ANTIGEN PRESENTING CELL TARGETED CANCER VACCINES
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.
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), β-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:
(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:
(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:
(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:
(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 — 12E12.3F3 (ATCC Accession No. PTA-9854), anti-CD40 — 12B4.2C10 (ATCC Submission No. HS446, Accession No. ______), and anti-CD40 — 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:
(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:
(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 — 12E12.3F3 (ATCC Accession No. PTA-9854), anti-CD40 — 12B4.2C10 (ATCC Submission No. HS446, Accession No. ______), and anti-CD40 — 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), β-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:
(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:
(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:
(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:
(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:
(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), β-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 — 12E12.3F3 (ATCC Accession No. PTA-9854), anti-CD40 — 12B4.2C10 (ATCC Submission No. HS446, Accession No. ______), and anti-CD40 — 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), β-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:
(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:
(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:
(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:
(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 αCD40-(PL-Ag)x or αCD40-(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 αCD40-(PL-Ag)x or αCD40-(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.