leu Arg Val Leu Ser Phe Ile Lys 35 40 45 Gly Thr Lys
Val Leu Pro Arg Gly Lys Leu Ser Thr 50 55 60 6860PRTArtificial
SequenceSynthetic peptide. 68Arg Gly Val Gln Ile Ala Ser Asn Glu
Asn Met Glu Thr Met Glu Ser 1 5 10 15 Ser Thr Leu Glu Leu Arg Ser
Arg Tyr Trp Ala Ile Arg Thr Arg Ser 20 25 30 Gly Gly Asn Thr Asn
Gln Gln Arg Ala Ser Ala Gly Gln Ile Ser Ile 35 40 45 Gln Pro Thr
Phe Ser Val Gln Arg Asn Leu Pro Phe 50 55 60 6960PRTArtificial
SequenceSynthetic peptide. 69Asp Arg Thr Thr Ile Met Ala Ala Phe
Asn Gly Asn Thr Glu Gly Arg 1 5 10 15 Thr Ser Asp Met Arg Thr Glu
Ile Ile Arg Met Met Glu Ser Ala Arg 20 25 30 Pro Glu Asp Val Ser
Phe Gln Gly Arg Gly Val Phe Glu Leu Ser Asp 35 40 45 Glu Lys Ala
Ala Ser Pro Ile Val Pro Ser Phe Asp 50 55 60 7018PRTArtificial
SequenceSynthetic peptide. 70Met Ser Asn Glu Gly Ser Tyr Phe Phe
Gly Asp Asn Ala Glu Glu Tyr 1 5 10 15 Asp Asn 7117PRTArtificial
SequenceSynthetic peptide. 71Gly Lys Trp Val Arg Glu Leu Val Leu
Tyr Asp Lys Glu Glu Ile Arg 1 5 10 15 Arg 7217PRTArtificial
SequenceSynthetic peptide. 72Arg Thr Gly Met Asp Pro Arg Met Cys
Ser Leu Met Gln Gly Ser Thr 1 5 10 15 Leu 7317PRTArtificial
SequenceSynthetic peptide. 73Met Cys Asn Ile Leu Lys Gly Lys Phe
Gln Thr Ala Ala Gln Lys Ala 1 5 10 15 Met 7460PRTArtificial
SequenceSynthetic peptide. 74Met Trp Val Pro Val Val Phe Leu Thr
Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala Ala Pro Leu Ile Leu Ser
Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25 30 Lys His Ser Gln Pro
Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala 35 40 45 Val Cys Gly
Gly Val Leu Val His Pro Gln Trp Val 50 55 60 7560PRTArtificial
SequenceSynthetic peptide. 75Leu Thr Ala Ala His Cys Ile Arg Asn
Lys Ser Val Ile Leu Leu Gly 1 5 10 15 Arg His Ser Leu Phe His Pro
Glu Asp Thr Gly Gln Val Phe Gln Val 20 25 30 Ser His Ser Phe Pro
His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn 35 40 45 Arg Phe Leu
Arg Pro Gly Asp Asp Ser Ser His Asp 50 55 60 7660PRTArtificial
SequenceSynthetic peptide. 76Leu Met Leu Leu Arg Leu Ser Glu Pro
Ala Glu Leu Thr Asp Ala Val 1 5 10 15 Lys Val Met Asp Leu Pro Thr
Gln Glu Pro Ala Leu Gly Thr Thr Cys 20 25 30 Tyr Ala Ser Gly Trp
Gly Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro 35 40 45 Lys Lys Leu
Gln Cys Val Asp Leu His Val Ile Ser 50 55 60 7760PRTArtificial
SequenceSynthetic peptide. 77Asn Asp Val Cys Ala Gln Val His Pro
Gln Lys Val Thr Lys Phe Met 1 5 10 15 Leu Cys Ala Gly Arg Trp Thr
Gly Gly Lys Ser Thr Cys Ser Gly Asp 20 25 30 Ser Gly Gly Pro Leu
Val Cys Asn Gly Val Leu Gln Gly Ile Thr Ser 35 40 45 Trp Gly Ser
Glu Pro Cys Ala Leu Pro Glu Arg Pro 50 55 60 7821PRTArtificial
SequenceSynthetic peptide. 78Ser Leu Tyr Thr Lys Val Val His Tyr
Arg Lys Trp Ile Lys Asp Thr 1 5 10 15 Ile Val Ala Asn Pro 20
7915PRTArtificial SequenceSynthetic peptide. 79Ala Pro Leu Ile Leu
Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 1 5 10 15 8015PRTArtificial
SequenceSynthetic peptide. 80Glu Cys Glu Lys His Ser Gln Pro Trp
Gln Val Leu Val Ala Ser 1 5 10 15 8115PRTArtificial
sequenceSynthetic peptide. 81Gly Asp Asp Ser Ser His Asp Leu Met
Leu Leu Arg Leu Ser Glu 1 5 10 15 8215PRTArtificial
SequenceSynthetic peptide. 82Ser His Asp Leu Met Leu Leu Arg Leu
Ser Glu Pro Ala Glu Leu 1 5 10 15 8315PRTArtificial
SequenceSynthetic peptide. 83Ser Gly Asp Ser Gly Gly Pro Leu Val
Cys Asn Gly Val Leu Gln 1 5 10 15 8415PRTArtificial
SequenceSynthetic peptide. 84Gly Ser Glu Pro Cys Ala Leu Pro Glu
Arg Pro Ser Leu Tyr Thr 1 5 10 15 8515PRTArtificial
SequenceSynthetic peptide. 85Glu Arg Pro Ser Leu Tyr Thr Lys Val
Val His Tyr Arg Lys Trp 1 5 10 15 8615PRTArtificial
SequenceSynthetic peptide. 86Val Val His Tyr Arg Lys Trp Ile Lys
Asp Thr Ile Val Ala Asn 1 5 10 15 8760PRTArtificial
SequenceSynthetic peptide. 87Met Arg Ser Tyr Arg Phe Ser Asp Tyr
Leu His Met Ser Val Ser Phe 1 5 10 15 Ser Asn Asp Met Asp Leu Phe
Cys Gly Glu Asp Ser Gly Val Phe Ser 20 25 30 Gly Glu Ser Thr Val
Asp Phe Ser Ser Ser Glu Val Asp Ser Trp Pro 35 40 45 Gly Asp Ser
Ile Ala Cys Phe Ile Glu Asp Glu Arg 50 55 60 8860PRTArtificial
SequenceSynthetic peptide. 88His Phe Val Pro Gly His Asp Tyr Leu
Ser Arg Phe Gln Thr Arg Ser 1 5 10 15 Leu Asp Ala Ser Ala Arg Glu
Asp Ser Val Ala Trp Ile Leu Lys Val 20 25 30 Gln Ala Tyr Tyr Asn
Phe Gln Pro Leu Thr Ala Tyr Leu Ala Val Asn 35 40 45 Tyr Met Asp
Arg Phe Leu Tyr Ala Arg Arg Leu Pro 50 55 60 8960PRTArtificial
SequenceSynthetic peptide. 89Glu Thr Ser Gly Trp Pro Met Gln Leu
Leu Ala Val Ala Cys Leu Ser 1 5 10 15 Leu Ala Ala Lys Met Glu Glu
Ile Leu Val Pro Ser Leu Phe Asp Phe 20 25 30 Gln Val Ala Gly Val
Lys Tyr Leu Phe Glu Ala Lys Thr Ile Lys Arg 35 40 45 Met Glu Leu
Leu Val Leu Ser Val Leu Asp Trp Arg 50 55 60 9060PRTArtificial
SequenceSynthetic peptide. 90Leu Arg Ser Val Thr Pro Phe Asp Phe
Ile Ser Phe Phe Ala Tyr Lys 1 5 10 15 Ile Asp Pro Ser Gly Thr Phe
Leu Gly Phe Phe Ile Ser His Ala Thr 20 25 30 Glu Ile Ile Leu Ser
Asn Ile Lys Glu Ala Ser Phe Leu Glu Tyr Trp 35 40 45 Pro Ser Ser
Ile Ala Ala Ala Ala Ile Leu Cys Val 50 55 60 9160PRTArtificial
SequenceSynthetic peptide. 91Ala Asn Glu Leu Pro Ser Leu Ser Ser
Val Val Asn Pro His Glu Ser 1 5 10 15 Pro Glu Thr Trp Cys Asp Gly
Leu Ser Lys Glu Lys Ile Val Arg Cys 20 25 30 Tyr Arg Leu Met Lys
Ala Met Ala Ile Glu Asn Asn Arg Leu Asn Thr 35 40 45 Pro Lys Val
Ile Ala Lys Leu Arg Val Ser Val Arg 50 55 60 9239PRTArtificial
SequenceSynthetic peptide. 92Ala Ser Ser Thr Leu Thr Arg Pro Ser
Asp Glu Ser Ser Phe Ser Ser 1 5 10 15 Ser Ser Pro Cys Lys Arg Arg
Lys Leu Ser Gly Tyr Ser Trp Val Gly 20 25 30 Asp Glu Thr Ser Thr
Ser Asn 35 9315PRTArtificial SequenceSynthetic peptide. 93Asp Arg
Val Leu Arg Ala Met Leu Lys Ala Glu Glu Thr Cys Ala 1 5 10 15
9415PRTArtificial SequenceSynthetic peptide. 94Arg Ala Met Leu Lys
Ala Glu Glu Thr Cys Ala Pro Ser Val Ser 1 5 10 15 9515PRTArtificial
SequenceSynthetic peptide. 95Thr Cys Ala Pro Ser Val Ser Tyr Phe
Lys Cys Val Gln Lys Glu 1 5 10 15 96587PRTArtificial
SequenceSynthetic peptide. 96Glu Val Lys Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala
Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val
Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile
Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Ala Lys Thr Lys
Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195
200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro
Ser Val Phe 225 230 235 240 Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275
280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395
400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
Gly Lys Ala Ser 435 440 445 Gln Thr Pro Thr Asn Thr Ile Ser Val Thr
Pro Thr Asn Asn Ser Thr 450 455 460 Pro Thr Asn Asn Ser Asn Pro Lys
Pro Asn Pro Ala Ser Gly Phe Asp 465 470 475 480 His Arg Asp Ser Lys
Val Ser Leu Gln Glu Lys Asn Cys Glu Pro Val 485 490 495 Val Pro Asn
Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser 500 505 510 Pro
Pro Pro Tyr Ser Pro Ala Ser Thr Asn Gly Ser Ile Thr Val Ala 515 520
525 Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr Pro Ser Ala
530 535 540 Ala Ala Ser Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly
Tyr Pro 545 550 555 560 Lys Lys Gly His Gly His Ser Tyr Thr Thr Ala
Glu Glu Ala Ala Gly 565 570 575 Ile Gly Ile Leu Thr Val Ile Leu Gly
Ala Ser 580 585 97656PRTArtificial SequenceSynthetic peptide. 97Glu
Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu
Trp Val 35 40 45 Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr
Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Arg Leu Lys Ser
Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Leu Pro
Phe His Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr
Val Ser Ser Ala Lys Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145
150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala
Pro Glu Phe Glu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265
270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390
395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys Ala Ser 435 440 445 Gln Thr Pro Thr Asn Thr Ile Ser Val
Thr Pro Thr Asn Asn Ser Thr 450 455 460 Pro Thr Asn Asn Ser Asn Pro
Lys Pro Asn Pro Ala Ser Gly Phe Asp 465 470 475 480 His Arg Asp Ser
Lys Val Ser Leu Gln Glu Lys Asn Cys Glu Pro Val 485 490 495 Val Pro
Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser 500 505 510
Pro Pro Pro Tyr Ser Pro Ala Ser Thr Asn Gly Ser Ile Thr Val Ala 515
520 525 Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr Pro Ser
Ala 530 535 540 Ala Ala Ser Met Pro Arg Glu Asp Ala His Phe Ile Tyr
Gly Tyr Pro 545 550 555 560 Lys Lys Gly His Gly His Ser Tyr Thr Thr
Ala Glu Glu Ala Ala Gly 565 570 575 Ile Gly Ile Leu Thr Val Ile Leu
Gly Ala Ser Thr Val Thr Pro Thr 580 585 590 Ala Thr Ala Thr Pro Ser
Ala Ile Val Thr Thr Ile Thr Pro Thr Ala 595 600 605 Thr Thr Lys Pro
Ala Ser Val Leu Leu Leu Ile Gly Cys Trp Tyr Cys 610 615 620 Arg Arg
Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser Leu His Val 625 630 635
640 Gly Thr Gln Cys Ala Leu Thr Arg Arg Cys Pro Gln Glu Gly Ala Ser
645 650 655 98593PRTArtificial SequenceSynthetic peptide. 98Gln Val
Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu
Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr
Ala Asp Ala Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr
Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala
Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150
155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395
400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys Ala Ser Gln Thr Pro Thr
Asn Thr Ile Ser Val Thr 450 455 460 Pro Thr Asn Asn Ser Thr Pro Thr
Asn Asn Ser Asn Pro Lys Pro Asn 465 470 475 480 Pro Ala Ser Gly Phe
Asp His Arg Asp Ser Lys Val Ser Leu Gln Glu 485 490 495 Lys Asn Cys
Glu Pro Val Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys 500 505 510 Leu
Ser Ala Glu Gln Ser Pro Pro Pro Tyr Ser Pro Ala Ser Thr Asn 515 520
525 Gly Ser Ile Thr Val Ala Ala Thr Ala Pro Thr Val Thr Pro Thr Val
530 535 540 Asn Ala Thr Pro Ser Ala Ala Ala Ser Met Pro Arg Glu Asp
Ala His 545 550 555 560 Phe Ile Tyr Gly Tyr Pro Lys Lys Gly His Gly
His Ser Tyr Thr Thr 565 570 575 Ala Glu Glu Ala Ala Gly Ile Gly Ile
Leu Thr Val Ile Leu Gly Ala 580 585 590 Ser 99660PRTArtificial
SequenceSynthetic peptide. 99Gln Val Thr Leu Lys Glu Ser Gly Pro
Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Leu Ser
Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala
His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60 Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val 65 70
75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp Ala Ala Thr Tyr
Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr Gly Tyr Gly
Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195
200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300 Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315
320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440
445 Gly Lys Ala Ser Gln Thr Pro Thr Asn Thr Ile Ser Val Thr Pro Thr
450 455 460 Asn Asn Ser Thr Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn
Pro Ala 465 470 475 480 Ser Gly Phe Asp His Arg Asp Ser Lys Val Ser
Leu Gln Glu Lys Asn 485 490 495 Cys Glu Pro Val Val Pro Asn Ala Pro
Pro Ala Tyr Glu Lys Leu Ser 500 505 510 Ala Glu Gln Ser Pro Pro Pro
Tyr Ser Pro Ala Ser Thr Asn Gly Ser 515 520 525 Ile Thr Val Ala Ala
Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala 530 535 540 Thr Pro Ser
Ala Ala Ala Ser Met Pro Arg Glu Asp Ala His Phe Ile 545 550 555 560
Tyr Gly Tyr Pro Lys Lys Gly His Gly His Ser Tyr Thr Thr Ala Glu 565
570 575 Glu Ala Ala Gly Ile Gly Ile Leu Thr Val Ile Leu Gly Ala Ser
Thr 580 585 590 Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val
Thr Thr Ile 595 600 605 Thr Pro Thr Ala Thr Thr Lys Pro Ala Ser Val
Leu Leu Leu Ile Gly 610 615 620 Cys Trp Tyr Cys Arg Arg Arg Asn Gly
Tyr Arg Ala Leu Met Asp Lys 625 630 635 640 Ser Leu His Val Gly Thr
Gln Cys Ala Leu Thr Arg Arg Cys Pro Gln 645 650 655 Glu Gly Ala Ser
660 100630DNAArtificial SequenceSynthetic oligonucleotide.
100aacaccgaca acaacagatg atctggatgc agctagtggg tttgatcatc
gggacagcaa 60agtgtctctt caagagaaaa actgtgaacc tgtggttccc aatgctccac
ctgcttatga 120gaaactctct gcagaacagt caccaccacc
ttattcacct gctagtacca acggcagcat 180caccgtggcc gccaccgccc
ccaccgtgac ccccaccgtg aacgccaccc ccagcgccgc 240cgctagtatg
ccaagagaag atgctcactt catctatggt taccccaaga aggggcacgg
300ccactcttac accacggctg aagaggccgc tgggatcggc atcctgacag
tgatcctggg 360agctagtacc gtgaccccca ccgccaccgc cacccccagc
gccatcgtga ccaccatcac 420ccccaccgcc accaccaagc ccgctagtgt
cttactgctc atcggctgtt ggtattgtag 480aagacgaaat ggatacagag
ccttgatgga taaaagtctt catgttggca ctcaatgtgc 540cttaacaaga
agatgcccac aagaagggtg agcggccgca tcgaagagct cggtacccgg
600ggatcctcta gagtcgacct gcaggcatgc 63010160PRTArtificial
SequenceSynthetic peptide. 101Gly Phe Asp His Arg Asp Ser Lys Val
Ser Leu Gln Glu Lys Asn Cys 1 5 10 15 Glu Pro Val Val Pro Asn Ala
Pro Pro Ala Tyr Glu Lys Leu Ser Ala 20 25 30 Glu Gln Ser Pro Pro
Pro Tyr Ser Pro Ala Ser Thr Asn Gly Ser Ile 35 40 45 Thr Val Ala
Ala Thr Ala Pro Thr Val Thr Pro Thr 50 55 60 10260PRTArtificial
SequenceSynthetic peptide. 102Val Asn Ala Thr Pro Ser Ala Ala Ala
Ser Met Pro Arg Glu Asp Ala 1 5 10 15 His Phe Ile Tyr Gly Tyr Pro
Lys Lys Gly His Gly His Ser Tyr Thr 20 25 30 Thr Ala Glu Glu Ala
Ala Gly Ile Gly Ile Leu Thr Val Ile Leu Gly 35 40 45 Ala Ser Thr
Val Thr Pro Thr Ala Thr Ala Thr Pro 50 55 60 10357PRTArtificial
SequenceSynthetic peptide. 103Ser Ala Ile Val Thr Thr Ile Thr Pro
Thr Ala Thr Thr Lys Pro Ala 1 5 10 15 Ser Val Leu Leu Leu Ile Gly
Cys Trp Tyr Cys Arg Arg Arg Asn Gly 20 25 30 Tyr Arg Ala Leu Met
Asp Lys Ser Leu His Val Gly Thr Gln Cys Ala 35 40 45 Leu Thr Arg
Arg Cys Pro Gln Glu Gly 50 55 10441PRTArtificial SequenceSynthetic
peptide. 104Gly Phe Asp His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys
Asn Cys 1 5 10 15 Glu Pro Val Val Pro Asn Ala Pro Pro Ala Tyr Glu
Lys Leu Ser Ala 20 25 30 Glu Gln Ser Pro Pro Pro Tyr Ser Pro 35 40
10529PRTArtificial SequenceSynthetic peptide. 105Ala Ser Thr Asn
Gly Ser Ile Thr Val Ala Ala Thr Ala Pro Thr Val 1 5 10 15 Thr Pro
Thr Val Asn Ala Thr Pro Ser Ala Ala Ala Ser 20 25
10638PRTArtificial SequenceSynthetic peptide. 106Met Pro Arg Glu
Asp Ala His Phe Ile Tyr Gly Tyr Pro Lys Lys Gly 1 5 10 15 His Gly
His Ser Tyr Thr Thr Ala Glu Glu Ala Ala Gly Ile Gly Ile 20 25 30
Leu Thr Val Ile Leu Gly 35 10729PRTArtificial SequenceSynthetic
peptide. 107Ala Ser Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala
Ile Val 1 5 10 15 Thr Thr Ile Thr Pro Thr Ala Thr Thr Lys Pro Ala
Ser 20 25 10840PRTArtificial SequenceSynthetic peptide. 108Val Leu
Leu Leu Ile Gly Cys Trp Tyr Cys Arg Arg Arg Asn Gly Tyr 1 5 10 15
Arg Ala Leu Met Asp Lys Ser Leu His Val Gly Thr Gln Cys Ala Leu 20
25 30 Thr Arg Arg Cys Pro Gln Glu Gly 35 40 109110PRTArtificial
SequenceSynthetic peptide. 109Gly Phe Asp His Arg Asp Ser Lys Val
Ser Leu Gln Glu Lys Asn Cys 1 5 10 15 Glu Pro Val Val Pro Asn Ala
Pro Pro Ala Tyr Glu Lys Leu Ser Ala 20 25 30 Glu Gln Ser Pro Pro
Pro Tyr Ser Pro Ala Ser Thr Asn Gly Ser Ile 35 40 45 Thr Val Ala
Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr 50 55 60 Pro
Ser Ala Ala Ala Ser Met Pro Arg Glu Asp Ala His Phe Ile Tyr 65 70
75 80 Gly Tyr Pro Lys Lys Gly His Gly His Ser Tyr Thr Thr Ala Glu
Glu 85 90 95 Ala Ala Gly Ile Gly Ile Leu Thr Val Ile Leu Gly Ala
Ser 100 105 110 110675PRTArtificial SequenceSynthetic peptide.
110Met Asn Leu Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly
1 5 10 15 Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu
Val Gln 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr Ser
Gly Phe Thr Phe 35 40 45 Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln
Thr Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Tyr Ile Asn Ser
Gly Gly Gly Ser Thr Tyr Tyr Pro 65 70 75 80 Asp Thr Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu
Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr
Cys Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp 115 120 125
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Lys Gly Pro 130
135 140 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 210 215 220 His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 225 230 235 240 Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 245 250
255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp 275 280 285 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335 Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 340 345 350 Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365 Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375
380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys 420 425 430 Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 435 440 445 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly 450 455 460 Lys Ala Ser Gln Thr
Pro Thr Asn Thr Ile Ser Val Thr Pro Thr Asn 465 470 475 480 Asn Ser
Thr Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn Pro Ala Ser 485 490 495
Gly Phe Asp His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys 500
505 510 Glu Pro Val Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser
Ala 515 520 525 Glu Gln Ser Pro Pro Pro Tyr Ser Pro Ala Ser Thr Asn
Gly Ser Ile 530 535 540 Thr Val Ala Ala Thr Ala Pro Thr Val Thr Pro
Thr Val Asn Ala Thr 545 550 555 560 Pro Ser Ala Ala Ala Ser Met Pro
Arg Glu Asp Ala His Phe Ile Tyr 565 570 575 Gly Tyr Pro Lys Lys Gly
His Gly His Ser Tyr Thr Thr Ala Glu Glu 580 585 590 Ala Ala Gly Ile
Gly Ile Leu Thr Val Ile Leu Gly Ala Ser Thr Val 595 600 605 Thr Pro
Thr Ala Thr Ala Thr Pro Ser Ala Ile Val Thr Thr Ile Thr 610 615 620
Pro Thr Ala Thr Thr Lys Pro Ala Ser Val Leu Leu Leu Ile Gly Cys 625
630 635 640 Trp Tyr Cys Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp
Lys Ser 645 650 655 Leu His Val Gly Thr Gln Cys Ala Leu Thr Arg Arg
Cys Pro Gln Glu 660 665 670 Gly Ala Ser 675 111606PRTArtificial
SequenceSynthetic peptide. 111Met Asn Leu Gly Leu Ser Leu Ile Phe
Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Lys Leu
Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu
Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe 35 40 45 Ser Asp Tyr
Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu
Trp Val Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro 65 70
75 80 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Arg Leu Lys Ser Glu Asp
Thr Ala Met 100 105 110 Tyr Tyr Cys Ala Arg Arg Gly Leu Pro Phe His
Ala Met Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Ser Val Thr Val Ser
Ser Ala Lys Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr 145 150 155 160 Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195
200 205 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
Asp 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr 225 230 235 240 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Glu Gly Gly Pro 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp 275 280 285 Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300 Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 305 310 315
320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys 340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr 355 360 365 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr 370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415 Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 420 425 430 Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440
445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
450 455 460 Lys Ala Ser Gln Thr Pro Thr Asn Thr Ile Ser Val Thr Pro
Thr Asn 465 470 475 480 Asn Ser Thr Pro Thr Asn Asn Ser Asn Pro Lys
Pro Asn Pro Ala Ser 485 490 495 Gly Phe Asp His Arg Asp Ser Lys Val
Ser Leu Gln Glu Lys Asn Cys 500 505 510 Glu Pro Val Val Pro Asn Ala
Pro Pro Ala Tyr Glu Lys Leu Ser Ala 515 520 525 Glu Gln Ser Pro Pro
Pro Tyr Ser Pro Ala Ser Thr Asn Gly Ser Ile 530 535 540 Thr Val Ala
Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr 545 550 555 560
Pro Ser Ala Ala Ala Ser Met Pro Arg Glu Asp Ala His Phe Ile Tyr 565
570 575 Gly Tyr Pro Lys Lys Gly His Gly His Ser Tyr Thr Thr Ala Glu
Glu 580 585 590 Ala Ala Gly Ile Gly Ile Leu Thr Val Ile Leu Gly Ala
Ser 595 600 605 112661PRTArtificial SequenceSynthetic peptide.
112Met Asp Leu Val Leu Lys Arg Cys Leu Leu His Leu Ala Val Ile Gly
1 5 10 15 Ala Leu Leu Ala Val Gly Ala Thr Lys Val Pro Arg Asn Gln
Asp Trp 20 25 30 Leu Gly Val Ser Arg Gln Leu Arg Thr Lys Ala Trp
Asn Arg Gln Leu 35 40 45 Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu
Asp Cys Trp Arg Gly Gly 50 55 60 Gln Val Ser Leu Lys Val Ser Asn
Asp Gly Pro Thr Leu Ile Gly Ala 65 70 75 80 Asn Ala Ser Phe Ser Ile
Ala Leu Asn Phe Pro Gly Ser Gln Lys Val 85 90 95 Leu Pro Asp Gly
Gln Val Ile Trp Val Asn Asn Thr Ile Ile Asn Gly 100 105 110 Ser Gln
Val Trp Gly Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp 115 120 125
Ala Cys Ile Phe Pro Asp Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser 130
135 140 Gln Lys Arg Ser Phe Val Tyr Val Trp Lys Thr Trp Gly Gln Tyr
Trp 145 150 155 160 Gln Val Leu Gly Gly Pro Val Ser Gly Leu Ser Ile
Gly Thr Gly Arg 165 170 175 Ala Met Leu Gly Thr His Thr Met Glu Val
Thr Val Tyr His Arg Arg 180 185 190 Gly Ser Arg Ser Tyr Val Pro Leu
Ala His Ser Ser Ser Ala Phe Thr 195 200 205 Ile Thr Asp Gln Val Pro
Phe Ser Val Ser Val Ser Gln Leu Arg Ala 210 215 220 Leu Asp Gly Gly
Asn Lys His Phe Leu Arg Asn Gln Pro Leu Thr Phe 225 230 235 240 Ala
Leu Gln Leu His Asp Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu 245 250
255 Ser Tyr Thr Trp Asp Phe Gly Asp Ser Ser Gly Thr Leu Ile Ser Arg
260 265 270 Ala Leu Val Val Thr His Thr Tyr Leu Glu Pro Gly Pro Val
Thr Ala 275 280 285 Gln Val Val Leu Gln Ala Ala Ile Pro Leu Thr Ser
Cys Gly Ser Ser 290 295 300 Pro Val Pro Gly Thr Thr Asp Gly His Arg
Pro Thr Ala Glu Ala Pro 305 310 315 320 Asn Thr Thr Ala Gly Gln Val
Pro Thr Thr Glu Val Val Gly Thr Thr 325 330 335 Pro Gly Gln Ala Pro
Thr Ala Glu Pro Ser Gly Thr Thr Ser Val Gln 340 345 350 Val Pro Thr
Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro Thr 355 360 365 Ala
Glu Ser Thr Gly Met Thr Pro Glu Lys Val Pro Val Ser Glu Val 370 375
380 Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu Ala Thr Gly Met
385 390 395 400 Thr Pro Ala
Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 405 410 415 Gln
Val Thr Thr Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu Pro 420 425
430 Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser Thr Glu
435 440 445 Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu Asp Gly Thr Ala
Thr Leu 450 455 460 Arg Leu Val Lys Arg Gln Val Pro Leu Asp Cys Val
Leu Tyr Arg Tyr 465 470 475 480 Gly Ser Phe Ser Val Thr Leu Asp Ile
Val Gln Gly Ile Glu Ser Ala 485 490 495 Glu Ile Leu Gln Ala Val Pro
Ser Gly Glu Gly Asp Ala Phe Glu Leu 500 505 510 Thr Val Ser Cys Gln
Gly Gly Leu Pro Lys Glu Ala Cys Met Glu Ile 515 520 525 Ser Ser Pro
Gly Cys Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val 530 535 540 Leu
Pro Ser Pro Ala Cys Gln Leu Val Leu His Gln Ile Leu Lys Gly 545 550
555 560 Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu Ala Asp Thr Asn
Ser 565 570 575 Leu Ala Val Val Ser Thr Gln Leu Ile Met Pro Gly Gln
Glu Ala Gly 580 585 590 Leu Gly Gln Val Pro Leu Ile Val Gly Ile Leu
Leu Val Leu Met Ala 595 600 605 Val Val Leu Ala Ser Leu Ile Tyr Arg
Arg Arg Leu Met Lys Gln Asp 610 615 620 Phe Ser Val Pro Gln Leu Pro
His Ser Ser Ser His Trp Leu Arg Leu 625 630 635 640 Pro Arg Ile Phe
Cys Ser Cys Pro Ile Gly Glu Asn Ser Pro Leu Leu 645 650 655 Ser Gly
Gln Gln Val 660 1139PRTArtificial SequenceSynthetic peptide. 113Ile
Met Asp Gln Val Pro Phe Ser Val 1 5 1149PRTArtificial
SequenceSynthetic peptide. 114Ile Thr Asp Gln Val Pro Phe Ser Val 1
5 1159PRTArtificial SequenceSynthetic peptide. 115Tyr Leu Glu Pro
Gly Pro Val Thr Val 1 5 1169PRTArtificial SequenceSynthetic
peptide. 116Tyr Leu Glu Pro Gly Pro Val Thr Ala 1 5
1179PRTArtificial SequenceSynthetic peptide. 117Lys Thr Trp Gly Gln
Tyr Trp Gln Val 1 5 1181160PRTArtificial SequenceSynthetic peptide.
118Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser
Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg
Leu Glu Trp Val 35 40 45 Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr
Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Arg Leu
Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly
Leu Pro Phe His Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser
Val Thr Val Ser Ser Ala Lys Thr Lys Gly Pro Ser Val Phe 115 120 125
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130
135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250
255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly Lys Ala Ser 435 440 445 Asp Thr Thr Glu Pro Ala Thr
Pro Thr Thr Pro Val Thr Thr Pro Thr 450 455 460 Thr Thr Lys Val Pro
Arg Asn Gln Asp Trp Leu Gly Val Ser Arg Gln 465 470 475 480 Leu Arg
Thr Lys Ala Trp Asn Arg Gln Leu Tyr Pro Glu Trp Thr Glu 485 490 495
Ala Gln Arg Leu Asp Cys Trp Arg Gly Gly Gln Val Ser Leu Lys Val 500
505 510 Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala Asn Ala Ser Phe Ser
Ile 515 520 525 Ala Leu Asn Phe Pro Gly Ser Gln Lys Val Leu Pro Asp
Gly Gln Val 530 535 540 Ile Trp Val Asn Asn Thr Ile Ile Asn Gly Ser
Gln Val Trp Gly Gly 545 550 555 560 Gln Pro Val Tyr Pro Gln Glu Thr
Asp Asp Ala Cys Ile Phe Pro Asp 565 570 575 Gly Gly Pro Cys Pro Ser
Gly Ser Trp Ser Gln Lys Arg Ser Phe Val 580 585 590 Tyr Val Trp Lys
Thr Trp Gly Gln Tyr Trp Gln Val Leu Gly Gly Pro 595 600 605 Val Ser
Gly Leu Ser Ile Gly Thr Gly Arg Ala Met Leu Gly Thr His 610 615 620
Thr Met Glu Val Thr Val Tyr His Arg Arg Gly Ser Gln Ser Tyr Val 625
630 635 640 Pro Leu Ala His Ser Ser Ser Ala Phe Thr Ile Thr Asp Gln
Val Pro 645 650 655 Phe Ser Val Ser Val Ser Gln Leu Arg Ala Leu Asp
Gly Gly Asn Lys 660 665 670 His Phe Leu Arg Asn Gln Ala Ser Thr Asn
Gly Ser Ile Thr Val Ala 675 680 685 Ala Thr Ala Pro Thr Val Thr Pro
Thr Val Asn Ala Thr Pro Ser Ala 690 695 700 Ala Ala Ser Gly Thr Thr
Asp Gly His Arg Pro Thr Thr Glu Ala Pro 705 710 715 720 Asn Thr Thr
Ala Gly Gln Val Pro Thr Thr Glu Val Val Gly Thr Thr 725 730 735 Pro
Gly Gln Ala Pro Thr Ala Glu Pro Ser Gly Thr Thr Ser Val Gln 740 745
750 Val Pro Thr Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro Thr
755 760 765 Ala Glu Ser Thr Gly Met Thr Pro Glu Lys Val Pro Val Ser
Glu Val 770 775 780 Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu
Ala Thr Gly Met 785 790 795 800 Thr Pro Ala Glu Val Ser Ile Val Val
Leu Ser Gly Thr Thr Ala Ala 805 810 815 Ala Ser Thr Val Thr Pro Thr
Ala Thr Ala Thr Pro Ser Ala Ile Val 820 825 830 Thr Thr Ile Thr Pro
Thr Ala Thr Thr Lys Pro Ala Ser Gln Val Thr 835 840 845 Thr Thr Glu
Trp Val Glu Thr Thr Ala Arg Glu Leu Pro Ile Pro Glu 850 855 860 Pro
Glu Gly Pro Asp Ala Ser Ser Ile Met Ser Thr Glu Ser Ile Thr 865 870
875 880 Gly Ser Leu Gly Pro Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu
Val 885 890 895 Lys Arg Gln Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr
Gly Ser Phe 900 905 910 Ser Val Thr Leu Asp Ile Val Gln Ala Ser Thr
Asn Gly Ser Ile Thr 915 920 925 Val Ala Ala Thr Ala Pro Thr Val Thr
Pro Thr Val Asn Ala Thr Pro 930 935 940 Ser Ala Ala Ala Ser Gly Ile
Glu Ser Ala Glu Ile Leu Gln Ala Val 945 950 955 960 Pro Ser Gly Glu
Gly Asp Ala Phe Glu Leu Thr Val Ser Cys Gln Gly 965 970 975 Gly Leu
Pro Lys Glu Ala Cys Met Glu Ile Ser Ser Pro Gly Cys Gln 980 985 990
Pro Pro Ala Gln Arg Leu Cys Gln Pro Val Leu Pro Ser Pro Ala Cys 995
1000 1005 Gln Leu Val Leu His Gln Ile Leu Lys Gly Gly Ser Gly Thr
Tyr 1010 1015 1020 Cys Leu Asn Val Ser Leu Ala Asp Thr Asn Ser Leu
Ala Val Val 1025 1030 1035 Ser Thr Gln Leu Ile Val Pro Gly Ile Leu
Leu Thr Gly Gln Glu 1040 1045 1050 Ala Gly Leu Gly Gln Ala Ser Thr
Val Thr Pro Thr Ala Thr Ala 1055 1060 1065 Thr Pro Ser Ala Ile Val
Thr Thr Ile Thr Pro Thr Ala Thr Thr 1070 1075 1080 Lys Pro Ala Ser
Pro Leu Thr Phe Ala Leu Gln Leu His Asp Pro 1085 1090 1095 Ser Gly
Tyr Leu Ala Glu Ala Asp Leu Ser Tyr Thr Trp Asp Phe 1100 1105 1110
Gly Asp Ser Ser Gly Thr Leu Ile Ser Arg Ala Leu Val Val Thr 1115
1120 1125 His Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala Gln Val Val
Leu 1130 1135 1140 Gln Ala Ala Ile Pro Leu Thr Ser Cys Gly Ser Ser
Pro Val Pro 1145 1150 1155 Ala Ser 1160 1191161PRTArtificial
SequenceSynthetic peptide. 119Arg Leu Gln Leu Gln Glu Ser Gly Pro
Gly Leu Leu Lys Pro Ser Val 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Asp Ser Val Ala Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly
Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly
Thr Ile Asn Phe Ser Gly Asn Met Tyr Tyr Ser Pro Ser 50 55 60 Leu
Arg Ser Arg Val Thr Met Ser Ala Asp Met Ser Glu Asn Ser Phe 65 70
75 80 Tyr Leu Lys Leu Asp Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr 85 90 95 Cys Ala Ala Gly His Leu Val Met Gly Phe Gly Ala His
Trp Gly Gln 100 105 110 Gly Lys Leu Val Ser Val Ser Pro Ala Ser Thr
Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195
200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly
Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315
320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala 435 440
445 Ser Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr Thr Pro
450 455 460 Thr Thr Thr Lys Val Pro Arg Asn Gln Asp Trp Leu Gly Val
Ser Arg 465 470 475 480 Gln Leu Arg Thr Lys Ala Trp Asn Arg Gln Leu
Tyr Pro Glu Trp Thr 485 490 495 Glu Ala Gln Arg Leu Asp Cys Trp Arg
Gly Gly Gln Val Ser Leu Lys 500 505 510 Val Ser Asn Asp Gly Pro Thr
Leu Ile Gly Ala Asn Ala Ser Phe Ser 515 520 525 Ile Ala Leu Asn Phe
Pro Gly Ser Gln Lys Val Leu Pro Asp Gly Gln 530 535 540 Val Ile Trp
Val Asn Asn Thr Ile Ile Asn Gly Ser Gln Val Trp Gly 545 550 555 560
Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp Ala Cys Ile Phe Pro 565
570 575 Asp Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser Gln Lys Arg Ser
Phe 580 585 590 Val Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp Gln Val
Leu Gly Gly 595 600 605 Pro Val Ser Gly Leu Ser Ile Gly Thr Gly Arg
Ala Met Leu Gly Thr 610 615 620 His Thr Met Glu Val Thr Val Tyr His
Arg Arg Gly Ser Gln Ser Tyr 625 630 635 640 Val Pro Leu Ala His Ser
Ser Ser Ala Phe Thr Ile Thr Asp Gln Val 645 650 655 Pro Phe Ser Val
Ser Val Ser Gln Leu Arg Ala Leu Asp Gly Gly Asn 660 665 670 Lys His
Phe Leu Arg Asn Gln Ala Ser Thr Asn Gly Ser Ile Thr Val 675 680 685
Ala Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr Pro Ser 690
695 700 Ala Ala Ala Ser Gly Thr Thr Asp Gly His Arg Pro Thr Thr Glu
Ala 705 710 715 720 Pro Asn Thr Thr Ala Gly Gln Val Pro Thr Thr Glu
Val Val Gly Thr 725 730 735 Thr Pro Gly Gln Ala Pro Thr Ala Glu Pro
Ser Gly Thr Thr Ser Val 740 745 750 Gln Val Pro Thr Thr Glu Val Ile
Ser Thr Ala Pro Val Gln Met Pro 755 760 765 Thr Ala Glu Ser Thr Gly
Met Thr Pro Glu Lys Val Pro Val Ser Glu 770 775 780 Val Met Gly Thr
Thr Leu
Ala Glu Met Ser Thr Pro Glu Ala Thr Gly 785 790 795 800 Met Thr Pro
Ala Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala 805 810 815 Ala
Ala Ser Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile 820 825
830 Val Thr Thr Ile Thr Pro Thr Ala Thr Thr Lys Pro Ala Ser Gln Val
835 840 845 Thr Thr Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu Pro
Ile Pro 850 855 860 Glu Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser
Thr Glu Ser Ile 865 870 875 880 Thr Gly Ser Leu Gly Pro Leu Leu Asp
Gly Thr Ala Thr Leu Arg Leu 885 890 895 Val Lys Arg Gln Val Pro Leu
Asp Cys Val Leu Tyr Arg Tyr Gly Ser 900 905 910 Phe Ser Val Thr Leu
Asp Ile Val Gln Ala Ser Thr Asn Gly Ser Ile 915 920 925 Thr Val Ala
Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr 930 935 940 Pro
Ser Ala Ala Ala Ser Gly Ile Glu Ser Ala Glu Ile Leu Gln Ala 945 950
955 960 Val Pro Ser Gly Glu Gly Asp Ala Phe Glu Leu Thr Val Ser Cys
Gln 965 970 975 Gly Gly Leu Pro Lys Glu Ala Cys Met Glu Ile Ser Ser
Pro Gly Cys 980 985 990 Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val
Leu Pro Ser Pro Ala 995 1000 1005 Cys Gln Leu Val Leu His Gln Ile
Leu Lys Gly Gly Ser Gly Thr 1010 1015 1020 Tyr Cys Leu Asn Val Ser
Leu Ala Asp Thr Asn Ser Leu Ala Val 1025 1030 1035 Val Ser Thr Gln
Leu Ile Val Pro Gly Ile Leu Leu Thr Gly Gln 1040 1045 1050 Glu Ala
Gly Leu Gly Gln Ala Ser Thr Val Thr Pro Thr Ala Thr 1055 1060 1065
Ala Thr Pro Ser Ala Ile Val Thr Thr Ile Thr Pro Thr Ala Thr 1070
1075 1080 Thr Lys Pro Ala Ser Pro Leu Thr Phe Ala Leu Gln Leu His
Asp 1085 1090 1095 Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu Ser Tyr
Thr Trp Asp 1100 1105 1110 Phe Gly Asp Ser Ser Gly Thr Leu Ile Ser
Arg Ala Leu Val Val 1115 1120 1125 Thr His Thr Tyr Leu Glu Pro Gly
Pro Val Thr Ala Gln Val Val 1130 1135 1140 Leu Gln Ala Ala Ile Pro
Leu Thr Ser Cys Gly Ser Ser Pro Val 1145 1150 1155 Pro Ala Ser 1160
1202142DNAArtificial SequenceSynthetic oligonucleotide.
120gatacaacag aacctgcaac acctacaaca cctgtaacaa caccgacaac
aacaaaagta 60cccagaaacc aggactggct tggtgtctca aggcaactca gaaccaaagc
ctggaacagg 120cagctgtatc cagagtggac agaagcccag agacttgact
gctggagagg tggtcaagtg 180tccctcaagg tcagtaatga tgggcctaca
ctgattggtg caaatgcctc cttctctatt 240gccttgaact tccctggaag
ccaaaaggta ttgccagatg ggcaggttat ctgggtcaac 300aataccatca
tcaatgggag ccaggtgtgg ggaggacagc cagtgtatcc ccaggaaact
360gacgatgcct gcatcttccc tgatggtgga ccttgcccat ctggctcttg
gtctcagaag 420agaagctttg tttatgtctg gaagacctgg ggccaatact
ggcaagttct agggggccca 480gtgtctgggc tgagcattgg gacaggcagg
gcaatgctgg gcacacacac catggaagtg 540actgtctacc atcgccgggg
atcccagagc tatgtgcctc ttgctcattc cagctcagcc 600ttcaccatta
ctgaccaggt gcctttctcc gtgagcgtgt cccagttgcg ggccttggat
660ggagggaaca agcacttcct gagaaatcag gctagtacca acggcagcat
caccgtggcc 720gccaccgccc ccaccgtgac ccccaccgtg aacgccaccc
ccagcgccgc cgctagtggc 780accacagatg ggcacaggcc aactgcagag
gcccctaaca ccacagctgg ccaagtgcct 840actacagaag ttgtgggtac
tacacctggt caggcgccaa ctgcagagcc ctctggaacc 900acatctgtgc
aggtgccaac cactgaagtc ataagcactg cacctgtgca gatgccaact
960gcagagagca caggtatgac acctgagaag gtgccagttt cagaggtcat
gggtaccaca 1020ctggcagaga tgtcaactcc agaggctaca ggtatgacac
ctgcagaggt atcaattgtg 1080gtgctttctg gaaccacagc tgcagctagt
accgtgaccc ccaccgccac cgccaccccc 1140agcgccatcg tgaccaccat
cacccccacc gccaccacca agcccgctag tcaggtaaca 1200actacagagt
gggtggagac cacagctaga gagctaccta tccctgagcc tgaaggtcca
1260gatgccagct caatcatgtc tacggaaagt attacaggtt ccctgggccc
cctgctggat 1320ggtacagcca ccttaaggct ggtgaagaga caagtccccc
tggattgtgt tctgtatcga 1380tatggttcct tttccgtcac cctggacatt
gtccaggcta gtaccaacgg cagcatcacc 1440gtggccgcca ccgcccccac
cgtgaccccc accgtgaacg ccacccccag cgccgccgct 1500agtggtattg
aaagtgccga gatcctgcag gctgtgccgt ccggtgaggg ggatgcattt
1560gagctgactg tgtcctgcca aggcgggctg cccaaggaag cctgcatgga
gatctcatcg 1620ccagggtgcc agccccctgc ccagcggctg tgccagcctg
tgctacccag cccagcctgc 1680cagctggttc tgcaccagat actgaagggt
ggctcgggga catactgcct caatgtgtct 1740ctggctgata ccaacagcct
ggcagtggtc agcacccagc ttatcgtgcc tgggattctt 1800ctcacaggtc
aagaagcagg ccttgggcag taagctagta ccgtgacccc caccgccacc
1860gccaccccca gcgccatcgt gaccaccatc acccccaccg ccaccaccaa
gcccgctagt 1920cctctgacct ttgccctcca gctccatgac cctagtggct
atctggctga agctgacctc 1980tcctacacct gggactttgg agacagtagt
ggaaccctga tctctcgggc acytgtggtc 2040actcatactt acctggagcc
tggcccagtc actgcccagg tggtcctgca ggctgccatt 2100cctctcacct
cctgtggctc ctccccagtt ccagctagct ga 2142121690DNAArtificial
SequenceSynthetic oligonucleotide. 121gatacaacag aacctgcaac
acctacaaca cctgtaacaa caccgacaac aacaaaagta 60cccagaaacc aggactggct
tggtgtctca aggcaactca gaaccaaagc ctggaacagg 120cagctgtatc
cagagtggac agaagcccag agacttgact gctggagagg tggtcaagtg
180tccctcaagg tcagtaatga tgggcctaca ctgattggtg caaatgcctc
cttctctatt 240gccttgaact tccctggaag ccaaaaggta ttgccagatg
ggcaggttat ctgggtcaac 300aataccatca tcaatgggag ccaggtgtgg
ggaggacagc cagtgtatcc ccaggaaact 360gacgatgcct gcatcttccc
tgatggtgga ccttgcccat ctggctcttg gtctcagaag 420agaagctttg
tttatgtctg gaagacctgg ggccaatact ggcaagttct agggggccca
480gtgtctgggc tgagcattgg gacaggcagg gcaatgctgg gcacacacac
catggaagtg 540actgtctacc atcgccgggg atcccagagc tatgtgcctc
ttgctcattc cagctcagcc 600ttcaccatta ctgaccaggt gcctttctcc
gtgagcgtgt cccagttgcg ggccttggat 660ggagggaaca agcacttcct
gagaaatcag 690122230PRTArtificial SequenceSynthetic peptide. 122Asp
Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr Thr Pro Thr 1 5 10
15 Thr Thr Lys Val Pro Arg Asn Gln Asp Trp Leu Gly Val Ser Arg Gln
20 25 30 Leu Arg Thr Lys Ala Trp Asn Arg Gln Leu Tyr Pro Glu Trp
Thr Glu 35 40 45 Ala Gln Arg Leu Asp Cys Trp Arg Gly Gly Gln Val
Ser Leu Lys Val 50 55 60 Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala
Asn Ala Ser Phe Ser Ile 65 70 75 80 Ala Leu Asn Phe Pro Gly Ser Gln
Lys Val Leu Pro Asp Gly Gln Val 85 90 95 Ile Trp Val Asn Asn Thr
Ile Ile Asn Gly Ser Gln Val Trp Gly Gly 100 105 110 Gln Pro Val Tyr
Pro Gln Glu Thr Asp Asp Ala Cys Ile Phe Pro Asp 115 120 125 Gly Gly
Pro Cys Pro Ser Gly Ser Trp Ser Gln Lys Arg Ser Phe Val 130 135 140
Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp Gln Val Leu Gly Gly Pro 145
150 155 160 Val Ser Gly Leu Ser Ile Gly Thr Gly Arg Ala Met Leu Gly
Thr His 165 170 175 Thr Met Glu Val Thr Val Tyr His Arg Arg Gly Ser
Gln Ser Tyr Val 180 185 190 Pro Leu Ala His Ser Ser Ser Ala Phe Thr
Ile Thr Asp Gln Val Pro 195 200 205 Phe Ser Val Ser Val Ser Gln Leu
Arg Ala Leu Asp Gly Gly Asn Lys 210 215 220 His Phe Leu Arg Asn Gln
225 230 123327DNAArtificial SequenceSynthetic oligonucleotide.
123ggcaccacag atgggcacag gccaactgca gaggccccta acaccacagc
tggccaagtg 60cctactacag aagttgtggg tactacacct ggtcaggcgc caactgcaga
gccctctgga 120accacatctg tgcaggtgcc aaccactgaa gtcataagca
ctgcacctgt gcagatgcca 180actgcagaga gcacaggtat gacacctgag
aaggtgccag tttcagaggt catgggtacc 240acactggcag agatgtcaac
tccagaggct acaggtatga cacctgcaga ggtatcaatt 300gtggtgcttt
ctggaaccac agctgca 327124109PRTArtificial SequenceSynthetic
peptide. 124Gly Thr Thr Asp Gly His Arg Pro Thr Ala Glu Ala Pro Asn
Thr Thr 1 5 10 15 Ala Gly Gln Val Pro Thr Thr Glu Val Val Gly Thr
Thr Pro Gly Gln 20 25 30 Ala Pro Thr Ala Glu Pro Ser Gly Thr Thr
Ser Val Gln Val Pro Thr 35 40 45 Thr Glu Val Ile Ser Thr Ala Pro
Val Gln Met Pro Thr Ala Glu Ser 50 55 60 Thr Gly Met Thr Pro Glu
Lys Val Pro Val Ser Glu Val Met Gly Thr 65 70 75 80 Thr Leu Ala Glu
Met Ser Thr Pro Glu Ala Thr Gly Met Thr Pro Ala 85 90 95 Glu Val
Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 100 105
125225DNAArtificial SequenceSynthetic oligonucleotide.
125caggtaacaa ctacagagtg ggtggagacc acagctagag agctacctat
ccctgagcct 60gaaggtccag atgccagctc aatcatgtct acggaaagta ttacaggttc
cctgggcccc 120ctgctggatg gtacagccac cttaaggctg gtgaagagac
aagtccccct ggattgtgtt 180ctgtatcgat atggttcctt ttccgtcacc
ctggacattg tccag 22512675PRTArtificial SequenceSynthetic
oligonucleotide. 126Gln Val Thr Thr Thr Glu Trp Val Glu Thr Thr Ala
Arg Glu Leu Pro 1 5 10 15 Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser
Ser Ile Met Ser Thr Glu 20 25 30 Ser Ile Thr Gly Ser Leu Gly Pro
Leu Leu Asp Gly Thr Ala Thr Leu 35 40 45 Arg Leu Val Lys Arg Gln
Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr 50 55 60 Gly Ser Phe Ser
Val Thr Leu Asp Ile Val Gln 65 70 75 127327DNAArtificial
SequenceSynthetic oligonucleotide. 127ggtattgaaa gtgccgagat
cctgcaggct gtgccgtccg gtgaggggga tgcatttgag 60ctgactgtgt cctgccaagg
cgggctgccc aaggaagcct gcatggagat ctcatcgcca 120gggtgccagc
cccctgccca gcggctgtgc cagcctgtgc tacccagccc agcctgccag
180ctggttctgc accagatact gaagggtggc tcggggacat actgcctcaa
tgtgtctctg 240gctgatacca acagcctggc agtggtcagc acccagctta
tcgtgcctgg gattcttctc 300acaggtcaag aagcaggcct tgggcag
327128109PRTArtificial SequenceSynthetic peptide. 128Gly Ile Glu
Ser Ala Glu Ile Leu Gln Ala Val Pro Ser Gly Glu Gly 1 5 10 15 Asp
Ala Phe Glu Leu Thr Val Ser Cys Gln Gly Gly Leu Pro Lys Glu 20 25
30 Ala Cys Met Glu Ile Ser Ser Pro Gly Cys Gln Pro Pro Ala Gln Arg
35 40 45 Leu Cys Gln Pro Val Leu Pro Ser Pro Ala Cys Gln Leu Val
Leu His 50 55 60 Gln Ile Leu Lys Gly Gly Ser Gly Thr Tyr Cys Leu
Asn Val Ser Leu 65 70 75 80 Ala Asp Thr Asn Ser Leu Ala Val Val Ser
Thr Gln Leu Ile Val Pro 85 90 95 Gly Ile Leu Leu Thr Gly Gln Glu
Ala Gly Leu Gly Gln 100 105 129219DNAArtificial SequenceSynthetic
oligonucleotide. 129cctctgacct ttgccctcca gctccatgac cctagtggct
atctggctga agctgacctc 60tcctacacct gggactttgg agacagtagt ggaaccctga
tctctcgggc acytgtggtc 120actcatactt acctggagcc tggcccagtc
actgcccagg tggtcctgca ggctgccatt 180cctctcacct cctgtggctc
ctccccagtt ccagctagc 21913073PRTArtificial SequenceSynthetic
peptide. 130Pro Leu Thr Phe Ala Leu Gln Leu His Asp Pro Ser Gly Tyr
Leu Ala 1 5 10 15 Glu Ala Asp Leu Ser Tyr Thr Trp Asp Phe Gly Asp
Ser Ser Gly Thr 20 25 30 Leu Ile Ser Arg Ala Xaa Val Val Thr His
Thr Tyr Leu Glu Pro Gly 35 40 45 Pro Val Thr Ala Gln Val Val Leu
Gln Ala Ala Ile Pro Leu Thr Ser 50 55 60 Cys Gly Ser Ser Pro Val
Pro Ala Ser 65 70 131438PRTArtificial SequenceSynthetic peptide.
131Met Ala Leu Arg Val Thr Arg Asn Ser Lys Ile Asn Ala Glu Asn Lys
1 5 10 15 Ala Lys Ile Asn Met Ala Gly Ala Lys Arg Val Pro Thr Ala
Pro Ala 20 25 30 Ala Thr Ser Lys Pro Gly Leu Arg Pro Arg Thr Ala
Leu Gly Asp Ile 35 40 45 Gly Asn Lys Val Ser Glu Gln Leu Gln Ala
Lys Met Pro Met Lys Lys 50 55 60 Glu Ala Lys Pro Ser Ala Thr Gly
Lys Val Ile Asp Lys Lys Leu Pro 65 70 75 80 Lys Pro Leu Glu Lys Val
Pro Met Leu Val Pro Val Pro Val Ser Glu 85 90 95 Pro Val Pro Glu
Pro Glu Pro Glu Pro Glu Pro Glu Pro Val Lys Glu 100 105 110 Glu Lys
Leu Ser Pro Glu Pro Ile Leu Val Asp Thr Ala Ser Pro Ser 115 120 125
Pro Met Glu Thr Ser Gly Cys Ala Pro Ala Glu Glu Asp Leu Cys Gln 130
135 140 Ala Phe Ser Asp Val Ile Leu Ala Val Asn Asp Val Asp Ala Glu
Asp 145 150 155 160 Gly Ala Asp Pro Asn Leu Cys Ser Glu Tyr Val Lys
Asp Ile Tyr Ala 165 170 175 Tyr Leu Arg Gln Leu Glu Glu Glu Gln Ala
Val Arg Pro Lys Tyr Leu 180 185 190 Leu Gly Arg Glu Val Thr Gly Asn
Met Arg Ala Ile Leu Ile Asp Trp 195 200 205 Leu Val Gln Val Gln Met
Lys Phe Arg Leu Leu Gln Glu Thr Met Tyr 210 215 220 Met Thr Val Ser
Ile Ile Asp Arg Phe Met Gln Asn Asn Cys Val Pro 225 230 235 240 Lys
Lys Met Leu Gln Leu Val Gly Val Thr Ala Met Phe Ile Ala Ser 245 250
255 Lys Tyr Glu Glu Met Tyr Pro Pro Glu Ile Gly Asp Phe Ala Phe Val
260 265 270 Thr Asp Asn Thr Tyr Thr Lys His Gln Ile Arg Gln Met Glu
Met Lys 275 280 285 Ile Leu Arg Ala Leu Asn Phe Gly Leu Gly Arg Pro
Leu Pro Leu His 290 295 300 Phe Leu Arg Arg Ala Ser Lys Ile Gly Glu
Val Asp Val Glu Gln His 305 310 315 320 Thr Leu Ala Lys Tyr Leu Met
Glu Thr Met Leu Asp Tyr Asp Met Val 325 330 335 His Phe Pro Pro Ser
Gln Ile Ala Ala Gly Ala Phe Cys Leu Ala Leu 340 345 350 Lys Ile Leu
Asp Asn Gly Glu Trp Thr Pro Thr Leu Gln His Tyr Leu 355 360 365 Ser
Tyr Thr Glu Glu Ser Leu Leu Pro Val Met Gln His Leu Ala Lys 370 375
380 Asn Val Val Met Val Asn Gln Gly Leu Thr Lys His Met Thr Val Lys
385 390 395 400 Asn Lys Tyr Ala Thr Ser Lys His Ala Lys Ile Ser Thr
Leu Pro Gln 405 410 415 Leu Asn Ser Ala Leu Val Gln Asp Leu Ala Lys
Ala Val Ala Lys Val 420 425 430 His His His His His His 435
13250PRTArtificial SequenceSynthetic peptide. 132Met Glu Met Lys
Ile Leu Arg Ala Leu Asn Phe Gly Leu Gly Arg Pro 1 5 10 15 Leu Pro
Leu His Phe Leu Arg Arg Ala Ser Lys Ile Gly Glu Val Asp 20 25 30
Val Glu Gln His Thr Leu Ala Lys Tyr Leu Met Glu Leu Thr Met Leu 35
40 45 Asp Tyr 50 13336PRTArtificial SequenceSynthetic peptide.
133Asp Trp Leu Val Gln Val Gln Met Lys Phe Arg Leu Leu Gln Glu Thr
1 5 10 15 Met Tyr Met Thr Val Ser Ile Ile Asp Arg Phe Met Gln Asn
Asn Cys 20 25 30 Val Pro Lys Lys 35 134594PRTArtificial
SequenceSynthetic peptide. 134Glu Val Lys Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala
Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val
Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile
Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr
Tyr Cys 85 90 95 Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Ala Lys Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly
Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310
315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser 435
440 445 Gln Thr Pro Thr Asn Thr Ile Ser Val Thr Pro Thr Asn Asn Ser
Thr 450 455 460 Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn Pro Ala Ser
Asp Trp Leu 465 470 475 480 Val Gln Val Gln Met Lys Phe Arg Leu Leu
Gln Glu Thr Met Tyr Met 485 490 495 Thr Val Ser Ile Ile Asp Arg Phe
Met Gln Asn Asn Cys Val Pro Lys 500 505 510 Lys Ala Ser Met Glu Met
Lys Ile Leu Arg Ala Leu Asn Phe Gly Leu 515 520 525 Gly Arg Pro Leu
Pro Leu His Phe Leu Arg Arg Ala Ser Lys Ile Gly 530 535 540 Glu Val
Asp Val Glu Gln His Thr Leu Ala Lys Tyr Leu Met Glu Leu 545 550 555
560 Thr Met Leu Asp Tyr Ala Ser Thr Asn Asp Ser Ile Thr Val Ala Ala
565 570 575 Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr Pro Ser
Ala Ala 580 585 590 Ala Ser 1351842DNAArtificial SequenceSynthetic
oligonucleotide. 135atgaacttgg ggctcagctt gattttcctt gtccttgttt
taaaaggtgt ccagtgtgaa 60gtgaagctgg tggagtctgg gggaggctta gtgcagcccg
gagggtccct gaaactctcc 120tgtgcaacct ctggattcac tttcagtgac
tattacatgt attgggttcg ccagactcca 180gagaagaggc tggagtgggt
cgcatacatt aattctggtg gtggtagcac ctattatcca 240gacactgtaa
agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg
300caaatgagcc ggctgaagtc tgaggacaca gccatgtatt actgtgcaag
acgggggtta 360ccgttccatg ctatggacta ttggggtcaa ggaacctcag
tcaccgtctc ctcagccaaa 420acgaagggcc catccgtctt ccccctggcg
ccctgctcca ggagcacctc cgagagcaca 480gccgccctgg gctgcctggt
caaggactac ttccccgaac cggtgacggt gtcgtggaac 540tcaggcgccc
tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc
600tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcacgaa
gacctacacc 660tgcaacgtag atcacaagcc cagcaacacc aaggtggaca
agagagttga gtccaaatat 720ggtcccccat gcccaccctg cccagcacct
gagttcgaag ggggaccatc agtcttcctg 780ttccccccaa aacccaagga
cactctcatg atctcccgga cccctgaggt cacgtgcgtg 840gtggtggacg
tgagccagga agaccccgag gtccagttca actggtacgt ggatggcgtg
900gaggtgcata atgccaagac aaagccgcgg gaggagcagt tcaacagcac
gtaccgtgtg 960gtcagcgtcc tcaccgtcct gcaccaggac tggctgaacg
gcaaggagta caagtgcaag 1020gtctccaaca aaggcctccc gtcctccatc
gagaaaacca tctccaaagc caaagggcag 1080ccccgagagc cacaggtgta
caccctgccc ccatcccagg aggagatgac caagaaccag 1140gtcagcctga
cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag
1200agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 1260tccttcttcc tctacagcag gctaaccgtg gacaagagca
ggtggcagga ggggaatgtc 1320ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacacagaa gagcctctcc 1380ctgtctctgg gtaaagctag
tcagaccccc accaacacca tcagcgtgac ccccaccaac 1440aacagcaccc
ccaccaacaa cagcaacccc aagcccaacc ccgctagtga ctggctagta
1500caggttcaaa tgaaattcag gttgttgcag gagaccatgt acatgactgt
ctccattatt 1560gatcggttca tgcagaataa ttgtgtgccc aagaaggcta
gtatggaaat gaagattcta 1620agagctttaa actttggtct gggtcggcct
ctacctttgc acttccttcg gagagcatct 1680aagattggag aggttgatgt
cgagcaacat actttggcca aatacctgat ggaactaact 1740atgttggact
atgctagtac caacgacagc atcaccgtgg ccgccaccgc ccccaccgtg
1800acccccaccg tgaacgccac ccccagcgcc gccgctagct ga
1842136542PRTArtificial SequenceSynthetic peptide. 136Glu Val Lys
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45 Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp
Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Arg Leu Lys Ser Glu Asp
Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Leu Pro Phe His
Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser
Ser Ala Lys Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu
Phe Glu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280
285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405
410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys Ala Ser 435 440 445 Gln Thr Pro Thr Asn Thr Ile Ser Val Thr Pro
Thr Asn Asn Ser Thr 450 455 460 Pro Thr Asn Asn Ser Asn Pro Lys Pro
Asn Pro Ala Ser Asp Trp Leu 465 470 475 480 Val Gln Val Gln Met Lys
Phe Arg Leu Leu Gln Glu Thr Met Tyr Met 485 490 495 Thr Val Ser Ile
Ile Asp Arg Phe Met Gln Asn Asn Cys Val Pro Lys 500 505 510 Lys Ala
Ser Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile 515 520 525
Val Thr Thr Ile Thr Pro Thr Ala Thr Thr Lys Pro Ala Ser 530 535 540
1371686DNAArtificial SequenceSynthetic oligonucleotide.
137atgaacttgg ggctcagctt gattttcctt gtccttgttt taaaaggtgt
ccagtgtgaa 60gtgaagctgg tggagtctgg gggaggctta gtgcagcccg gagggtccct
gaaactctcc 120tgtgcaacct ctggattcac tttcagtgac tattacatgt
attgggttcg ccagactcca 180gagaagaggc tggagtgggt cgcatacatt
aattctggtg gtggtagcac ctattatcca 240gacactgtaa agggccgatt
caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagcc
ggctgaagtc tgaggacaca gccatgtatt actgtgcaag acgggggtta
360ccgttccatg ctatggacta ttggggtcaa ggaacctcag tcaccgtctc
ctcagccaaa 420acgaagggcc catccgtctt ccccctggcg ccctgctcca
ggagcacctc cgagagcaca 480gccgccctgg gctgcctggt caaggactac
ttccccgaac cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg
cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600tactccctca
gcagcgtggt gaccgtgccc tccagcagct tgggcacgaa gacctacacc
660tgcaacgtag atcacaagcc cagcaacacc aaggtggaca agagagttga
gtccaaatat 720ggtcccccat gcccaccctg cccagcacct gagttcgaag
ggggaccatc agtcttcctg 780ttccccccaa aacccaagga cactctcatg
atctcccgga cccctgaggt cacgtgcgtg 840gtggtggacg tgagccagga
agaccccgag gtccagttca actggtacgt ggatggcgtg 900gaggtgcata
atgccaagac aaagccgcgg gaggagcagt tcaacagcac gtaccgtgtg
960gtcagcgtcc tcaccgtcct gcaccaggac tggctgaacg gcaaggagta
caagtgcaag 1020gtctccaaca aaggcctccc gtcctccatc gagaaaacca
tctccaaagc caaagggcag 1080ccccgagagc cacaggtgta caccctgccc
ccatcccagg aggagatgac caagaaccag 1140gtcagcctga cctgcctggt
caaaggcttc taccccagcg acatcgccgt ggagtgggag 1200agcaatgggc
agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc
1260tccttcttcc tctacagcag gctaaccgtg gacaagagca ggtggcagga
ggggaatgtc 1320ttctcatgct ccgtgatgca tgaggctctg cacaaccact
acacacagaa gagcctctcc 1380ctgtctctgg gtaaagctag tcagaccccc
accaacacca tcagcgtgac ccccaccaac 1440aacagcaccc ccaccaacaa
cagcaacccc aagcccaacc ccgctagtga ctggctagta 1500caggttcaaa
tgaaattcag gttgttgcag gagaccatgt acatgactgt ctccattatt
1560gatcggttca tgcagaataa ttgtgtgccc aagaaggcta gtaccgtgac
ccccaccgcc 1620accgccaccc ccagcgccat cgtgaccacc atcaccccca
ccgccaccac caagcccgct 1680agctga 1686138556PRTArtificial
SequenceSynthetic peptide. 138Glu Val Lys Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala
Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val
Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile
Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Ala Lys Thr Lys
Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195
200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro
Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315
320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser 435 440
445 Gln Thr Pro Thr Asn Thr Ile Ser Val Thr Pro Thr Asn Asn Ser Thr
450 455 460 Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn Pro Ala Ser Met
Glu Met 465 470 475 480 Lys Ile Leu Arg Ala Leu Asn Phe Gly Leu Gly
Arg Pro Leu Pro Leu 485 490 495 His Phe Leu Arg Arg Ala Ser Lys Ile
Gly Glu Val Asp Val Glu Gln 500 505 510 His Thr Leu Ala Lys Tyr Leu
Met Glu Leu Thr Met Leu Asp Tyr Ala 515 520 525 Ser Thr Asn Gly Ser
Ile Thr Val Ala Ala Thr Ala Pro Thr Val Thr 530 535 540 Pro Thr Val
Asn Ala Thr Pro Ser Ala Ala Ala Ser 545 550 555
1391728DNAArtificial SequenceSynthetic oligonucleotide.
139atgaacttgg ggctcagctt gattttcctt gtccttgttt taaaaggtgt
ccagtgtgaa 60gtgaagctgg tggagtctgg gggaggctta gtgcagcccg gagggtccct
gaaactctcc 120tgtgcaacct ctggattcac tttcagtgac tattacatgt
attgggttcg ccagactcca 180gagaagaggc tggagtgggt cgcatacatt
aattctggtg gtggtagcac ctattatcca 240gacactgtaa agggccgatt
caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagcc
ggctgaagtc tgaggacaca gccatgtatt actgtgcaag acgggggtta
360ccgttccatg ctatggacta ttggggtcaa ggaacctcag tcaccgtctc
ctcagccaaa 420acgaagggcc catccgtctt ccccctggcg ccctgctcca
ggagcacctc cgagagcaca 480gccgccctgg gctgcctggt caaggactac
ttccccgaac cggtgacggt gtcgtggaac 540tcaggcgccc
tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc
600tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcacgaa
gacctacacc 660tgcaacgtag atcacaagcc cagcaacacc aaggtggaca
agagagttga gtccaaatat 720ggtcccccat gcccaccctg cccagcacct
gagttcgaag ggggaccatc agtcttcctg 780ttccccccaa aacccaagga
cactctcatg atctcccgga cccctgaggt cacgtgcgtg 840gtggtggacg
tgagccagga agaccccgag gtccagttca actggtacgt ggatggcgtg
900gaggtgcata atgccaagac aaagccgcgg gaggagcagt tcaacagcac
gtaccgtgtg 960gtcagcgtcc tcaccgtcct gcaccaggac tggctgaacg
gcaaggagta caagtgcaag 1020gtctccaaca aaggcctccc gtcctccatc
gagaaaacca tctccaaagc caaagggcag 1080ccccgagagc cacaggtgta
caccctgccc ccatcccagg aggagatgac caagaaccag 1140gtcagcctga
cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag
1200agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 1260tccttcttcc tctacagcag gctaaccgtg gacaagagca
ggtggcagga ggggaatgtc 1320ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacacagaa gagcctctcc 1380ctgtctctgg gtaaagctag
tcagaccccc accaacacca tcagcgtgac ccccaccaac 1440aacagcaccc
ccaccaacaa cagcaacccc aagcccaacc ccgctagtat ggaaatgaag
1500attctaagag ctttaaactt tggtctgggt cggcctctac ctttgcactt
ccttcggaga 1560gcatctaaga ttggagaggt tgatgtcgag caacatactt
tggccaaata cctgatggaa 1620ctaactatgt tggactatgc tagtaccaac
ggcagcatca ccgtggccgc caccgccccc 1680accgtgaccc ccaccgtgaa
cgccaccccc agcgccgccg ctagctga 1728140294PRTArtificial
SequenceSynthetic peptide. 140Glu His Gln Leu Leu Cys Cys Glu Val
Glu Thr Ile Arg Arg Ala Tyr 1 5 10 15 Pro Asp Ala Asn Leu Leu Asn
Asp Arg Val Leu Arg Ala Met Leu Lys 20 25 30 Ala Glu Glu Thr Cys
Ala Pro Ser Val Ser Tyr Phe Lys Cys Val Gln 35 40 45 Lys Glu Val
Leu Pro Ser Met Arg Lys Ile Val Ala Thr Trp Met Leu 50 55 60 Glu
Val Cys Glu Glu Gln Lys Cys Glu Glu Glu Val Phe Pro Leu Ala 65 70
75 80 Met Asn Tyr Leu Asp Arg Phe Leu Ser Leu Glu Pro Val Lys Lys
Ser 85 90 95 Arg Leu Gln Leu Leu Gly Ala Thr Cys Met Phe Val Ala
Ser Lys Met 100 105 110 Lys Glu Thr Ile Pro Leu Thr Ala Glu Lys Leu
Cys Ile Tyr Thr Asp 115 120 125 Asn Ser Ile Arg Pro Glu Glu Leu Leu
Gln Met Glu Leu Leu Leu Val 130 135 140 Asn Lys Leu Lys Trp Asn Leu
Ala Ala Met Thr Pro His Asp Phe Ile 145 150 155 160 Glu His Phe Leu
Ser Lys Met Pro Glu Ala Glu Glu Asn Lys Gln Ile 165 170 175 Ile Arg
Lys His Ala Gln Thr Phe Val Ala Leu Cys Ala Thr Asp Val 180 185 190
Lys Phe Ile Ser Asn Pro Pro Ser Met Val Ala Ala Gly Ser Val Val 195
200 205 Ala Ala Val Gln Gly Leu Asn Leu Arg Ser Pro Asn Asn Phe Leu
Ser 210 215 220 Tyr Tyr Arg Leu Thr Arg Phe Leu Ser Arg Val Ile Lys
Cys Asp Pro 225 230 235 240 Asp Cys Leu Arg Ala Cys Gln Glu Gln Ile
Glu Ala Leu Leu Glu Ser 245 250 255 Ser Leu Arg Gln Ala Gln Gln Asn
Met Asp Pro Lys Ala Ala Glu Glu 260 265 270 Glu Glu Glu Glu Glu Glu
Glu Val Asp Leu Ala Cys Thr Pro Thr Asp 275 280 285 Val Arg Asp Val
Asp Ile 290 14148PRTArtificial SequenceSynthetic peptide. 141Met
Glu His Gln Leu Leu Cys Cys Glu Val Glu Thr Ile Arg Arg Ala 1 5 10
15 Tyr Pro Asp Ala Asn Leu Leu Asn Asp Arg Val Leu Arg Ala Met Leu
20 25 30 Lys Ala Glu Glu Thr Cys Ala Pro Ser Val Ser Tyr Phe Lys
Cys Val 35 40 45 14295PRTArtificial SequenceSynthetic peptide.
142Gln Lys Glu Val Leu Pro Ser Met Arg Lys Ile Val Ala Thr Trp Met
1 5 10 15 Leu Glu Val Cys Glu Glu Gln Lys Cys Glu Glu Glu Val Phe
Pro Leu 20 25 30 Ala Met Asn Tyr Leu Asp Arg Phe Leu Ser Leu Glu
Pro Val Lys Lys 35 40 45 Ser Arg Leu Gln Leu Leu Gly Ala Thr Cys
Met Phe Val Ala Ser Lys 50 55 60 Met Lys Glu Thr Ile Pro Leu Thr
Ala Glu Lys Leu Cys Ile Tyr Thr 65 70 75 80 Asp Asn Ser Ile Arg Pro
Glu Glu Leu Leu Gln Met Glu Leu Leu 85 90 95 14360PRTArtificial
SequenceSynthetic peptide. 143Leu Val Asn Lys Leu Lys Trp Asn Leu
Ala Ala Met Thr Pro His Asp 1 5 10 15 Phe Ile Glu His Phe Leu Ser
Lys Met Pro Glu Ala Glu Glu Asn Lys 20 25 30 Gln Ile Ile Arg Lys
His Ala Gln Thr Phe Val Ala Leu Cys Ala Thr 35 40 45 Asp Val Lys
Phe Ile Ser Asn Pro Pro Ser Met Val 50 55 60 14492PRTArtificial
SequenceSynthetic peptide. 144Ala Ala Gly Ser Val Val Ala Ala Val
Gln Gly Leu Asn Leu Arg Ser 1 5 10 15 Pro Asn Asn Phe Leu Ser Tyr
Tyr Arg Leu Thr Arg Phe Leu Ser Arg 20 25 30 Val Ile Lys Cys Asp
Pro Asp Cys Leu Arg Ala Cys Gln Glu Gln Ile 35 40 45 Glu Ala Leu
Leu Glu Ser Ser Leu Arg Gln Ala Gln Gln Asn Met Asp 50 55 60 Pro
Lys Ala Ala Glu Glu Glu Glu Glu Glu Glu Glu Glu Val Asp Leu 65 70
75 80 Ala Cys Thr Pro Thr Asp Val Arg Asp Val Asp Ile 85 90
14527PRTArtificial SequenceSynthetic peptide. 145Gln Thr Pro Thr
Asn Thr Ile Ser Val Thr Pro Thr Asn Asn Ser Thr 1 5 10 15 Pro Thr
Asn Asn Ser Asn Pro Lys Pro Asn Pro 20 25 146560PRTArtificial
SequenceSynthetic peptide. 146Gln Val Thr Leu Lys Glu Ser Gly Pro
Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Leu Ser
Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala
His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60 Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val 65 70
75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp Ala Ala Thr Tyr
Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr Gly Tyr Gly
Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195
200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300 Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315
320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415 Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445 Ser Leu Gly Lys Ala Ser Gln Thr Pro Thr Asn Thr Ile Ser Val Thr
450 455 460 Pro Thr Asn Asn Ser Thr Pro Thr Asn Asn Ser Asn Pro Lys
Pro Asn 465 470 475 480 Pro Ala Ser Met Glu His Gln Leu Leu Cys Cys
Glu Val Glu Thr Ile 485 490 495 Arg Arg Ala Tyr Pro Asp Ala Asn Leu
Leu Asn Asp Arg Val Leu Arg 500 505 510 Ala Met Leu Lys Ala Glu Glu
Thr Cys Ala Pro Ser Val Ser Tyr Phe 515 520 525 Lys Cys Val Ala Ser
Thr Asn Gly Ser Ile Thr Val Ala Ala Thr Ala 530 535 540 Pro Thr Val
Thr Pro Thr Val Asn Ala Thr Pro Ser Ala Ala Ala Ser 545 550 555 560
147759PRTArtificial SequenceSynthetic peptide. 147Gln Val Thr Leu
Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu
Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30
Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35
40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro
Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser
Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp
Ala Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Ser His Tyr Tyr Gly
Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110 Asp Val Trp Gly Ala Gly Thr
Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125 Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165
170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro
Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290
295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410
415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 435 440 445 Ser Leu Gly Lys Ala Ser Gln Thr Pro Thr Asn Thr
Ile Ser Val Thr 450 455 460 Pro Thr Asn Asn Ser Thr Pro Thr Asn Asn
Ser Asn Pro Lys Pro Asn 465 470 475 480 Pro Ala Ser Gln Lys Glu Val
Leu Pro Ser Met Arg Lys Ile Val Ala 485 490 495 Thr Trp Met Leu Glu
Val Cys Glu Glu Gln Lys Cys Glu Glu Glu Val 500 505 510 Phe Pro Leu
Ala Met Asn Tyr Leu Asp Arg Phe Leu Ser Leu Glu Pro 515 520 525 Val
Lys Lys Ser Arg Leu Gln Leu Leu Gly Ala Thr Cys Met Phe Val 530 535
540 Ala Ser Lys Met Lys Glu Thr Ile Pro Leu Thr Ala Glu Lys Leu Cys
545 550 555 560 Ile Tyr Thr Asp Asn Ser Ile Arg Pro Glu Glu Leu Leu
Gln Met Glu 565 570 575 Leu Leu Leu Val Asn Lys Leu Lys Trp Asn Leu
Ala Ala Met Thr Pro 580 585 590 His Asp Phe Ile Glu His Phe Leu Ser
Lys Met Pro Glu Ala Glu Glu 595 600 605 Asn Lys Gln Ile Ile Arg Lys
His Ala Gln Thr Phe Val Ala Leu Cys 610 615 620 Ala Thr Asp Val Lys
Phe Ile Ser Asn Pro Pro Ser Met Val Ala Ala 625 630 635 640 Gly Ser
Val Val Ala Ala Val Gln Gly Leu Asn Leu Arg Ser Pro Asn 645 650 655
Asn Phe Leu Ser Tyr Tyr Arg Leu Thr Arg Phe Leu Ser Arg Val Ile 660
665 670 Lys Cys Asp Pro Asp Cys Leu Arg Ala Cys Gln Glu Gln Ile Glu
Ala 675 680 685 Leu Leu Glu Ser Ser Leu Arg Gln Ala Gln Gln Asn Met
Asp Pro Lys 690 695 700 Ala Ala Glu Glu Glu Glu Glu Glu Glu Glu Glu
Val Asp Leu Ala Cys 705 710 715 720 Thr Pro Thr Asp Val Arg Asp Val
Asp Ile Ala Ser Thr Asn Gly Ser 725 730 735 Ile Thr Val Ala Ala Thr
Ala Pro Thr Val Thr Pro Thr Val Asn Ala 740 745 750 Thr Pro Ser Ala
Ala Ala Ser 755 148468PRTArtificial SequenceSynthetic peptide.
148Met Asn Leu Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly
1 5 10 15 Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu
Val Gln 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr Ser
Gly Phe Thr Phe 35 40 45 Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln
Thr Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Tyr Ile Asn Ser
Gly Gly Gly Ser Thr Tyr Tyr Pro 65 70 75 80 Asp Thr Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu
Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr
Cys Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp 115 120 125
Gly
Gln Gly Thr Ser Val Thr Phe Val Ser Ser Ala Lys Thr Lys Gly 130 135
140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr Tyr Thr Cys Asn Val 210 215 220 Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 225 230 235 240 Tyr Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly 245 250 255
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260
265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu 275 280 285 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 340 345 350 Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385
390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu 450 455 460 Gly Lys Ala Ser 465
149234PRTArtificial SequenceSynthetic peptide. 149Met Met Ser Ser
Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln 1 5 10 15 Gly Thr
Arg Cys Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser 20 25 30
Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly 35
40 45 Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
Val 50 55 60 Lys Leu Leu Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly
Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr
Ser Leu Thr Ile Gly 85 90 95 Asn Leu Glu Pro Glu Asp Ile Ala Thr
Tyr Tyr Cys Gln Gln Phe Asn 100 105 110 Lys Leu Pro Pro Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165
170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 225 230 150203PRTArtificial SequenceSynthetic peptide. 150Met
Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10
15 Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr
Thr Phe 35 40 45 Thr Asp Tyr Val Leu His Trp Val Lys Gln Lys Pro
Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn
Asp Gly Thr Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr
Leu Thr Ser Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Glu Leu
Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala
Arg Gly Tyr Pro Ala Tyr Ser Gly Tyr Ala Met Asp 115 120 125 Tyr Trp
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 130 135 140
Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn 145
150 155 160 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro
Glu Pro 165 170 175 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Lys Gly Glu Phe
Val 195 200 151234PRTArtificial SequenceSynthetic peptide. 151Met
Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln 1 5 10
15 Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser
20 25 30 Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
Gln Asp 35 40 45 Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
Asp Gly Thr Val 50 55 60 Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu
His Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Tyr Ser Leu Thr Ile Ser 85 90 95 Asn Leu Glu Gln Glu Asp
Ile Ala Thr Tyr Phe Cys His His Gly Asn 100 105 110 Thr Leu Pro Trp
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Ala Asp
Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 130 135 140
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 145
150 155 160 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu
Arg Gln 165 170 175 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser
Lys Asp Ser Thr 180 185 190 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr
Lys Asp Glu Tyr Glu Arg 195 200 205 His Asn Ser Tyr Thr Cys Glu Ala
Thr His Lys Thr Ser Thr Ser Pro 210 215 220 Ile Val Lys Ser Phe Asn
Arg Asn Glu Cys 225 230 152235PRTArtificial SequenceSynthetic
peptide. 152Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser
Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln
Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr
Met Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met Tyr Arg
Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr
Gly Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser
Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr 100 105 110
His Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115
120 125 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser
Glu 130 135 140 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu
Asn Asn Phe 145 150 155 160 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys
Ile Asp Gly Ser Glu Arg 165 170 175 Gln Asn Gly Val Leu Asn Ser Trp
Thr Asp Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Met Ser Ser
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 195 200 205 Arg His Asn Ser
Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 210 215 220 Pro Ile
Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 153196PRTArtificial
SequenceSynthetic peptide. 153Met Gly Trp Ser Trp Ile Phe Leu Phe
Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val Leu Ser Glu Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45 Thr Gly Tyr
Tyr Met His Trp Val Lys Gln Ser His Val Lys Ser Leu 50 55 60 Glu
Trp Ile Gly Arg Ile Asn Pro Tyr Asn Gly Ala Thr Ser Tyr Asn 65 70
75 80 Gln Asn Phe Lys Asp Lys Ala Ser Leu Thr Val Asp Lys Ser Ser
Ser 85 90 95 Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser Glu Asp
Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Glu Asp Tyr Val Tyr Trp
Gly Gln Gly Thr Thr 115 120 125 Leu Thr Val Ser Ser Ala Lys Thr Thr
Pro Pro Ser Val Tyr Pro Leu 130 135 140 Ala Pro Gly Ser Ala Ala Gln
Thr Asn Ser Met Val Thr Leu Gly Cys 145 150 155 160 Leu Val Lys Gly
Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser 165 170 175 Gly Ser
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Lys 180 185 190
Gly Glu Phe Val 195 154238PRTArtificial SequenceSynthetic peptide.
154Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala
1 5 10 15 Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu
Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu 35 40 45 Val His Ser Asn Gly Asn Thr Tyr Leu His
Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Ala 85 90 95 Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 100 105 110 Ser Gln
Ser Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125
Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 130
135 140 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe
Leu 145 150 155 160 Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp
Lys Ile Asp Gly 165 170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser
Trp Thr Asp Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Met Ser
Ser Thr Leu Thr Leu Thr Lys Asp 195 200 205 Glu Tyr Glu Arg His Asn
Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210 215 220 Ser Thr Ser Pro
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235
155705DNAArtificial SequenceSynthetic oligonucleotide.
155atgatgtcct ctgctcagtt ccttggtctc ctgttgctct gttttcaagg
taccagatgt 60gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctaggaga
cagagtcacc 120atcagttgca gtgcaagtca gggcattagc aattatttaa
actggtatca gcagaaacca 180gatggaactg ttaaactcct gatctattac
acatcaattt tacactcagg agtcccatca 240aggttcagtg gcagtgggtc
tgggacagat tattctctca ccatcggcaa cctggaacct 300gaagatattg
ccacttacta ttgtcagcag tttaataagc ttcctccgac gttcggtgga
360ggcaccaaac tcgagatcaa acgaactgtg gctgcaccat ctgtcttcat
cttcccgcca 420tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 480cccagagagg ccaaagtaca gtggaaggtg
gataacgccc tccaatcggg taactcccag 540gagagtgtca cagagcagga
cagcaaggac agcacctaca gcctcagcag caccctgacg 600ctgagcaaag
cagactacga gaaacacaaa gtctatgcct gcgaagtcac ccatcagggc
660ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag
7051561404DNAArtificial SequenceSynthetic oligonucleotide.
156atgaacttgg ggctcagctt gattttcctt gtccttgttt taaaaggtgt
ccagtgtgaa 60gtgaagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct
gaaactctcc 120tgtgcaacct ctggattcac tttcagtgac tattacatgt
attgggttcg ccagactcca 180gagaagaggc tggagtgggt cgcatacatt
aattctggtg gtggtagcac ctattatcca 240gacactgtaa agggccgatt
caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagcc
ggctgaagtc tgaggacaca gccatgtatt actgtgcaag acgggggtta
360ccgttccatg ctatggacta ttggggtcaa ggaacctcag tcaccgtctc
ctcagccaaa 420acgaagggcc catccgtctt ccccctggcg ccctgctcca
ggagcacctc cgagagcaca 480gccgccctgg gctgcctggt caaggactac
ttccccgaac cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg
cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600tactccctca
gcagcgtggt gaccgtgccc tccagcagct tgggcacgaa gacctacacc
660tgcaacgtag atcacaagcc cagcaacacc aaggtggaca agagagttga
gtccaaatat 720ggtcccccat gcccaccctg cccagcacct gagttcgaag
ggggaccatc agtcttcctg 780ttccccccaa aacccaagga cactctcatg
atctcccgga cccctgaggt cacgtgcgtg 840gtggtggacg tgagccagga
agaccccgag gtccagttca actggtacgt ggatggcgtg 900gaggtgcata
atgccaagac aaagccgcgg gaggagcagt tcaacagcac gtaccgtgtg
960gtcagcgtcc tcaccgtcct gcaccaggac tggctgaacg gcaaggagta
caagtgcaag 1020gtctccaaca aaggcctccc gtcctccatc gagaaaacca
tctccaaagc caaagggcag 1080ccccgagagc cacaggtgta caccctgccc
ccatcccagg aggagatgac caagaaccag 1140gtcagcctga cctgcctggt
caaaggcttc taccccagcg acatcgccgt ggagtgggag 1200agcaatgggc
agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc
1260tccttcttcc tctacagcag gctaaccgtg gacaagagca ggtggcagga
ggggaatgtc 1320ttctcatgct ccgtgatgca tgaggctctg cacaaccact
acacacagaa gagcctctcc 1380ctgtctctgg gtaaagctag ctga
14041571410DNAArtificial SequenceSynthetic oligonucleotide.
157atggaatgga gttggatatt tctctttctt ctgtcaggaa ctgcaggtgt
ccactctgag 60gtccagctgc agcagtctgg acctgagctg gtaaagcctg gggcttcagt
gaagatgtcc 120tgcaaggctt ctggatacac attcactgac tatgttttgc
actgggtgaa acagaagcct 180gggcagggcc ttgagtggat tggatatatt
aatccttaca atgatggtac taagtacaat 240gagaagttca aaggcaaggc
cacactgact tcagacaaat cctccagcac agcctacatg 300gagctcagca
gcctgacctc tgaggactct gcggtctatt actgtgcaag gggctatccg
360gcctactctg ggtatgctat ggactactgg ggtcaaggaa cctcagtcac
cgtctcctca 420gccaaaacga agggcccatc cgtcttcccc ctggcgccct
gctccaggag cacctccgag 480agcacagccg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 540tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc
660tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag
agttgagtcc 720aaatatggtc ccccatgccc accctgccca gcacctgagt
tcgaaggggg accatcagtc 780ttcctgttcc ccccaaaacc caaggacact
ctcatgatct cccggacccc tgaggtcacg 840tgcgtggtgg tggacgtgag
ccaggaagac cccgaggtcc agttcaactg gtacgtggat 900ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac
960cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa
ggagtacaag 1020tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga
aaaccatctc caaagccaaa 1080gggcagcccc gagagccaca ggtgtacacc
ctgcccccat cccaggagga gatgaccaag 1140aaccaggtca gcctgacctg
cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1200tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
1260gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg
gcaggagggg 1320aatgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac acagaagagc 1380ctctccctgt ctctgggtaa agctagctga
1410158708DNAArtificial SequenceSynthetic oligonucleotide.
158atggattttc
aagtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60aggggacaaa
ttgttctcac ccagtctcca gcaatcctgt ctgcatctcc aggggagaag
120gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgtacaggta
ccagcagaag 180ccaggatcct cacccaaacc ctggatttat ggcacatcca
acctggcttc tggagtccct 240gctcgcttca gtggcagtgg atctgggacc
tcttattctc tcacaatcag cagcatggag 300gctgaagatg ctgccactta
ttactgccag caatatcata gttacccgct cacgttcggt 360gctgggacca
agctcgagat caaacgaact gtggctgcac catctgtctt catcttcccg
420ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 480tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 540caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 600acgctgagca aagcagacta
cgagaaacac aaagtctatg cctgcgaagt cacccatcag 660ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgttag 708159705DNAArtificial
SequenceSynthetic oligonucleotide. 159atgatgtcct ctgctcagtt
ccttggtctc ctgttgctct gttttcaagg taccagatgt 60gatatccaga tgacacagac
tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 120atcagttgca
gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca
180gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg
agtcccatca 240aggttcagtg gcagtgggtc tggaacagat tattctctca
ccattagcaa cctggagcaa 300gaagatattg ccacttactt ttgccatcat
ggtaatacgc ttccgtggac gttcggtgga 360ggcaccaagc tcgagatcaa
acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
480cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 540gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 600ctgagcaaag cagactacga gaaacacaaa
gtctatgcct gcgaagtcac ccatcagggc 660ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gttag 7051601389DNAArtificial
SequenceSynthetic oligonucleotide. 160atgggatgga gctggatctt
tctctttctc ctgtcaggaa ctgcaggtgt cctctctgag 60gtccagctgc aacagtctgg
acctgagctg gtgaagcctg gggcttcagt gaagatatcc 120tgcaaggctt
ctggttactc attcactggc tactacatgc actgggtgaa gcaaagccat
180gtaaagagcc ttgagtggat tggacgtatt aatccttaca atggtgctac
tagctacaac 240cagaatttca aggacaaggc cagcttgact gtagataagt
cctccagcac agcctacatg 300gagctccaca gcctgacatc tgaggactct
gcagtctatt actgtgcaag agaggactac 360gtctactggg gccaaggcac
cactctcaca gtctcctcag ccaaaacgaa gggcccatcc 420gtcttccccc
tggcgccctg ctccaggagc acctccgaga gcacagccgc cctgggctgc
480ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg
cgccctgacc 540agcggcgtgc acaccttccc ggctgtccta cagtcctcag
gactctactc cctcagcagc 600gtggtgaccg tgccctccag cagcttgggc
acgaagacct acacctgcaa cgtagatcac 660aagcccagca acaccaaggt
ggacaagaga gttgagtcca aatatggtcc cccatgccca 720ccctgcccag
cacctgagtt cgaaggggga ccatcagtct tcctgttccc cccaaaaccc
780aaggacactc tcatgatctc ccggacccct gaggtcacgt gcgtggtggt
ggacgtgagc 840caggaagacc ccgaggtcca gttcaactgg tacgtggatg
gcgtggaggt gcataatgcc 900aagacaaagc cgcgggagga gcagttcaac
agcacgtacc gtgtggtcag cgtcctcacc 960gtcctgcacc aggactggct
gaacggcaag gagtacaagt gcaaggtctc caacaaaggc 1020ctcccgtcct
ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agagccacag
1080gtgtacaccc tgcccccatc ccaggaggag atgaccaaga accaggtcag
cctgacctgc 1140ctggtcaaag gcttctaccc cagcgacatc gccgtggagt
gggagagcaa tgggcagccg 1200gagaacaact acaagaccac gcctcccgtg
ctggactccg acggctcctt cttcctctac 1260agcaggctaa ccgtggacaa
gagcaggtgg caggagggga atgtcttctc atgctccgtg 1320atgcatgagg
ctctgcacaa ccactacaca cagaagagcc tctccctgtc tctgggtaaa
1380gctagctga 1389161717DNAArtificial SequenceSynthetic
oligonucleotide. 161atgaagttgc ctgttaggct gttggtgctg atgttctgga
ttcctgcttc cagcagtgat 60gttgtgatga cccaaactcc actctccctg cctgtcagtc
ttggagatca agcctccatc 120tcttgcagat ctagtcagag ccttgtacac
agtaatggaa acacctattt acattggtac 180ctgcagaagc caggccagtc
tccaaagctc ctgatctaca aagtttccaa ccgattttct 240ggggtcccag
acaggttcag tggcagtgga tcagggacag atttcgcact caagatcagt
300agagtggagg ctgaggatct gggagtttat ttctgctctc aaagtacaca
tgttccgtgg 360acgttcggtg gaggcaccaa gctcgagatc aaacgaactg
tggctgcacc atctgtcttc 420atcttcccgc catctgatga gcagttgaaa
tctggaactg cctctgttgt gtgcctgctg 480aataacttct atcccagaga
ggccaaagta cagtggaagg tggataacgc cctccaatcg 540ggtaactccc
aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc
600agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctatgc
ctgcgaagtc 660acccatcagg gcctgagctc gcccgtcaca aagagcttca
acaggggaga gtgttag 717
* * * * *